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Granulin Is a Soluble Cofactor for Toll-like Receptor 9 Signaling
Abstract
Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling.
... Progranulin is expressed in both the embryo and placenta, where it modulates embryo growth [1] and implantation [2], as well as placenta formation [3]. In adult tissues, progranulin regulates tissue regeneration [4,5], promotes angiogenesis [6], modulates the immune response [7,8] and is implicated in host defense against bacterial infections [8,9]. In addition, progranulin is a key neurotrophic factor as, in fact, it promotes neuronal survival and neurite growth [10,11], modulates neuroinflammation [12] and regulates lysosome function in neurons [13,14]. ...
... Progranulin can bind several membrane proteins and cell membrane receptors, such as sortilin [13], prosaposin [55], tumor-necrosis factor receptor (TNFR) 1 and 2 [7], DR3 [56], four Notch receptors [57], DLK1 [58], EphA2 [59], RET [48] and Toll-like receptor (TLR)9 [9], and these interactions are highly context-dependent. ...
... Finally, progranulin binds to both TLR9 and CpG oligonucleotides (CpG-ODNs) in immune cells and endosomes, favoring TLR9 and CpG-ODNs interaction and potentiating the innate immune response to bacterial infections [9]. Notably, it has been reported that progranulin can activate other receptor-tyrosine kinases, including members of the Eph family, such as EphA4 and EphB2 [48,59], EGFR [48,59,65], ErbB2 [48] and RYK [65]. ...
Progranulin is a pleiotropic growth factor with important physiological roles in embryogenesis and maintenance of adult tissue homeostasis. While-progranulin deficiency is associated with a broad range of pathological conditions affecting the brain, such as frontotemporal dementia and neuronal ceroid lipofuscinosis, progranulin upregulation characterizes many tumors, including brain tumors, multiple myeloma, leiomyosarcoma, mesothelioma and epithelial cancers such as ovarian, liver, breast, bladder, adrenal, prostate and kidney carcinomas. The increase of progranulin levels in tumors might have diagnostic and prognostic significance. In cancer, progranulin has a pro-tumorigenic role by promoting cancer cell proliferation, migration, invasiveness, anchorage-independent growth and resistance to chemotherapy. In addition, progranulin regulates the tumor microenvironment, affects the function of cancer-associated fibroblasts, and modulates tumor immune surveillance. However, the molecular mechanisms of progranulin oncogenic function are not fully elucidated. In bladder cancer, progranulin action relies on the activation of its functional signaling receptor EphA2. Notably, more recent data suggest that progranulin can also modulate a functional crosstalk between multiple receptor-tyrosine kinases, demonstrating a more complex and context-dependent role of progranulin in cancer. Here, we will review what is currently known about the function of progranulin in tumors, with a focus on its molecular mechanisms of action and regulation.
... Interestingly, CpG PS-ASO-protein interactions have been shown to play a role TLR9 signaling. For example, PS-ASOs bound to either HMGB1 or Granulin can more productively activate TLR9 as a complex than with the PS-ASO administered alone (24)(25)(26). Given the extent of known PS-ASO-plasma protein interactions (27), we have been interested to determine whether any other plasma protein-PS-ASO complexes play a role in innate immune activation and to understand the molecular events that are involved in this process. ...
... Interactions between PS-ASOs and plasma proteins have been documented (27), and the effects of PS-ASO-protein interactions in TLR9 activation has also been described for some proteins. For example, HMGB1 and Granulin have been documented to interact with CpG PS-ASOs to enhance innate immune responses (24,25), while human serum albumin (HSA)-PS-ASO-TLR9 interactions lead to decreases in innate immune responses (41). Consistently, we found that reduction of HMGB1 and Granulin by siRNA in our two model cell systems decreased the innate immune response of the non-CpG PS-ASOs and we also confirm the co-localization of PS-ASOs with granulin in cells (Supplementary Figure S4). ...
... We next tested if these novel innate immune-stimulatory PS-ASO-protein complexes enter cells and are co-present in the endocytic organelles, as has been shown for other protein-CpG PS-ASO complexes (24). We added various Cy3-PS-ASOs with Alexa 647-labeled, purified proteins of interest, and administered lysotracker to perform live-cell imaging in Bjab cells. ...
Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary—in part—as a function of 2′modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO–protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.
... The GRN acts as an essential role in defense infection in the innate immunity. 8 The innate immunity belongs to the immune system, has been considered as the first powerful defense against the invasion of various pathogenic microorganisms and foreign bodies. 9 GRN assists in recruiting of CpG oligonucleotides (CpG-ODNs) in macrophages by binding to Toll-like receptor 9 (TLR9), reinforcing innate immunity against bacteria invasion. ...
... 9 GRN assists in recruiting of CpG oligonucleotides (CpG-ODNs) in macrophages by binding to Toll-like receptor 9 (TLR9), reinforcing innate immunity against bacteria invasion. 8,10,11 For example, PGRN can exacerbate SLE severity by enhancing TLR9 signaling. 12 Therefore, PGRN may be the primary regulators of inflammation and autoimmunity and may serve as biomarkers as well as indicating a promising and novel therapeutic concept to treat diverse inflammatory immunemediated diseases. ...
... 59,60 Concretely, when PGRN is cleaved into GRN in tissues by elastase, GRN not only acts as a reinforcing agent for a combination between CpG-ODNs and TLR9, but also heavily promotes CpG-ODNs delivery to the localization of TLR9 like endolysosomal compartments. 8 In addition to the vital role of TLR9 in bacterial defense mentioned above, TLR9 also regulates NF-κB nuclear translocation 61-65 which is critical for inflammatory cytokine production and involved in autophagy in many immune-mediated diseases such as RA. 66 However, whether PGRN may mediate TLR9 signaling pathway in RA has not been reported. ...
Progranulin (PGRN), a secretory glycoprotein consisting of 593 amino acid residues, is a key actor and regulator of multiple system functions such as innate immune response and inflammation, as well as tissue regeneration. Recently, there is emerging evidence that PGRN is protective in the development of a variety of immune-mediated diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) by regulating signaling pathways known to be critical for immunology, particularly the tumor necrosis factor alpha/TNF receptor (TNF-α/TNFR) signaling pathway. Whereas, the role of PGRN in psoriasis, systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) is controversial. This review summarizes the immunological functions of PGRN and its role in the pathogenesis of several immune-mediated diseases, in order to provide new ideas for developing therapeutic strategies for these diseases.
... Finally, we explored the effect of AZP2006 on Toll-like receptor 9 (TLR9) activity. Into the lysosome in fact, PGRN can be cleaved in granulins that in turn are implicated in pro-inflammatory action through the activation of TLR9 33,34 . ...
... AZP2006 was given orally by daily gavage and improvement was measured at the spatial working memory (spontaneous alternation in the Y-maze) and in spatial learning and memory (NOR test) (see design in Supplementary Fig. 4A). Aβ [25][26][27][28][29][30][31][32][33][34][35] injection and/or treatment with AZP2006 from D0, D01 or D04 showed no effect on speed, anxiety, or stereotypic behavior in the open-field procedure in mice measured during session 1 at D08 ( Supplementary Fig. 5). ...
... Once in the lysosome, PGRN can be cleaved into individual granulin (GRN) peptides 12 and PSAP into individual saposins (A, B, C, and D) which in turn, serve as activators of lysosomal sphingolipid metabolizing enzymes 46 . Once released, GRN are cofactors that potentiate TLR9-driven signaling and pro-inflammatory responses 34 (Fig. 6). ...
Progranulin (PGRN) is a protein with multiple functions including the regulation of neuroinflammation, neuronal survival, neurite and synapsis growth. Although the mechanisms of action of PGRN are currently unknown, its potential therapeutic application in treating neurodegenerative diseases is huge. Thus, strategies to increase PGRN levels in patients could provide an effective treatment. In the present study, we investigated the effects of AZP2006, a lysotropic molecule now in phase 2a clinical trial in Progressive Supranuclear Palsy patients, for its ability to increase PGRN level and promote neuroprotection. We showed for the first time the in vitro and in vivo neuroprotective effects of AZP2006 in neurons injured with Aβ1–42 and in two different pathological animal models of Alzheimer’s disease (AD) and aging. Thus, the chronic treatment with AZP2006 was shown to reduce the loss of central synapses and neurons but also to dramatically decrease the massive neuroinflammation associated with the animal pathology. A deeper investigation showed that the beneficial effects of AZP2006 were associated with PGRN production. Also, AZP2006 binds to PSAP (the cofactor of PGRN) and inhibits TLR9 receptors normally responsible for proinflammation when activated. Altogether, these results showed the high potential of AZP2006 as a new putative treatment for AD and related diseases.
... RNAse T2-depleted microglia further show lysosomes that are engorged with Aβ and other protein substrates as well as with undigested apoptotic material and they adopt an inflammatory signature (Hamilton et al., 2020). In addition, PLD3 directly interacts with PGRN and PGRN has been identified as a co-receptor for TLR9 through binding CpG-ODNs and transporting them to endolysosomes for interaction (Park et al., 2011). In this way, PGRN enables macrophages to respond to very low amounts of CpG-ODNs (15 nM) that would otherwise not elicit TLR9 activation ((Park et al., 2011), Fig. 2). ...
... In addition, PLD3 directly interacts with PGRN and PGRN has been identified as a co-receptor for TLR9 through binding CpG-ODNs and transporting them to endolysosomes for interaction (Park et al., 2011). In this way, PGRN enables macrophages to respond to very low amounts of CpG-ODNs (15 nM) that would otherwise not elicit TLR9 activation ((Park et al., 2011), Fig. 2). In agreement, depletion of PGRN in phagocytes lowers production of TNFα and IL-6 upon CpG-ODNs addition (Park et al., 2011). ...
... In this way, PGRN enables macrophages to respond to very low amounts of CpG-ODNs (15 nM) that would otherwise not elicit TLR9 activation ((Park et al., 2011), Fig. 2). In agreement, depletion of PGRN in phagocytes lowers production of TNFα and IL-6 upon CpG-ODNs addition (Park et al., 2011). PGRN deficiency significantly upregulates the global ratio of CD68 + microglia in the brain of APPswe/PSEN1ΔE9 mice, with a specific increase near Aβ plaques, and promotes the transcription of the TYROBP network of inflammation-related genes (C1qA, CD22, CD68, TREM2, TYROBP/DAP12). ...
Microglia, the brain-resident immune cells, play an essential role in the upkeep of brain homeostasis. They actively adapt into specific activation states based on cues from the microenvironment. One of these encompasses the activated response microglia (ARMs) phenotype. It arises along a healthy aging process and in a range of neurodegenerative diseases, including Alzheimer’s disease (AD). As the phenotype is characterized by an increased lipid metabolism, phagocytosis rate, lysosomal protease content and secretion of neuroprotective agents, it leaves to reason that the phenotype is adapted in an attempt to restore homeostasis. This is important to the conundrum of inflammatory processes. Inflammation per se may not be deleterious; it is only when microglial reactions become chronic or the microglial subtype is made dysfunctional by (multiple) risk proteins with single-nucleotide polymorphisms that microglial involvement becomes deleterious instead of beneficial. Interestingly, the ARMs up- and downregulate many late-onset AD-associated risk factor genes, the products of which are particularly active in the endolysosomal system. Hence, in this review, we focus on how the endolysosomal system is placed at the crossroad of inflammation and microglial capacity to keep pace with degradation.
... It is now known that extracellular PGRN is endocytosed into the lysosome via a sortilin-mediated pathway to be processed by cathepsins into GRNs (43), which are thus speculated to possess lysosomal functions (44,45). GRNs also function in a plethora of roles in normal cell biology (29,46,47) but possess opposing inflammatory properties to PGRN; while PGRN is anti-inflammatory, GRNs show pro-inflammatory properties (42). During inflammation, PGRN secreted from activated microglia and astrocytes undergoes extracellular proteolysis by neutrophil elastases and other proteases to generate GRNs (48). ...
... Unlike GRNs generated in the lysosomes, the fate of those formed extracellularly, especially during inflammation, remains unclear, although transport to the cytosol has remained a possibility (47,51,64). The ambiguity surrounding the precise localization of GRNs in pathophysiology has also hindered understanding of their cellular functions (45,65). ...
... Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is recognized for its roles in a variety of physiologic and disease processes, including immunomodulation (Jian et al., 2013a), cell growth, wound healing , host defense (Park et al., 2011) and inflammation (Park et al., 2011;Tang et al., 2011;. PGRN acts as an anti-inflammation molecule by direct binding to TNF receptors (Tang et al., 2011;Jian et al., 2013b). ...
... Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is recognized for its roles in a variety of physiologic and disease processes, including immunomodulation (Jian et al., 2013a), cell growth, wound healing , host defense (Park et al., 2011) and inflammation (Park et al., 2011;Tang et al., 2011;. PGRN acts as an anti-inflammation molecule by direct binding to TNF receptors (Tang et al., 2011;Jian et al., 2013b). ...
... This phenomenon could be potentially associated with PGRN overexpression across a broad spectrum of cancers. Recently, PGRN was found to be an essential secreted cofactor that activates and potentiates Toll-like receptor 9 (TLR9)-driven responses [67]. TLR9 is an innate immune receptor responsible for the recognition of the unmethylated CpG-DNA of bacterial, viral, and parasitic origin, as well as self-DNA in immune complexes [68]. ...
Glioblastoma multiforme (GBM) represents the most common and aggressive malignant form of brain tumour in adults and is characterized by an extremely poor prognosis with dismal survival rates. Currently, expanding concepts concerning the pathophysiology of GBM are inextricably linked with neuroinflammatory phenomena. On account of this fact, the identification of novel pathomechanisms targeting neuroinflammation seems to be crucial in terms of yielding successful individual therapeutic strategies. In recent years, the pleiotropic growth factor progranulin (PGRN) has attracted significant attention in the neuroscience and oncological community regarding its neuroimmunomodulatory and oncogenic functions. This review of the literature summarizes and updates contemporary knowledge about PGRN, its associated receptors and signalling pathway involvement in GBM pathogenesis, indicating possible cellular and molecular mechanisms with potential diagnostic, prognostic and therapeutic targets in order to yield successful individual therapeutic strategies. After a review of the literature, we found that there are possible PGRN-targeted therapeutic approaches for implementation in GBM treatment algorithms both in preclinical and future clinical studies. Furthermore, PGRN-targeted therapies exerted their highest efficacy in combination with other established chemotherapeutic agents, such as temozolomide. The results of the analysis suggested that the possible implementation of routine determinations of PGRN and its associated receptors in tumour tissue and biofluids could serve as a diagnostic and prognostic biomarker of GBM. Furthermore, promising preclinical applications of PGRN-related findings should be investigated in clinical studies in order to create new diagnostic and therapeutic algorithms for GBM treatment.
... Studies have suggested that CPG-ODNs may also enhance NETosis, making it a promising option for combating Stage I/II BRIs. [13][14][15][16] However, uncontrolled M1 polarization and excessive NET release usually lead to implant instability. [17] During the repair period (Stage III), M2 macrophages play a crucial role in tissue repair and implant integration by producing anti-inflammatory cytokines and promoting tissue regeneration. ...
Biofilm‐related infections (BRIs) present significant challenges owing to drug resistance, adverse immune responses, and implant failure; however, current approaches inadequately cater to the diverse therapeutic requirements at different stages of infection. To address this issue, we proposed a multi‐immunotherapy strategy in combination with sonodynamic therapy for the chronological treatment of BRIs. Macrophage membrane‐decorated targeting sonosensitive nanoadjuvants were fabricated to load cytosine‐phosphate‐guanine oligodeoxynucleotide (CPG‐ODN) or microRNA (miR)‐21‐5p. In the early stages of BRI (Stage I), CPG‐ODN‐loaded nanoadjuvants (CPG@HMPN@M) promoted the formation of neutrophil extracellular traps to capture and neutralize detached microbes. During the late stage of infection (Stage II), CPG‐ODNs redirected macrophage polarization into the M1 phase to combat infections via TLR9/Myd88/TRAF6 pathway. During these stages, CPG@HMPN@M generated singlet oxygen through sonodynamic processes, eradicating the biofilms under US irradiation. Once the BRIs were eliminated, miR‐21‐5p‐loaded nanoadjuvants (miR@HMPN@M) were delivered to the lesions to suppress excessive inflammation and promote tissue integration by evoking macrophage M2 polarization during the repair phase (Stage III) through PTEN/PI3K/Akt pathway. This innovative approach aimed to provide comprehensive treatment strategies for the chronological treatment of BRI by effectively eliminating infections, promoting tissue restoration, and implementing different immune regulations at different stages, thus demonstrating promising clinical value.
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... Moreover, PGRN's role in wound healing has been described in fibroblast accumulation during early granulation but not in the subsequent collagen deposition phase [22]. It is also known that PGRN is the co-factor for toll-like receptor 9 (TLR9) [23], and the expression of TLR9 in biopsies of fastprogressing IPFs was found to be significantly higher than in slow-progressing patients [24]. Besides, the TLR9 agonist cytosine-phosphorothioate-guanine (CpG) oligonucleotide was able to induce acute exacerbation in mouse lung transplanted with fibroblasts of rapid progressors [24]. ...
Diagnosing interstitial lung disease (ILD) can be a challenging process. New biomarkers may support diagnostic decisions. Elevated serum progranulin (PGRN) levels have been reported in liver fibrosis and dermatomyositis-associated acute interstitial pneumonia. Our aim was to assess the role of PGRN in the differential diagnosis of idiopathic pulmonary fibrosis (IPF) and other ILDs. Serum levels of PGRN were measured by enzyme-linked immunosorbent assay in stable IPF (n = 40), non-IPF ILD (n = 48) and healthy controls (n = 17). Patient characteristics, lung function, CO diffusion (DLCO), arterial blood gases, 6-min walk test, laboratory parameters and high-resolution (HR)CT pattern were assessed. In stable IPF, PGRN levels did not differ from healthy controls; however, serum PGRN levels were significantly higher in non-IPF ILD patients compared to healthy subjects and IPF (53.47 ± 15.38 vs. 40.99 ± 5.33 vs. 44.66 ± 7.77 ng/mL respectively; p < 0.01). The HRCT pattern of usual interstitial pneumonia (UIP) was associated with normal PGRN level, while for non-UIP patterns, significantly elevated PGRN level was measured. Elevated serum PGRN levels may be associated with non-IPF ILD, especially non-UIP patterns and might be helpful in cases of unclear radiological patterns in the differentiation between IPF and other ILDs.
... Although the full length PGRN is reported to have anti-inflammatory action, the cleaved granulin units are believed to be proinflammatory and can counteract the anti-inflammatory activity of PGRN [8,9]. These GRN peptides are able to increase the expression of interleukin 1 beta (IL-1β), IL-8, and tumor necrosis factor alpha (TNF-α) [10], and also to bind to toll-like receptor 9 (TLR9) activating its pathway (Figure 2A) [11]. During the inflammation process, neutrophils and macrophages secrete matrix metalloproteinases (MMP-9, MMP-12, MMP-14) [12,13], neutrophil elastase and proteinase 3 (PRTN3) [14]. ...
Progranulin (PGRN) is a glycoprotein formed by 593 amino acids encoded by the GRN gene. It has an important role in immunity and inflammatory response, as well as in tissue recovery. Its role in musculoskeletal inflammatory diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and intervertebral disc degeneration disease (IVDD), is, nowadays, an important target to investigate. The objective of this review is to systematically sum up all the recent findings concerning PGRN as a target in the development and resolution of the inflammatory diseases. PubMed was examined with the terms combinations (Progranulin) AND (Lupus Erythematosus, Systemic), (Progranulin) AND (Arthritis, Rheumatoid), and (Progranulin) AND (Intervertebral Disc Degeneration). PubMed was examined with the terms combinations (Atsttrin) AND (Lupus Erythematosus, Systemic), (Atsttrin) AND (Arthritis, Rheumatoid), and (Atsttrin) AND (Intervertebral Disc Degeneration). Moreover, research through Web of Science was performed searching the same items. The inclusion criteria were: studies whose main topic were progranulin, or atsttrin, with emphasis on the three selected diseases. On the other hand, the exclusion criteria were studies that only focused on diseases not related to RA, lupus or IVDD, in addition to the previous published literature reviews. Since few results were obtained, we did not filter by year. The records assessed for eligibility were 23, including all the studies with the information in state of art of progranulin and its capability to be a potential target or treatment for each one of the selected diseases. As these results are descriptive and not clinical trials, we did not perform risk of bias methods. Within these results, many studies have shown an anti-inflammatory activity of PGRN in RA. PGRN levels in serum and synovial fluids in RA patients were reported higher than controls. On the other hand, serum levels were directly correlated with SLE disease activity index, suggesting an important role of PGRN as a player in the progression of inflammatory diseases and a therapeutical approach for the recovery. This review has some limitations due to the small number of studies in this regard; therefore, we highlight the importance and the necessity of further investigation. No external funding was implicated in this systematical review.
... TLRs are immune receptors predominantly expressed on innate immune cells includ i ng monocytes, macrophages and pDCs 36 Recent studies have also shown a role for various molecules like granulin, DEC-205 and mannose receptor-1 in the uptake of CpG ODNs in innate immune cells [50][51][52] . Since most of these receptors are not yet reported to be expressed in CD4+T cells, it will be interesting to look at the differences in the uptake of different classes of ODNs in context of receptors utilized and the differences in conformation of various ODNs in CD4+ T cells. ...
CpG Oligodeoxynucleotides (ODNs) are established TLR9 ligands; however, their functional responses in CD4+ T cells are believed to be independent of TLR9 and MyD88. We studied ligand‐receptor interactions of ODN 2216 and TLR9 in human CD4+ T cells and assessed their consequences in terms of TLR9 signaling and cell phenotype. We demonstrated that the uptake of ODN 2216, a synthetic TLR9 agonist, is controlled by TLR9 signaling molecules and results in an increase in the expression of TLR9 signaling molecules, regulated via a feedback mechanism. Next, the uptake of ODN 2216 resulted in TLR9 signaling dependent but MyD88 independent increase in expression of TGF‐β. Finally, ODN 2216 treated CD4+ T cells showed an anti‐inflammatory phenotype that was similar to Th3 type of regulatory T cells. These Th3 like cells were able to suppress the proliferation of untreated CD4+ T cells. Collectively, our results demonstrate a direct and interdependent relationship between ODN 2216 uptake and TLR9 signaling in CD4+ T cells. Our findings thus pave the way for future research to explore direct modulation of adaptive immune cells, using innate immune ligands, to subvert exaggerated inflammatory responses.
... The mechanism for reduced clearance of pathogens by Grn −/− macrophages is likely due to the role of PGRN in controlling macrophage-associated inflammatory responses and defects in lysosome-mediated degradation pathway. For instance, PGRN binds to and enhances toll-like receptor 9 (TLR9) signaling upon the treatment of CpG oligonucleotides in macrophage to promote the TNFα secretion [27]. Furthermore, in collagen-induced inflammatory arthritis model, PGRN attenuates TNFα-induced MAPK and NF-κB signaling for anti-inflammatory effects by binding to tumor necrosis factor receptors (TNFRs) and antagonizing TNFα [28], though the interaction between PGRN and TNFR is disputed by other studies [29,30]. ...
Neuroimmune dysfunction is a cardinal feature of neurodegenerative diseases. But how immune dysregulation in the brain and peripheral organs contribute to neurodegeneration remains unclear. Here, we discuss the recent advances highlighting neuroimmune dysfunction as a key disease-driving factor in frontotemporal dementia (FTD). We provide an overview of the clinical observations supporting a high prevalence of autoimmune diseases in FTD patients with mutations in GRN or C9orf72. We then focus on a myriad of evidence from human genetic studies, mouse models, in vitro assays, and multi-omics platform, which indicate that haploinsufficiency in GRN and C9orf72 promotes neuroimmune dysfunction and contributes to neurodegeneration and premature death. These compelling data provide key insights to disease mechanisms, biomarker discovery, and therapeutic interventions for FTD (120 words).
... Although PGRN has been widely studied in autoimmune disorders, cancer, and neurodegenerative diseases [28][29][30][31], few studies have focused on immune cells. PGRN has been found to bind TLR9 and it plays an important role in innate immunity [32]. One study indicated that PGRN promotes CD4 + T cells to differentiate into Treg cells, enhancing the latter's function [6]. ...
T helper 2 (Th2) cytokine production by invariant natural killer T (iNKT) cells is involved in the development of asthma, but the regulation of Th2 cytokines in iNKT cells remains unknown. Although it is known that progranulin (PGRN) induces the production of Th2 cytokines in iNKT cells in vivo, the underlying mechanism is not clear. This study aims to investigate the role of PGRN in iNKT cells. The effects of PGRN on the differentiation of iNKT cells was detected by flow cytometry. Then stimulation of iNKT cells and airway resistance were carried out to evaluate the function of PGRN on iNKT cells. Furthermore, the mechanisms of PGRN in regulating iNKT cells was investigated by RT-PCR, WB, confocal and luciferase reporter assays. The absolute number of iNKT cells decreased in PGRN KO mice despite an increase in the percentage of iNKT cells. Furthermore, analyzing the subsets of iNKT cells, we found that NKT2 cells and their IL-4 production were reduced. Mechanistically, the decrease in NKT2 cells in the PGRN KO mice was caused by increased expression of enhancer of zeste homolog 2 (EZH2), that in turn caused increased degradation and altered nuclear localization of PLZF. Interestingly, PGRN signaling decreased expression of EZH2 and treatment of the PGRN KO mice with the EZH2 specific inhibitor GSK343 rescued the defect in NKT2 differentiation, IL-4 generation, and PLZF expression. Altogether, We have revealed a new pathway (PGRN-EZH2-PLZF), which regulates the Th2 responses of iNKT cells and provides a potentially new target for asthma treatment.
... The activated proinflammatory response was also evidenced by the early (3 hpi) upregulation of genes for several tumor necrosis alpha (Tnfα) receptors, interleukins (Ils), and their receptors and interferon (Ifn) and related proteins by RBCs and WBCs with a common (il12β, il10 receptor β, and il20) and cell-typespecific pattern (RBCs: il17a/f1; WBCs: several Tnf receptors, Il1 receptor-like, ifnc1, ifna2, and irf1) followed by a strong upregulation of genes for Il1β, two receptors for Il1β, Il8 precursor, progranulin and granulin in B samples at 12 hpi ( Table 2). Granulins are multifunctional proteins produced after proteolytic processing of progranulin (Bateman et al., 1990) that enhance the production of proinflammatory cytokines such as Tnfα and Il8 (Park et al., 2011). Related to these results, we highlight that Il1β is the main proinflammatory cytokine in both humans and fish (Zou and Secombes, 2016) and that Ifna has been related to the immune response against virus (Zou and Secombes, 2016). ...
Vibrio vulnificus is a marine zoonotic pathogen associated with fish farms that is considered a biomarker of climate change. Zoonotic strains trigger a rapid death of their susceptible hosts (fish or humans) by septicemia that has been linked to a cytokine storm in mice. Therefore, we hypothesize that V. vulnificus also causes fish death by triggering a cytokine storm in which red blood cells (RBCs), as nucleated cells in fish, could play an active role. To do it, we used the eel immersion infection model and then analyzed the transcriptome in RBCs, white BCs, and whole blood using an eel-specific microarray platform. Our results demonstrate that V. vulnificus triggers an acute but atypical inflammatory response that occurs in two main phases. The early phase (3 h post-infection [hpi]) is characterized by the upregulation of several genes for proinflammatory cytokines related to the mucosal immune response ( il17a/f1 and il20 ) along with genes for antiviral cytokines ( il12β ) and antiviral factors ( ifna and ifnc ). In contrast, the late phase (12 hpi) is based on the upregulation of genes for typical inflammatory cytokines ( il1β ), endothelial destruction ( mmp9 and hyal2 ), and, interestingly, genes related to an RNA-based immune response ( sidt1 ). Functional assays revealed significant proteolytic and hemolytic activity in serum at 12 hpi that would explain the hemorrhages characteristic of this septicemia in fish. As expected, we found evidence that RBCs are transcriptionally active and contribute to this atypical immune response, especially in the short term. Based on a selected set of marker genes, we propose here an in vivo RT-qPCR assay that allows detection of early sepsis caused by V. vulnificus. Finally, we develop a model of sepsis that could serve as a basis for understanding sepsis caused by V. vulnificus not only in fish but also in humans.
... In contrast apolipoprotein A1 (ApoA1), secretory leukocyte protease inhibitor (SLPI) and high-density lipoprotein (HDL) are known as PGRN proteolysis and cleavage suppressors [26,27]. Active PGRN plays a role in cell biology as autocrine and paracrine by binding to sortilin (SORT1), tumor necrosis factor receptor 1/2 (TNFR1/2), tumor necrosis factor receptor superfamily member 25 (TNFRSF25), ephrin type-A receptor 2 (EPHA2) and toll-like receptor 9 (TLR9) [28][29][30][31][32]. The PGRN expression profile varies under neoplastic and non-neoplastic conditions, which is certainly biologically significant according to the ongoing research in pre-and clinical field. ...
Breast cancer constitute a common type of oncological disease with a highlighted mortality rate. In recent years, researchers have introduced progranulin (PGRN) as an novel potential biomarker and associated its function with higher risk factor for development of breast cancer. The present review article collects evidence on the association of PGRN with clinicopathological features and drug resistance in the patients with breast cancer. The results of this study suggested the use of routine determination of PGRN in the clinic as a reliable biomarker for screening people at high risk or as early indication of breast cancer. Targeting PGRN and its associated signaling pathways and receptors, such as sortilin (SORT1), could also cover a novel therapeutic strategy in the breast cancer.
... Several putative PGRN receptors and binding proteins have been proposed. Among them are heparan sulfate proteoglycans (Gonzalez et al., 2003;Yip et al., 2014), EphA2 (Neill et al., 2016), Tumor necrosis factor receptor 2 (Tang et al., 2011), Toll-like receptor-9 (Park et al., 2011), notch receptors (Altmann et al., 2016), components of lysosomal trafficking such as sortilin (Hu et al., 2010) and prosaposin (Zhou et al., 2015), and lysosomal enzymes involved in lipid and protein metabolism (Paushter et al., 2018). Reconciling these diverse binding proteins into an integrated model of PGRN action is challenging, particularly since PGRN may interact with more than one membrane protein at the same time. ...
Progranulin (PGRN) is critical in supporting a healthy CNS. Its haploinsufficiency results in frontotemporal dementia, while in experimental models of age-related neurodegenerative diseases, the targeted expression of PGRN greatly slows the onset of disease phenotypes. Nevertheless, much remains unclear about how PGRN affects its target cells. In previous studies we found that PGRN showed a remarkable ability to support the survival of NSC-34 motor neuron cells under conditions that would otherwise lead to their apoptosis. Here we used the same model to investigate other phenotypes of PGRN expression in NSC-34 cells. PGRN significantly influenced morphological differentiation, resulting in cells with enlarged cell bodies and extended projections. At a molecular level this correlated with pathways associated with the cytoskeleton and synaptic differentiation. Depletion of PGRN led to increased expression of several neurotrophic receptors, which may represent a homeostatic mechanism to compensate for loss of neurotrophic support from PGRN. The exception was RET, a neurotrophic tyrosine receptor kinase, which, when PGRN levels are high, shows increased expression and enhanced tyrosine phosphorylation. Other receptor tyrosine kinases also showed higher tyrosine phosphorylation when PGRN was elevated, suggesting a generalized enhancement of receptor activity. PGRN was found to bind to multiple plasma membrane proteins, including RET, as well as proteins in the ER/Golgi apparatus/lysosome pathway. Understanding how these various pathways contribute to PGRN action may provide routes toward improving neuroprotective therapies.
... pDCs have a unique capacity to produce a large amount of IFN in response to nucleic acids 3 and display specialized molecular features that control IRF7 activation directly downstream of TLR7 and TLR9. In addition to granulin 23 , which favors CpG ODN capture, other molecules, including the solute carrier protein superfamily member Slc15a4 or the biogenesis of lysosome-related organelles complexes (BLOC) proteins are required by pDCs, but not by conventional DCs to respond to DNA 24,25 . pDCs seem, therefore, to have developed a specific regulation of their endocytic compartments, that controls TLR access to different endosome subsets to achieve a coordinated and commensurate DNA detection response to potential threats. ...
... Additionally, GRN has been shown to interact with the toll-like receptor 9 (Park et al., 2011), a key regulator of the immune response to foreign DNA. GRN also bound the extracellular domain of Notch receptors and affected Notch target gene expression (Altmann et al., 2016). ...
Frontotemporal dementia (FTD) is a neurodegenerative disease, leading to behavioral changes and language difficulties. Heterozygous loss-of-function mutations in progranulin (GRN) induce haploinsufficiency of the protein and are associated with up to one-third of all genetic FTD cases worldwide. While the loss of GRN is primarily associated with neurodegeneration, the biological functions of the secreted growth factor-like protein are more diverse, ranging from wound healing, inflammation, vasculogenesis, and metabolic regulation to tumor cell growth and metastasis. To date, no disease-modifying treatments exist for FTD, but different therapeutic approaches to boost GRN levels in the central nervous system are currently being developed (including AAV-mediated GRN gene delivery as well as anti-SORT1 antibody therapy). In this review, we provide an overview of the multifaceted regulation of GRN levels and the corresponding therapeutic avenues. We discuss the opportunities, advantages, and potential drawbacks of the diverse approaches. Additionally, we highlight the therapeutic potential of elevating GRN levels beyond patients with loss-of-function mutations in GRN.
... Ligand recognition by TLR9 results in the activation of the mitogenactivated protein kinase (MAPK), AKT, and NF-κB signaling pathways (Xu et al., 2003;Das et al., 2015), which promotes the secretion of proinflammatory cytokines (Gazzinelli and Denkers, 2006;Kawai and Akira, 2009). Macrophages are a key component of the innate immune system, and many PRRs, such as TLRs, localize to either the cell membrane (TLR1, TLR2, TLR4, TLR5, and TLR6) or endosomes (TLR3, TLR4, TLR7, TLR8, and TLR9) of these cells (Park et al., 2011;Cui et al., 2014;Liu et al., 2017). Activated macrophages can produce a wide array of cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, which initiate the inflammatory response (Sodhi and Pandey, 2011). ...
Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase–quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)–free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis–stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis–induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.
... For tumor cells, GP88 might function as an autocrine growth factor [25], while in the context of immune cells, its anti-inflammatory function might be predominant [50]. Moreover, there are several points at which GP88 can be active in the immune system with an anti-tumorigenic activity: (i) GP88 is involved in the activation of innate immunity by serving as a soluble cofactor for TLR9 e.g., in macrophages [51]. (ii) GP88 promotes the Th2 (T-helper 2 cells) response [52], and can induce expression of Th2-like cytokines, IL-4 and IL-5 [53]. ...
Urothelial bladder cancer (BCa) is the ninth most commonly diagnosed cancer worldwide and accounts for approximately 3% of global cancer diagnoses. We are interested in prognostic markers that may characterize tumor cells (TCs) and immune cells (ICs) and their relationship in BCa. A potential candidate marker that meets these criteria is progranulin (GP88), which is expressed separately in TCs and ICs. We analyzed GP88 expression by immunohistochemistry (IHC) in 196 muscle-invasive BCa samples using a tissue microarray. The immunoreactive score for GP88 staining in TCs and the percentage of GP88-positive ICs was determined. An easy cutoff for the staining status of TCs (positive vs. negative) and ICs (0% vs. > 0%) and, more generally, negative vs. positive GP88 staining could be applied. We detected 93 patients (47.4%) and 92 patients (46.9%) with GP88-positive TCs or ICs, respectively. The IHC results were correlated with clinicopathological and survival data. Positive GP88 staining in TCs appeared to be an independent poor prognostic factor for disease-specific survival (DSS) (RR (relative risk) = 1.74; p = 0.009) and recurrence-free survival (RFS) (RR = 1.92; p = 0.002). In contrast, negative GP88 staining in ICs was an independent negative predictor for overall survival (OS) (RR = 2.18; p < 0.001), DSS (RR = 2.84; p < 0.001) and RFS (RR = 2.91; p < 0.001) in multivariate Cox’s regression analysis. When combining GP88 staining in TCs and ICs, a specific combination of GP88-positive TCs and GP88-negative ICs was associated with a 2.54-fold increased risk of death, a 4.21-fold increased risk of disease-specific death and a 4.81-fold increased risk of recurrence compared to GP88-negative TCs and GP88-positive ICs. In summary, GP88 positivity in TCs is a negative prognostic factor for DSS and RFS. In addition, GP88 positivity can mark ICs that are associated with a good prognosis (OS, DSS and RFS). The combination of GP88 staining in TCs and ICs appears to be a significant independent prognostic biomarker in muscle-invasive BCa.
... It rapidly endocytoses and delivers PGRN to lysosomes (Hu et al., 2010;Paushter et al., 2018). Additionally, several other potential PGRN receptors have been reported, including tumor necrosis factor (TNF) receptor (TNFR) (Tang et al., 2011), Toll-like receptor 9 (Park et al., 2011), the prosaposin (PSAP)/cation-independent mannose-6-phosphate receptor (CI-M6PR) complex (Zhou et al., 2015), the PSAP/low-density lipoprotein receptor-related protein 1 (LRP1) complex (Paushter et al., 2018), and the ephrin type-A receptor 2 (EphA2) (Neill et al., 2016). ...
Long‐term or severe lack of protective factors is important in the pathogenesis of neurodegenerative dementia. Progranulin (PGRN), a neurotrophic factor expressed mainly in neurons and microglia, has various neuroprotective effects such as anti‐inflammatory effects, promoting neuron survival and neurite growth, and participating in normal lysosomal function. Mutations in the PGRN gene (GRN) have been found in several neurodegenerative dementias, including frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Herein, PGRN deficiency and PGRN hydrolytic products (GRNs) in the pathological changes related to dementia, including aggregation of tau and TAR DNA‐binding protein 43 (TDP‐43), amyloid‐β (Aβ) overproduction, neuroinflammation, lysosomal dysfunction, neuronal death, and synaptic deficit have been summarized. Furthermore, as some therapeutic strategies targeting PGRN have been developed in various models, we highlighted PGRN as a potential anti‐neurodegeneration target in dementia.
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... Granulins which activate the TLR 9 [67], the leucine-rich repeat-containing protein 59 (LRRC 59) which has a role in TLR trafficking from the ER [68] and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1 which catalyses IP3 and DAG production [69] all exhibited decreased expression in this study. The expressions of heterogeneous nuclear ribonucleoprotein A0 and Nuclear Receptor Coactivator 5, both with roles in the binding and endocytosis of bacterial DNA [70] and Ras-related protein Rab-11A, the downregulation of which has previously been implicated in the vascular leakage observed in pulmonary microvessels during microbial sepsis [71], were also found to be reduced. ...
Amoebic Gill Disease (AGD), caused by the ectoparasite Paramoeba perurans is characterised by hyperplasia of the gill epithelium and lamellar fusion. In this study, the initial host response of naïve Atlantic salmon (Salmo salar) inoculated with P. perurans was investigated. Using gel-free proteomic techniques and mass spectrometry gill and serum samples were analysed at 7 timepoints (2, 3, 4, 7, 9, 11 and 14 days) post-inoculation with P. perurans. Differential expression of immune related proteins was assessed by comparison of protein expression from each time point against naïve controls. Few host immune molecules associated with innate immunity showed increased expression in response to gill colonisation by amoebae. Furthermore, many proteins with roles in immune signalling, phagocytosis and T-cell proliferation were found to be inhibited upon disease progression. Initially, various immune factors demonstrated the anticipated increase in expression in response to infection in the serum while some immune inhibition became apparent at the later stages of disease progression. Taken together, the pro-immune trend observed in serum, the lack of a robust early immune response in the gill and the diversity of those proteins in the gill whose altered expression negatively impact the immune response, support the concept of a pathogen-derived suppression of the host response.
... The DNA curvature inducing proteins, including HMGB1 and histones H2A and H2C significantly enhance the TLR9 binding of the DNA (174). The cysteine rich protein granulin serving as a co-receptor for CpG DNA also coordinates their delivery to the endosomes or endolysosomes and promotes the interaction between CpG DNA and c-terminal domain of TLR9 to make ensure the TLR9 signaling activation (178,179). Thus, cytosolic HMGB1 and granulin bind to the CpG DNA and increase their potency to bind to endosomal TLR9 and activation ( Figure 1). Hence, TLR9 recognizes curved DNA backbones with increased curvature independent of its sequence length. ...
The immune system has evolved to protect the host from the pathogens and allergens surrounding their environment. The immune system develops in such a way to recognize self and non-self and develops self-tolerance against self-proteins, nucleic acids, and other larger molecules. However, the broken immunological self-tolerance leads to the development of autoimmune or autoinflammatory diseases. Pattern-recognition receptors (PRRs) are expressed by immunological cells on their cell membrane and in the cytosol. Different Toll-like receptors (TLRs), Nod-like receptors (NLRs) and absent in melanoma-2 (AIM-2)-like receptors (ALRs) forming inflammasomes in the cytosol, RIG (retinoic acid-inducible gene)-1-like receptors (RLRs), and C-type lectin receptors (CLRs) are some of the PRRs. The DNA-sensing receptor cyclic GMP–AMP synthase (cGAS) is another PRR present in the cytosol and the nucleus. The present review describes the role of ALRs (AIM2), TLR9, and cGAS in recognizing the host cell DNA as a potent damage/danger-associated molecular pattern (DAMP), which moves out to the cytosol from its housing organelles (nucleus and mitochondria). The introduction opens with the concept that the immune system has evolved to recognize pathogens, the idea of horror autotoxicus, and its failure due to the emergence of autoimmune diseases (ADs), and the discovery of PRRs revolutionizing immunology. The second section describes the cGAS-STING signaling pathway mediated cytosolic self-DNA recognition, its evolution, characteristics of self-DNAs activating it, and its role in different inflammatory conditions. The third section describes the role of TLR9 in recognizing self-DNA in the endolysosomes during infections depending on the self-DNA characteristics and various inflammatory diseases. The fourth section discusses about AIM2 (an ALR), which also binds cytosolic self-DNA (with 80–300 base pairs or bp) that inhibits cGAS-STING-dependent type 1 IFN generation but induces inflammation and pyroptosis during different inflammatory conditions. Hence, this trinity of PRRs has evolved to recognize self-DNA as a potential DAMP and comes into action to guard the cellular galaxy. However, their dysregulation proves dangerous to the host and leads to several inflammatory conditions, including sterile-inflammatory conditions autoinflammatory and ADs.
... PGRN is synthesized by macrophages and is cleaved by extracellular proteases, such as proteinase 3 (PR3) and elastase, into granulin (GRN), 20 which ranges in molecular weight from 6 to 25 kDa. Moreover, PGRN and/or GRN act as soluble cofactors for toll-like receptor 9 (TLR9) signaling and enhance it.28 Serum PGRN levels are significantly elevated in SLE patients in parallel with the disease activity. ...
Objectives
Dermatomyositis (DM) is an autoimmune disease that presents with a wide variety of clinical manifestations. Patients with DM or clinically amyopathic dermatomyositis (CADM) with antimelanoma differentiation‐associated protein 5 (anti‐MDA5) antibodies are frequently associated with interstitial pneumonia, especially rapidly progressive interstitial lung disease. Progranulin (PGRN) is an autocrine growth factor involved in inflammation. Elevated serum PGRN levels have been reported in the patients with systemic lupus erythematosus and rheumatoid arthritis. However, they have not been precisely studied in DM/CADM. We assessed PGRN levels as a potential biomarker for DM/CADM with anti‐MDA5 antibodies.
Methods
Twenty‐four DM/CADM patients with anti‐MDA5 antibodies, 12 patients without anti‐MDA5 antibodies, and eight healthy volunteers were enrolled. We measured serum PGRN levels and compared them between the anti‐MDA5 antibody‐positive and antibody‐negative groups. They were also measured before and after the start of treatment in anti‐MDA5 antibody‐positive patients. We examined the correlations between serum PGRN levels and laboratory data such as serum KL‐6 and ferritin levels.
Results
Patients with DM/CADM, especially those with anti‐MDA5 antibodies, had significantly higher serum PGRN levels than healthy individuals. They decreased after the start of treatment. Serum PGRN levels were positively correlated with serum KL‐6 and ferritin levels, and anti‐MDA5 antibody titers.
Conclusions
This is the first study to show that PGRN levels were significantly elevated in the sera of DM/CADM patients, particularly in those with anti‐MDA5 antibodies. PGRN may be a useful biomarker of disease activity. However, further studies are required to reveal the mechanisms of PGRN in DM/CADM more precisely.
... GRN acts as a soluble cofactor for the TLR9 signaling cascade, which regulates innate and adaptive immune responses [8]. Moreover, PGRN has been reported in association with systemic inflammation and autoimmunity [9]. ...
Background Systemic lupus erythematosus (SLE) is a systemic autoimmune disease. SLE has different presentations that are not specific to the disease itself. Diagnosis and assessment of disease activity are considered a challenge to patients and physician, as both need experienced clinician to evaluate symptoms and laboratory findings. So, it is very important to search for an accurate diagnostic marker for SLE activity. In the past few years, increasing evidence has shown that progranulin (PGRN) may play a role in regulation of adaptive and innate immunity, so it might be a marker for disease activity.
Aim To investigate the clinical significance of PGRN in patients with SLE regarding its role in pathogenesis and its correlation to clinical manifestations and disease activity.
Patients and methods This is a case–control study conducted on 96 participants. Group I included 56 patients with SLE subdivided according to SLE Disease Activity Index (SLEDAI) score into patients with active and inactive SLE. Group II included 40 apparently healthy volunteers as a control group. All patients were subjected to a thorough history; complete clinical examination; assessment of disease activity by using SLEDAI; and laboratory investigations, including complete blood count, liver function, kidney function, complete urine analysis, anti-nuclear antibody, anti-double-stranded DNA antibody, complement 3, complement 4, and serum PGRN.
Results There was a highly statistically significant increase in serum PGRN level in patients with SLE compared with healthy control group. Moreover, a positive correlation was found between PGRN levels and SLEDAI score.
Conclusion Patients with SLE with higher SLEDAI score have higher serum PGRN level. So, serum PGRN level might play a role in SLE pathogenesis and as diagnostic marker for SLE activity.
... Alternatively, the binding of PGRN to other cell surface receptors may modulate inflammation. PGRN has been reported to bind directly, or indirectly, to a number of transmembrane receptors including delta homolog 1 (DLK1) [149], SORT1 [89], Toll-like receptor 9 (TLR9) [150], Notch1 [93], EphA2 [92], prosaposin (PSAP) mediated binding to the cation-independent mannose-6-phosphate receptor (M6PR), low-density lipoprotein receptor-related protein 1 (LRP1) [151], tyrosine-protein kinase receptor (Tyro3) [147], and GFRA2 [37]. Besides SORT1, most of these interactions have only been reported once and have not been extensively investigated, especially in the CNS. ...
It has been more than a decade since heterozygous loss-of-function mutations in the progranulin gene (GRN) were first identified as an important genetic cause of frontotemporal lobar degeneration (FTLD). Due to the highly diverse biological functions of the progranulin (PGRN) protein, encoded by GRN, multiple possible disease mechanisms have been proposed. Early work focused on the neurotrophic properties of PGRN and its role in the inflammatory response. However, since the discovery of homozygous GRN mutations in patients with a lysosomal storage disorder, investigation into the possible roles of PGRN and its proteolytic cleavage products granulins, in lysosomal function and dysfunction, has taken center stage. In this chapter, we summarize the GRN mutational spectrum and its associated phenotypes followed by an in-depth discussion on the possible disease mechanisms implicated in FTLD-GRN. We conclude with key outstanding questions which urgently require answers to ensure safe and successful therapy development for GRN mutation carriers.
... PGRN is secreted extracellularly and proteolytically cleaved by enzymes such as matrix metalloproteinase-12 (MMP-12), 10 neutrophil elastase, and proteinase 3 11 releasing the individual GRNs. In physiology, a variety of roles have been attributed to GRNs such as innate immune response, 12 neurotrophic roles, 3 while other studies ascribe pro-inflammatory functions, opposing that of the precursor. 13 In addition to the extracellular processing of PGRN to generate GRNs, 13,14 recent studies have revealed that PGRN is also transported into the lysosome by sortillin-mediated pathway where it is processed by cysteine peptidase cathepsin-L 15,16 to produce GRNs . ...
Granulins (GRN 1-7) are short (~6 kDa), cysteine-rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These modules, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Our previous investigations into GRN-3 and GRN-5 reveal them to be fully disordered in the reduced form and implicate redox sensitive attributes to the proteins. Such redox-dependent modulation has become associated with proteins involved in oxidative stress regulation and maintaining metal-homeostasis within cells. To probe whether GRNs play a contributory role in such functions, we tested the metal binding potential of the reduced form of GRNs -3 and -5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu(II) and no other divalent cations. Binding of Cu(II) also partly triggered the oxidative multimerization of GRNs via uncoordinated cystines at both pH conditions. Furthermore, binding did not induce gain in secondary structure and the protein remained disordered. Overall, the results indicate that GRN-3 and -5 have a surprisingly strong affinity for Cu(II) in the pM range, comparable to known copper sequestering proteins. This data also hints at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu-induced ROS cytotoxicity in cells. Together, this report uncovers a metal-coordinating capability of GRNs for the first time, which could have profound significance in their structure and function.
... Furthermore granulins were able to bind synthetic DNA molecules and were considered as a central actor in the signaling of viral invasion 115 . Especially, granulins were described as soluble co-factor of the toll-like receptor signaling pathway in macrophages 116 . To our knowledge, the role of granulins in the immune system of invertebrates has only been studied in a context of snail infection by trematodes 117,118 . ...
Biological responses of zebra mussel Dreissena polymorpha are investigated to assess the impact of contaminants on aquatic organisms and ecosystems. In addition to concentrate chemical contaminants in their tissues, zebra mussels accumulate several microorganisms such as viruses, protozoa and bacteria. In order to understand the molecular mechanisms involved in the defence against microorganisms this study aims at identifying immune proteins from D. polymorpha hemolymph involved in defence against protozoa and viruses. For this purpose, hemolymph were exposed ex vivo to Cryptosporidium parvum and RNA poly I:C. Differential proteomics on both hemocytes and plasma revealed immune proteins modulated under exposures. Different patterns of response were observed after C. parvum and RNA poly I:C exposures. The number of modulated proteins per hemolymphatic compartments suggest that C. parvum is managed in cells while RNA poly I:C is managed in plasma after 4 h exposure. BLAST annotation and GO terms enrichment analysis revealed further characteristics of immune mechanisms. Results showed that many proteins involved in the recognition and destruction of microorganisms were modulated in both exposure conditions, while proteins related to phagocytosis and apoptosis were exclusively modulated by C. parvum. This differential proteomic analysis highlights in zebra mussels modulated proteins involved in the response to microorganisms, which reflect a broad range of immune mechanisms such as recognition, internalization and destruction of microorganisms. This study paves the way for the identification of new markers of immune processes that can be used to assess the impact of both chemical and biological contaminations on the health status of aquatic organisms.
... The GRN-A is required for chondrogenesis and sufficient for interaction with COMP [16]. GRNs interact with TLR9 to activate downstream NF-kB signaling pathway through M YD88 and TRAF6, thereby affecting the host's immune response to pathogens [36]. Our present study demonstrated that 45-GRN effectively accelerated the calcification of aortic valves with up-regulated TNF-α and apoptotic indicators. ...
Inflammation is implicated in the pathogenesis of calcific aortic valve disease (CAVD) which is a major contributor to cardiovascular mortality and lacks non-surgical treatment. The progranulin (PGRN) is an important immunomodulatory factor in a variety of inflammatory diseases, including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease and pneumonia. However, its role in calcification of aortic valve remains unknown.
We firstly found that PGRN was increased in calcified human aortic valve (AV) tissues. Interestingly, in addition to full-length PGRN (68KD), a much stronger band of approximately 45 KD was also significantly increased. The band of 45 KD (45-GRN), was present in wild type (WT) mouse MEFs and AV but absent in grn−/−MEFs, indicating that it was a specific degradation product derived from PGRN. 45-GRN was upregulated whereas PGRN was reduced in human valve interstitial cells (hVICs) under calcifying conditions which is induced by osteogenic medium (OM). In primary porcine VICs (pVICs), PGRN downregulated TNF-α and α-SMA which was accompanied by downregulation of RUNX2, OPN, OCN, alkaline phosphatase activity and calcium deposition, effects pointing to reduced inflammation, myofibroblastic and osteoblastic transition under calcifying conditions. We overexpressed a mimic of 45-GRN which contains p-G-F-B-A-C in pVICs. However, 45-GRN overexpression promoted OM-induced calcification through activating the Smad1/5/8, NF-κB and AKT signaling pathways. Inhibition of the three signaling pathways suppressed 45-GRN's effect on VICs phenotype transition. 45-GRN promoted TNF-α and expressed converse pathogenic signatures with PGRN during TNF-α stimulation.
Collectively, this study provides new insight into the pathogenesis of CAVD, indicating that PGRN is a stratagem in mitigating valve fibrosis/osteoblastic differentiation, and also presenting 45-GRN as a potential target for the treatment of CAVD.
... We next sought to examine the involvement of different cofactors and accessory molecules that are involved in CpG-ODN uptake and endosomal delivery. These included granulin, a cofactor that binds to CpG-ODN and helps in its delivery to endosomes 47 and also has the potential to bind to TLR9; SLC15A, a transporter protein involved in TLR9 and endosomal functions 48 ; and PRAT4A, a chaperone that is needed for proper trafficking of TLR9. 49 SK1-I pretreatment decreased the expression of all these genes induced by CpG-ODN ( Figure 5D), supporting the notion that SphK1 may be critical in CpG-ODN uptake and processing. ...
Plasmacytoid dendritic cells (pDCs) express Toll like receptors (TLRs) that modulate the immune response by production of type I interferons. Here, we report that sphingosine kinase 1 (SphK1) which produces the bioactive sphingolipid metabolite, sphingosine 1‐phosphate (S1P), plays a critical role in the pDC functions and interferon production. Although dispensable for the pDC development, SphK1 is essential for the pDC activation and production of type I IFN and pro‐inflammatory cytokines stimulated by TLR7/9 ligands. SphK1 interacts with TLRs and specific inhibition or deletion of SphK1 in pDCs mitigates uptake of CpG oligonucleotide ligands by TLR9 ligand. In the pristane‐induced murine lupus model, pharmacological inhibition of SphK1 or its genetic deletion markedly decreased the IFN signature, pDC activation, and glomerulonephritis. Moreover, increases in the SphK1 expression and S1P levels were observed in human lupus patients. Taken together, our results indicate a pivotal regulatory role for the SphK1/S1P axis in maintaining the balance between immunosurveillance and immunopathology and suggest that specific SphK1 inhibitors might be a new therapeutic avenue for the treatment of type I IFN‐linked autoimmune disorders.
... Moreover, previous studies have shown that PGRN is a lysosomal protein [12]. PGRN-deficiency suppresses Toll-like receptor 9 (TLR9) signaling [37]. TLR9 is expressed in microglia, and localized in the lysosomes [38], and its function is dependent on acidic environment [39]. ...
Our previous investigations revealed that progranulin (PGRN) is a lysosomal protein involved in hippocampal neurogenesis and neuroinflammation. However, the possible involvement of PGRN in regulating inflammatory response and mediating neuronal activity is still not well-defined. Here, we demonstrate that PGRN deficiency enhances the age-dependent increase of neuronal activity in the paraventricular nucleus (PVN) of the hypothalamus. Aging increased neuronal activity in the PVN of the hypothalamus, and PGRN deficiency enhanced the effects of age on hypothalamic neuronal activity. Aging increased the lysosomal biogenesis and inflammatory response in microglia, which was also aggravated in PGRN-knockout mice. Moreover, PGRN deficiency enhanced interleukin-1 beta and lysosomal genes levels. These results suggest that PGRN deficiency may enhance the age-dependent increase of neuronal activity possibly because PGRN facilitates immunological responses through regulating lysosomal function.
... Other proteins have been identified to interact with PGRN that were not assessed. These include phospholipase D3 (PLD3) that colocalizes with PGRN on neuritic plaques [73], and Toll-like receptor-9 whose signaling in macrophages is regulated by granulin [74]. However, we could detect PSAP binding to cathepsin D and BACE-1 in brain samples. ...
Abstract Progranulin (PGRN) is a protein encoded by the GRN gene with multiple identified functions including as a neurotrophic factor, tumorigenic growth factor, anti-inflammatory cytokine and regulator of lysosomal function. A single mutation in the human GRN gene resulting in reduced PGRN expression causes types of frontotemporal lobar degeneration resulting in frontotemporal dementia. Prosaposin (PSAP) is also a multifunctional neuroprotective secreted protein and regulator of lysosomal function. Interactions of PGRN and PSAP affect their functional properties. Their roles in Alzheimer’s disease (AD), the leading cause of dementia, have not been defined. In this report, we examined in detail the cellular expression of PGRN in middle temporal gyrus samples of a series of human brain cases (n = 45) staged for increasing plaque pathology. Immunohistochemistry showed PGRN expression in cortical neurons, microglia, cerebral vessels and amyloid beta (Aβ) plaques, while PSAP expression was mainly detected in neurons and Aβ plaques, and to a limited extent in astrocytes. We showed that there were increased levels of PGRN protein in AD cases and corresponding increased levels of PSAP. Levels of PGRN and PSAP protein positively correlated with amyloid beta (Aβ), with PGRN levels correlating with phosphorylated tau (serine 205) levels in these samples. Although PGRN colocalized with lysosomal-associated membrane protein-1 in neurons, most PGRN associated with Aβ plaques did not. Aβ plaques with PGRN and PSAP deposits were identified in the low plaque non-demented cases suggesting this was an early event in plaque formation. We did not observe PGRN-positive neurofibrillary tangles. Co-immunoprecipitation studies of PGRN from brain samples identified only PSAP associated with PGRN, not sortilin or other known PGRN-binding proteins, under conditions used. Most PGRN associated with Aβ plaques were immunoreactive for PSAP showing a high degree of colocalization of these proteins that did not change between disease groups. As PGRN supplementation has been considered as a therapeutic approach for AD, the possible involvement of PGRN and PSAP interactions in AD pathology needs to be further considered.
... Alveolar macrophages are critical for protection from respiratory failure and associated morbidity after influenza virus infection [22]. Furthermore, some evidence suggests that macrophage-secreted PGRN plays a central role in host defense [21,23], inflammatory response [24,25] and tumor growth [26]. Clodronate Liposomes (CL) is taken up by phagocytic macrophage (Mφ) and accumulates in the cytosol, resulting in Mφ death and depletion [27]. ...
Type I interferons (IFNs) play a critical role in host defense against influenza virus infection, and the mechanism of influenza virus to evade type I IFNs responses remains to be fully understood. Here, we found that progranulin (PGRN) was significantly increased both in vitro and in vivo during influenza virus infection. Using a PGRN knockdown assay and PGRN-deficient mice model, we demonstrated that influenza virus-inducing PGRN negatively regulated type I IFNs production by inhibiting the activation of NF-κB and IRF3 signaling. Furthermore, we showed that PGRN directly interacted with NF-κB essential modulator (NEMO) via its Grn CDE domains. We also verified that PGRN recruited A20 to deubiquitinate K63-linked polyubiquitin chains on NEMO at K264. In addition, we found that macrophage played a major source of PGRN during influenza virus infection, and PGRN neutralizing antibodies could protect against influenza virus-induced lethality in mice. Our data identify a PGRN-mediated IFN evasion pathway exploited by influenza virus with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of PGRN in innate immunity.
... PGRN deficiency triggers increased expression of proinflammatory cytokines, including TNF and IL-6, by macrophages and microglia (6,7). Elastase cleaves PGRN into smaller granulin peptides, which have been shown to augment TLR9 signaling by enhancing delivery of CpG-oligodeoxynucleotides (CpG-ODNs) (8). ...
NK cell-mediated regulation of antigen-specific T cells can contribute to and exacerbate chronic viral infection, but the protective mechanisms against NK cell-mediated attack on T cell immunity are poorly understood. Here, we show that progranulin (PGRN) can reduce NK cell cytotoxicity through reduction of NK cell expansion, granzyme B transcription, and NK cell-mediated lysis of target cells. Following infection with the lymphocytic choriomeningitis virus (LCMV), PGRN levels increased - a phenomenon dependent on the presence of macrophages and type I IFN signaling. Absence of PGRN in mice (Grn-/-) resulted in enhanced NK cell activity, increased NK cell-mediated killing of antiviral T cells, reduced antiviral T cell immunity, and increased viral burden, culminating in increased liver immunopathology. Depletion of NK cells restored antiviral immunity and alleviated pathology during infection in Grn-/- mice. In turn, PGRN treatment improved antiviral T cell immunity. Taken together, we identified PGRN as a critical factor capable of reducing NK cell-mediated attack of antiviral T cells.
Despite pigs being an important species in influenza A virus (IAV) epidemiology and a reliable model of human IAV infections, many aspects of the porcine pulmonary immune system remain poorly understood. Here, we characterized the single cell landscape of lung leukocytes of healthy pigs and then compared them to pigs infected with 2009 pandemic H1N1 IAV with or without oseltamivir antiviral therapy. Our data show conserved features as well as species-specific differences in cell types and cell states compared to human and mouse lung leukocytes. IAV infection induced a robust antiviral transcriptional response in multiple lymphoid and myeloid cell types, as well as distinct patterns of cell-cell cross talk. Oseltamivir treatment reduced these responses. Together our findings describe key events in the pulmonary anti-IAV response of pigs that open new avenues to develop IAV vaccines and therapies. They should also enable the better use of pigs as a model for human IAV infection and immunity.
Objective
This study aimed to investigate the possible role of granulin (GRN) in activating the TLR9-IFN-α pathway in renal tubular epithelial cells (RTECs) and explore clues that RTECs regulate the micro-environment of inflammatory response in lupus nephritis (LN).
Methods
Renal sections from 57 LN patients and 30 non-LN patients were sampled for histological study, and GRN overexpression RTECs were applied for cytological study.
Results
In the histological study, GRN is highly expressed in LN RTECs with tubulointerstitial inflammation (TII) and well co-localized with TLR9. ROC analysis suggested a potential relationship between GRN expression in RTECs and therapeutic response. Moreover, IFN-α also highly expressed in LN RTECs with TII, and the intensity of IFN-α is positively correlated with the co-localization intensity of GRN and TLR9. In the cytological study, LN serum, especially serum from LN with TII, activates the expression of TLR9 in RTECs, and GRN engages the interaction of TLR9 to activate the expression of IFN-α in RTECs. While TLR9 inhibitors can suppress the expression of IFN-α in RTECs, the degree of inhibition is dose-dependent.
Conclusion
The expression of GRN in RTECs is associated with interstitial inflammation and therapeutic response. GRN may mediate the activation of the TLR9-IFN-α pathway in RTECs and involve in the micro-environment of inflammatory response in LN.
Nile tilapia (Oreochromis niloticus) is one of the major food fish worldwide. The farming business, on the other hand, has faced considerable obstacles, such as disease infestations. Toll-like receptors (TLRs) play an important function in the activation of the innate immune system in response to infections. Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing TLRs. Here the UNC93B1 gene, which was cloned from Nile tilapia tissue for this investigation, had the same genetic structure as a homologous gene in humans and mice. Phylogenetic analysis revealed that Nile tilapia UNC93B1 clustered with UNC93B1 from other species and separately from the UNC93A clade. The gene structure of the Nile tilapia UNC93B1 was found to be identical to that of human UNC93B1. Our gene expression studies revealed that Nile tilapia UNC93B1 was highly expressed in the spleen, followed by other immune-related tissues such as the head kidney, gills, and intestine. Moreover, Nile tilapia UNC93B1 mRNA transcripts were up-regulated in vivo in the head kidney and spleen tissues from poly I:C and Streptococcus agalactiae injected Nile tilapia, as well as in vitro in LPS stimulated Tilapia head kidney (THK) cells. The Nile tilapia UNC93B1-GFP protein signal was detected in the cytosol of THK cells and was co-localized with endoplasmic reticulum and lysosome but not with mitochondria. Moreover, the results of a co-immunoprecipitation and immunostaining analysis showed that Nile tilapia UNC93B1 can be pulled down with fish-specific TLRs such as TLR18 and TLR25 from Nile tilapia, and was found to be co-localized with these fish-specific TLRs in the THK cells. Overall, our findings highlight the potential role of UNC93B1 as an accessory protein in fish-specific TLR signaling.
Les démences frontotemporales (DFT) et la sclérose latérale amyotrophique (SLA) sont deux maladies neurodégénératives faisant partie d'un même spectre clinique, génétique et neuropathologique. Afin d’identifier une voie biologique commune aux DFTs et SLA, nous nous sommes intéressés aux mécanismes d’activation microgliale. Deux axes d’étude principaux ont composé cette thèse : 1/ La protéine TDP-43, qui forme les inclusions majoritaires des DFT-SLA, est-elle capable d’activer les cellules microgliales ? Par quels mécanismes ? 2/ La perte de fonction des protéines C9ORF72 et PGRN a-t-elle des conséquences sur la réactivité microgliale en présence de TDP-43? Par l’utilisation de modèles in vitro de cultures primaires microgliales nous montrons que la protéine TDP-43 active la voie non canonique microgliale de l'inflammasome NLRP3. Celle-ci se produit suite à l’interaction de TDP-43 avec les récepteurs TLR2/4, et à son internalisation dans les cellules microgliales. Les cellules microgliales murines déficientes C9orf72-/- et GrnR493X/R493X sont hyper-réactives vis-à-vis d’une stimulation par TDP-43. Cet effet est reproduit dans un modèle « MDMi » de cellules microgliales dérivées de monocytes de patients DFT porteurs de mutation GRN ou C9ORF72. Enfin, nous montrons que cet effet peut être lié aux fonctions des protéines PGRN et C9ORF72 nécessaires au bon fonctionnement de la boucle de rétrocontrôle de l’activité de l’inflammasome par la voie autophagique. Nos données mettent en évidence une voie commune de neuroinflammation dans les DFTs et la SLA, et ouvrent de nouvelles pistes sur les processus neurodégénératifs de ces maladies.
Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To glean into the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN’s absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize, and modulate cytoplasmic TDP-43 inclusions in proteinopathies.
Objectives
This study aimed to detect the expression of progranulin (PGRN) and elucidate associations with clinical features in dermatomyositis (DM) patients with anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody.Methods
We enrolled 40 DM patients with anti-MDA5 antibody, 20 patients with antisynthetase syndrome (ASS; disease control), and 20 healthy individuals (healthy control, HC). The clinical features of patients with anti-MDA5 antibody and anti-histidyl-tRNA antibody were collected. The level of PGRN in the serum was tested by ELISA.ResultsThe PGRN levels in DM patients with anti-MDA5 antibody (166.74 ± 97.95 ng/ml) were significantly higher than those in patients with ASS (82.66 ± 40.50 ng/ml; p < 0.001) and in HC (42.34 ± 18.69 ng/ml; p < 0.001). Patients with rapid progressive interstitial lung disease (RP-ILD) in DM with anti-MDA5 antibody (213.57 ± 114.05 ng/ml) had higher levels of PGRN than those without RP-ILD (135.51 ± 72.41 ng/ml; p = 0.012). ROC analysis showed an AUC value at 0.715 (95% CI, 0.541–0.888) for diagnosis of RP-ILD in DM patients with anti-MDA5 antibody. The expression of PGRN was positively correlated with the levels of ALT, AST, CK, LDH and ferritin (all p < 0.05).Conclusions
Our results indicated PGRN had great potential as a valuable serum marker of RP-ILD in DM with anti-MDA5 antibody.
Key Points
The level of PGRN was elevated in DM patients with anti-MDA5 antibody, especially for those with RP-ILD.
Granulins (GRN 1‐7) are short (~6 kDa), cysteine‐rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These peptides, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Previously we showed that GRN‐3 and GRN‐5 are fully disordered in the reduced form implicating redox sensitive attributes to the proteins. Redox‐based modulations are often carried out by metalloproteins in mitigating oxidative stress and maintaining metal‐homeostasis within cells. To probe whether GRNs play a role in metal sequestration, we tested the metal binding propensity of the reduced forms of GRNs −3 and − 5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu and no other divalent metal cations, with a greater specificity for Cu(I). Binding of Cu did not result in a disorder‐to‐order structural transition but partly triggered the multimerization of GRNs via uncoordinated cystines at both pH conditions. Overall, the results indicate that GRNs −3 and − 5 have surprisingly strong affinity for Cu in the pM range, comparable to other known copper sequestering proteins. The results also hint at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu‐induced ROS cytotoxicity in cells. Together, this report uncovers metal‐coordinating property of GRNs for the first time, which may have profound significance in their structure and pathophysiological functions.
We have identified progranulin (PGRN) gene as a sex steroid-inducible gene in the brain, which is involved in masculinization of the brain during the perinatal period and facilitation of adult neurogenesis in the hippocampus. PGRN was first reported in early 1990s by different groups as a protein containing seven and a half cysteine-rich granulin domains and having growth promoting properties involved in wound healing and tumorigenesis. Later it was found that mutations of the PGRN gene were associated with neurodegenerative and lysosomal storage diseases such as frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis, respectively. We have also found that PGRN is located in lysosomes and deficiency of PGRN results in exacerbated neuroinflammatory responses and increased lysosomal biogenesis in microglia after traumatic brain injury. It is now recognized that PGRN may regulate activities of lysosomal enzymes as a molecular chaperone and play a crucial role in maintaining cellular protein homeostasis. Deeper understanding of molecular and functional properties of PGRN would provide new insights for developing mechanism-based therapeutic approaches for multiple disorders including cancers and neurodegenerative diseases.
Background & Aims
Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection (CDI) via activation of TLR9.
Methods
We performed studies with human colonocytes and monocytes and macrophages from wildtype and TLR9-knockout mice, incubated with TcdA or its antagonist (ODN TTAGGG), or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with an ELISA. We studied a transduction domain of TcdA (TcdA57-80), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle x-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight:length ratios. A TLR9 antagonist was tested in mice infected with C. difficile. We incubated human colon with an antagonist of TLR9 and measured TcdA-induced production of cytokines.
Results
The TcdA57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C. difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA57–80 domain organized DNA into a geometrically ordered structure that activated TLR9.
Conclusions
TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.
Background
Non‐invasive screening for liver fibrosis using transient elastography (TE) could be of value in the management of Gaucher disease (GD). Progranulin (PGRN) is a novel disease modifier in GD and an independent marker of liver fibrosis.
Objectives
We determined PGRN levels in pediatric patients with GD and assessed its role as a potential marker for disease severity and relation to liver stiffness by TE.
Methods
Fifty‐one GD patients (20 had type 1 and 31 had type 3) with a median age 9.5 years were compared to 40 age‐ and sex‐matched healthy controls and were studied focusing on visceral manifestations, neurological disease, hematological profile and PGRN levels as well as abdominal ultrasound and TE. Patients were on enzyme replacement therapy for various durations and those with viral hepatitis infection were excluded.
Results
By TE, 14 GD patients (27.5%) had elevated liver stiffness ≥7.0 kPa. Liver stiffness was significantly higher in type 1 GD patients than type 3 (p=0.002), in splenectomized patients (p=0.012) and those with dysphagia (p<0.001). Liver stiffness was positively correlated to age of onset of ERT (p<0.001). PGRN levels were significantly lower in GD patients compared with controls (p<0.001). PGRN was significantly lower in GD patients with squint (p=0.025), dysphagia (p=0.036) and elevated liver stiffness (p=0.015). PGRN was positively correlated to white blood cell count (r=0.455, p=0.002) and hemoglobin (r=0.546, p<0.001) while negatively correlated to severity score index (r=‐0.529, p<0.001), liver volume (r=‐0.298, p=0.034) and liver stiffness (r=‐0.652, p<0.001).
Conclusions
Serum PGRN levels were associated with clinical disease severity and elevated liver stiffness in pediatric GD patients.
Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host, parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp. affect their host are not fully understood. Herein, proteomic alterations of primary, bone marrow-derived BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, applying a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (combined total of 6189 mouse proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface.
Significance
Little is known on proteome changes that occur in primary macrophages after Leishmania donovani infection. This study describes a SILAC–based quantitative proteomics approach to characterise changes of bone marrow-derived macrophages infected with L. donovani promastigotes for 72 h. With the application of SILAC and the use of SAX and GEL fractionation methods, we have tested new routes for proteome quantification of primary macrophages. The protocols developed here can be applicable to other diseases and pathologies. Moreover, this study sheds important new light on the “proteomic reprogramming” of infected macrophages in response to L. donovani promastigotes that affects primary metabolism, cellular catabolic processes, and lysosomal/vacuole organisation. Thus, our study reveals key molecules and processes that act at the host/pathogen interface that may inform on new immuno- or chemotherapeutic interventions to combat leishmaniasis.
Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.
Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation
groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary–K1 fibroblasts defective in responses to bacterial
endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation
by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. We identified the lesion in one of the complementation groups in
the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with
MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in
a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the
wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2C95Y, functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling
complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally
affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of
the true ligand for TLR4.
In atherosclerosis and Alzheimer's disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-beta triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-beta trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-beta stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.
Progranulin (PGRN) is a widely expressed protein involved in diverse biological processes. Haploinsufficiency of PGRN in the human causes tau-negative, ubiquitin-positive frontotemporal dementia (FTD). However, the mechanisms are unknown. To explore the role of PGRN in vivo, we generated PGRN-deficient mice. Macrophages from these mice released less interleukin-10 and more inflammatory cytokines than wild type (WT) when exposed to bacterial lipopolysaccharide. PGRN-deficient mice failed to clear Listeria monocytogenes infection as quickly as WT and allowed bacteria to proliferate in the brain, with correspondingly greater inflammation than in WT. PGRN-deficient macrophages and microglia were cytotoxic to hippocampal cells in vitro, and PGRN-deficient hippocampal slices were hypersusceptible to deprivation of oxygen and glucose. With age, brains of PGRN-deficient mice displayed greater activation of microglia and astrocytes than WT, and their hippocampal and thalamic neurons accumulated cytosolic phosphorylated transactivation response element DNA binding protein-43. Thus, PGRN is a key regulator of inflammation and plays critical roles in both host defense and neuronal integrity. FTD associated with PGRN insufficiency may result from many years of reduced neutrotrophic support together with cumulative damage in association with dysregulated inflammation.
Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we show that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The carboxy-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG DNA and, when expressed in Tlr9(-/-) dendritic cells, restored CpG DNA-induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9.
Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.
Epithelin 1 and 2 were originally purified from rat kidneys based on their ability to inhibit the growth of A-431 human epidermoid carcinoma cells (Shoyab, M., McDonald, V.L., Byles, C., Todaro, G.J., and Plowman, G.D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7912-7916). This study presents the complete amino acid sequence of these two growth factors and the cloning of their cDNA from rat, mouse, and human sources. Epithelins 1 and 2 are 56- and 57-amino acid polypeptides, respectively, and share 47% amino acid sequence identity with the conserved spacing of 12 cysteine residues. Molecular cloning revealed that both proteins are encoded by a single precursor that contains 7 1/2 copies of this novel 12-cysteine motif, 2 of which represent the known active molecules. Recombinant expression in COS cells demonstrated that the epithelin 1 protein was mitogenic on rodent keratinocytes and fibroblasts. In contrast, epithelin 2 had no activity on these cells, but at high concentrations was capable of antagonizing the growth proliferative activities of epithelin 1. Northern analysis shows the epithelin mRNA to be expressed in many types of epithelial cells. The broad expression profile of epithelin transcripts, along with the opposing activities of the two mature protein products, implicates these factors as natural mediators of epithelial homeostasis.
Two proteins, termed epithelin 1 and epithelin 2, that inhibit the growth of A431 cells, derived from a human epidermal carcinoma of the vulva, have been purified from rat kidney. Epithelin 1 stimulates the proliferation of murine keratinocytes, whereas epithelin 2 inhibits the epithelin 1-elicited growth of these cells. Thus epithelin 1 and 2 behave as agonist and antagonist, respectively, for normal epithelial cells. Epithelins are low molecular mass (approximately 6 kDa), acid- and heat-stable, single-chain proteins containing approximately 20% cysteine. Some of these cysteines form disulfide linkage(s) that are essential for biological activity. The amino-terminal amino acid sequences of epithelin 1 and epithelin 2 have been determined. The two proteins showed no substantial sequence homology with other proteins. However, a significant homology was seen between the amino-terminal sequences of epithelin 1 and epithelin 2. Epithelins 1 and 2, therefore, appear to represent members of a distinct family of growth regulators.
Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.
Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory tract infections. This study investigated the ability of NTHi to bind lipopolysaccharide-binding
protein (LBP) derived from respiratory epithelial cells and the subsequent stimulation of transfected cells expressing membrane-bound
CD14 and toll-like receptor 2 (TLR2) or TLR4. In the absence of LBP, NTHi at high concentrations (100 bacteria/epithelial
cell) were required to induce signals through TLR2 and TLR4. Flow cytometry showed that NTHi in the stationary phase bound
more LBP than did log-phase bacteria. Of interest, as few as 1 LBP-bearing bacterium/cell induced strong signaling through
TLR4. In contrast, LBP bound to NTHi did not promote any increased signaling mediated by TLR2, compared with NTHi without
LBP. These data suggest that, upon NTHi infection, low numbers of bacteria binding LBP may activate TLR4-bearing cells, such
as alveolar macrophages, and consequently induce an inflammatory response
The imidazoquinoline compounds imiquimod and R-848 are low-molecular-weight immune response modifiers that can induce the synthesis of interferon-alpha and other cytokines in a variety of cell types. These compounds have potent anti-viral and anti-tumor properties; however, the mechanisms by which they exert their anti-viral activities remain unclear. Here we show that the imidazoquinolines activate immune cells via the Toll-like receptor 7 (TLR7)-MyD88-dependent signaling pathway. In response to the imidazoquinolines, neither MyD88- nor TLR7-deficient mice showed any inflammatory cytokine production by macrophages, proliferation of splenocytes or maturation of dendritic cells. Imidazoquinoline-induced signaling events were also abolished in both MyD88- and TLR7-deficient mice.
The Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns (PAMPs). TLR2 is essential for the signaling of a variety of PAMPs, including bacterial lipoprotein/lipopeptides, peptidoglycan, and GPI anchors. TLR6 associates with TLR2 and recognizes diacylated mycoplasmal lipopeptide along with TLR2. We report here that TLR1 associates with TLR2 and recognizes the native mycobacterial 19-kDa lipoprotein along with TLR2. Macrophages from TLR1-deficient (TLR1(-/-)) mice showed impaired proinflammatory cytokine production in response to the 19-kDa lipoprotein and a synthetic triacylated lipopeptide. In contrast, TLR1(-/-) cells responded normally to diacylated lipopeptide. TLR1 interacts with TLR2 and coexpression of TLR1 and TLR2 enhanced the NF-kappaB activation in response to a synthetic lipopeptide. Furthermore, lipoprotein analogs whose acylation was modified were preferentially recognized by TLR1. Taken together, TLR1 interacts with TLR2 to recognize the lipid configuration of the native mycobacterial lipoprotein as well as several triacylated lipopeptides.
Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent
TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed
in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma
membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated
to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated
TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated
by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.
Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.
Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA-containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.
Toll-like receptors (TLRs) are sensors for the detection of invading infectious agents and can initiate innate immune responses. Because the innate immune system induces an appropriate defense against different pathogens, different TLR signaling domains may have unique properties that are responsible for eliciting distinctive responses to different types of pathogens. To test this hypothesis, we created ligand-regulated TLR chimeric receptors composed of the extracellular region of TLR4 and the transmembrane and cytoplasmic regions of other TLRs and expressed these chimeras in macrophages lacking endogenous TLR4. Interestingly, the chimeras between TLR4 and either TLR3, TLR7, or TLR9 were localized completely intracellularly whereas other chimeras were expressed on the cell surface. Lipopolysaccharide (LPS), a ligand for these chimeras, induced the activation of nuclear factor kappa B and mitogen-activated protein kinases and the subsequent production of pro-inflammatory cytokines in macrophages expressing TLR4, TLR4/TLR5, or TLR4/TLR8 chimeras but not in macrophages expressing TLR4/TLR1, TLR4/TLR2, or TLR4/TLR6 chimeras. Co-expression of unresponsive chimeras in some combinations (chimeras with TLR1+TLR2 or TLR2+TLR6 but not TLR1+TLR6) resulted in LPS responsiveness, indicating functional complementarity. Furthermore, the pair of TLR2+TLR6 chimera required approximately 10-fold less LPS to induce the same responses compared with the TLR1+TLR2 pair. Finally, LPS induced effective interferon-beta production and subsequent Stat1 phosphorylation in macrophages expressing full-length TLR4 but not other cell surface TLR chimeras. These results suggest that the functions of TLRs are diversified not only in their extracellular regions for ligand recognition but also in their transmembrane and cytoplasmic regions for subcellular localization and signaling properties.
The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity. All TLRs use the adaptor MyD88 for signalling, but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5-/- mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5-/- mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.
Toll-like receptor 2 (TLR2) is required for the recognition of numerous molecular components of bacteria, fungi and protozoa. The breadth of the ligand repertoire seems unusual, even if one considers that TLR2 may form heteromers with TLRs 1 and 6 (ref. 12), and it is likely that additional proteins serve as adapters for TLR2 activation. Here we show that an N-ethyl-N-nitrosourea-induced nonsense mutation of Cd36 (oblivious) causes a recessive immunodeficiency phenotype in which macrophages are insensitive to the R-enantiomer of MALP-2 (a diacylated bacterial lipopeptide) and to lipoteichoic acid. Homozygous mice are hypersusceptible to Staphylococcus aureus infection. Cd36(obl) macrophages readily detect S-MALP-2, PAM(2)CSK(4), PAM(3)CSK(4) and zymosan, revealing that some--but not all--TLR2 ligands are dependent on CD36. Already known as a receptor for endogenous molecules, CD36 is also a selective and nonredundant sensor of microbial diacylglycerides that signal via the TLR2/6 heterodimer.
The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.
Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation.
Toll-like receptor (TLR) 3 and TLR7 are indispensable for host defense against viral infection by recognizing virus-derived RNAs and are localized to intracellular membranes via an unknown mechanism. We recently reported experiments with chimeric Toll-like receptors that suggested that the subcellular distribution of TLRs may be defined by their transmembrane and/or cytoplasmic domains. Here we demonstrate that the intracellular localization of TLR3 is achieved by a 23-amino acid sequence (Glu(727) to Asp(749)) present in the linker region between the transmembrane domain and Toll-interleukin 1 receptor resistance (TIR) domain. In contrast, the intracellular localization of TLR7 is achieved by its transmembrane domain. These elements also targeted a heterologous type I transmembrane protein CD25 to the intracellular compartment that contained TLR3 and TLR7. Despite their using distinct regulatory elements for intracellular localization, TLR3 was found to co-localize with TLR7. In addition, TLR3 and TLR7 were preferentially localized near phagosomes containing apoptotic cell particles. These findings reveal that TLR3 and TLR7 contain unique targeting sequences, which differentially lead them to the same intracellular compartments and adjacent to phagosomes containing apoptotic cell particles, where these receptors may access their ligands for the induction of immune responses against viral infection.
CpG-DNA or its synthetic analog CpG-ODN activates innate immunity through Toll-like receptor 9 (TLR9). However, the mechanism of TLR9 activation by CpG-DNA remains elusive. Here we have identified HMGB1 as a CpG-ODN-binding protein. HMGB1 interacts and preassociates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and hastens TLR9's redistribution to early endosomes in response to CpG-ODN. CpG-ODN stimulates macrophages and dendritic cells to secrete HMGB1; in turn, extracellular HMGB1 accelerates the delivery of CpG-ODNs to its receptor, leading to a TLR9-dependent augmentation of IL-6, IL-12, and TNFalpha secretion. Loss of HMGB1 leads to a defect in the IL-6, IL-12, TNFalpha, and iNOS response to CpG-ODN. However, lack of intracellular TLR9-associated HMGB1 can be compensated by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells and regulates inflammatory responses to CpG-DNA.
Neutrophil granulocytes form the body's first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex-mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.
Epithelin 1 and 2 were originally purified from rat kidneys based on their ability to inhibit the growth of A-431 human epidermoid carcinoma cells (Shoyab, M., McDonald, V. L., Byles, C., Todaro, G. J., and Plowman, G. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7912-7916). This study presents the complete amino acid sequence of these two growth factors and the cloning of their cDNAs from rat, mouse, and human sources. Epithelins 1 and 2 are 56- and 57-amino acid polypeptides, respectively, and share 47% amino acid sequence identity with the conserved spacing of 12 cysteine residues. Molecular cloning revealed that both proteins are encoded by a single precursor that contains 7 1/2 copies of this novel 12-cysteine motif, 2 of which represent the known active molecules. Recombinant expression in COS cells demonstrated that the epithelin 1 protein was mitogenic on rodent keratinocytes and fibroblasts. In contrast, epithelin 2 had no activity on these cells, but at high concentrations was capable of antagonizing the growth proliferative activities of epithelin 1. Northern analysis shows the epithelin mRNA to be expressed in many types of epithelial cells. The broad expression profile of epithelin transcripts, along with the opposing activities of the two mature protein products, implicates these factors as natural mediators of epithelial homeostasis.
Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite. this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation.
Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFβ and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.
The discovery of Toll-like receptors (TLRs) as components that recognize conserved structures in pathogens has greatly advanced understanding of how the body senses pathogen invasion, triggers innate immune responses and primes antigen-specific adaptive immunity. Although TLRs are critical for host defense, it has become apparent that loss of negative regulation of TLR signaling, as well as recognition of self molecules by TLRs, are strongly associated with the pathogenesis of inflammatory and autoimmune diseases. Furthermore, it is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Here we describe the recent advances that have been made by research into the role of TLR biology in host defense and disease.
We report the isolation and characterization of a novel class of leukocyte peptides with possible cytokine-like activities which we call granulins. They are cystine-rich with molecular weights of approximately 6 Kda, except for granulin D, which appears to be a dimer. We present the sequence of one member of this family, a 56 residue peptide, granulin A, and amino-terminal sequences for three other granulins from human peripheral leukocytes. A fifth related peptide was isolated and partially sequenced from rat bone marrow, suggesting that at least some of the granulin in peripheral leukocytes is preformed in the marrow. Rat granulin, and human granulin A, are closely related, showing that the granulin structures are highly conserved between species.
The granulin/epithelin protein motif has an unusual structure consisting of a parallel stack of beta-hairpins stapled together by six disulphide bonds. The new structure also contains a folding subdomain shared by small toxins, protease inhibitors as well as the EGF-like protein modules.
The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.
The innate immune system is a universal and ancient form of host defense against infection. Innate immune recognition relies on a limited number of germline-encoded receptors. These receptors evolved to recognize conserved products of microbial metabolism produced by microbial pathogens, but not by the host. Recognition of these molecular structures allows the immune system to distinguish infectious nonself from noninfectious self. Toll-like receptors play a major role in pathogen recognition and initiation of inflammatory and immune responses. Stimulation of Toll-like receptors by microbial products leads to the activation of signaling pathways that result in the induction of antimicrobial genes and inflammatory cytokines. In addition, stimulation of Toll-like receptors triggers dendritic cell maturation and results in the induction of costimulatory molecules and increased antigen-presenting capacity. Thus, microbial recognition by Toll-like receptors helps to direct adaptive immune responses to antigens derived from microbial pathogens.
The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.
Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.
Toll-like receptors (TLRs) sense infection by detecting molecular structures of microbial origin. TLR3, TLR7 and TLR9 recognize nucleic acids and are localized to intracellular compartments where they normally respond to viral nucleic acids. The purpose for this intracellular localization, however, is not clear. Here we describe a chimeric TLR9 receptor that localized to the cell surface and responded normally to synthetic TLR9 ligands but not to viral nucleic acids. However, the 'relocated' chimeric TLR9 receptor was able to recognize self DNA, which does not stimulate wild-type TLR9. These data demonstrated that intracellular localization of TLR9 was not required for ligand recognition. Instead, localization of the nucleic acid-sensing TLRs is critical in discriminating between self and nonself nucleic acid.
Bacterial cell walls contain lipoproteins/peptides, which are strong modulators of the innate immune system. Triacylated lipopeptides
are assumed to be recognized by TLR2/TLR1-, whereas diacylated lipopeptides use TLR2/TLR6 heteromers for signaling. Following
our initial discovery of TLR6-independent diacylated lipopeptides, we could now characterize di- and triacylated lipopeptides
(e.g. Pam2C-SK4, Pam3C-GNNDESNISFKEK), which have stimulatory activity in TLR1- and in TLR6-deficient mice. Furthermore, for the first time, we
present triacylated lipopeptides with short length ester-bound fatty acids (like PamOct2C-SSNASK4), which induce no response in TLR1-deficient cells. No differences in the phosphorylation of MAP kinases by lipopeptide analogs
having different TLR2-coreceptor usage were observed. Blocking experiments indicated that different TLR2 heteromers recognize
their specific lipopeptide ligands independently from each other. In summary, a triacylation pattern is necessary but not
sufficient to render a lipopeptide TLR1-dependent, and a diacylation pattern is necessary but not sufficient to render a lipopeptide
TLR6-dependent. Contrary to the current model, distinct lipopeptides are recognized by TLR2 in a TLR1- and TLR6-independent
manner.
Deoxycytidyl-deoxyguanosine [(CpG)3] oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFN-alphabeta) and IFN-alphabeta-dependent MHC-I cross-presentation of exogenous antigens by dendritic cells (DCs). A puzzle was presented by our observation that three ODN classes, CpG-A, CpG-B, and CpG-C, had similar efficacy for induction of IFN-alphabeta-dependent MHC-I antigen cross-presentation by myeloid DCs despite greatly differing for induction of IFN-alphabeta (CpG-A>CpG-C>CpG-B). All ODN classes similarly enhanced plasmacytoid DC (pDC) presentation of exogenous MHC-I-restricted peptide, although pDCs did not cross-process protein antigen. MHC-I and the transporter for antigen presentation were induced by all ODN classes or IFN-alpha. CpG-B ODNs were slightly more potent than CpG-A or CpG-C ODNs for induction of low levels of IFN-alphabeta but less efficacious at high concentrations than CpG-A or CpG-C ODNs. Low levels of IFN-alphabeta induced by CpG-B ODNs sufficed for full induction of MHC-I cross-presentation. Thus, CpG-B ODNs are slightly more potent but less efficacious than CpG-A and CpG-C ODNs for induction of IFN-alphabeta. High sensitivity to IFN-alphabeta allows CpG-B ODNs to be equally efficacious for induction of MHC-I cross-presentation. CpG-B ODNs may be effective for inducing therapeutic responses that require low levels of IFN-alphabeta and may avoid unnecessarily high induction of IFN-alphabeta.
Sexual differentiation of the brain in rodents is achieved by estrogens, which are converted from androgens in the brain, during the perinatal period. We have identified the progranulin (PGRN) gene as one of the sex steroid-inducible genes that may be involved in masculinization of the rat brain. In the present study, we generated a line of mice with targeted disruption of the PGRN gene, and investigated male sexual behaviour, aggression and anxiety. PGRN-deficient mice exhibited a decrease in ejaculation incidence, while the latency and frequency of both mount and intromission were unchanged. For the aggressive behaviour test, the resident-intruder paradigm was used, and PGRN-deficient mice exhibited enhanced aggressiveness. In wild-type mice, males exhibited lower levels of anxiety than females by the open field test, while male PGRN-deficient mice exhibited an elevated level of anxiety and sex difference in anxiety was not observed. In addition, mRNA expression of the serotonergic receptor 5-HT1A, which could be related to the inhibition of aggression and anxiety, was significantly reduced in the hippocampus of PGRN-deficient mice after aggressive encounters. On the other hand, deficiency of the PGRN gene did not affect serum testosterone concentrations. These results suggest that PGRN gene plays a role in establishing sexual dimorphic behaviours at least partially by modulating the brain serotonergic system.
Different HLA class I alleles display a distinctive dependence on tapasin for surface expression and Ag presentation. In this study, we show that the tapasin dependence of HLA class I alleles correlates to the nature of the amino acid residues present at the naturally polymorphic position 114. The tapasin dependence of HLA class I alleles bearing different residues at position 114 decreases in the order of acidity, with high tapasin dependence for acidic amino acids (aspartic acid and glutamic acid), moderate dependence for neutral amino acids (asparagine and glutamine), and low dependence for basic amino acids (histidine and arginine). A glutamic acid to histidine substitution at position 114 allows the otherwise tapasin-dependent HLA-B4402 alleles to load high-affinity peptides independently of tapasin and to have surface expression levels comparable to the levels seen in the presence of tapasin. The opposite substitution, histidine to glutamic acid at position 114, is sufficient to change the HLA-B2705 allele from the tapasin-independent to the tapasin-dependent phenotype. Furthermore, analysis of point mutants at position 114 reveals that tapasin plays a principal role in transforming the peptide-binding groove into a high-affinity, peptide-receptive conformation. The natural polymorphisms in HLA class I H chains that selectively affect tapasin-dependent peptide loading provide insights into the functional interaction of tapasin with MHC class I molecules.
Signalling by means of toll-like receptors (TLRs) is essential for the development of innate and adaptive immune responses. UNC93B1, essential for signalling of TLR3, TLR7 and TLR9 in both humans and mice, physically interacts with these TLRs in the endoplasmic reticulum (ER). Here we show that the function of the polytopic membrane protein UNC93B1 is to deliver the nucleotide-sensing receptors TLR7 and TLR9 from the ER to endolysosomes. In dendritic cells of 3d mice, which express an UNC93B1 missense mutant (H412R) incapable of TLR binding, neither TLR7 nor TLR9 exits the ER. Furthermore, the trafficking and signalling defects of the nucleotide-sensing TLRs in 3d dendritic cells are corrected by expression of wild-type UNC93B1. However, UNC93B1 is dispensable for ligand recognition and signal initiation by TLRs. To our knowledge, UNC93B1 is the first protein to be identified as a molecule specifically involved in trafficking of nucleotide-sensing TLRs. By inhibiting the interaction between UNC93B1 and TLRs it should be possible to achieve specific regulation of the nucleotide-sensing TLRs without compromising signalling via the cell-surface-disposed TLRs.
CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer Immunity The Function of Granulin on TLR9 Signaling 512 Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors
- C R Stewart
- L M Stuart
- K Wilkinson
- J M Gils
- J Deng
- A Halle
- K J Rayner
- L Boyer
- R Zhong
- W A Frazier
Stewart, C.R., Stuart, L.M., Wilkinson, K., van Gils, J.M., Deng, J., Halle, A., Rayner, K.J., Boyer, L., Zhong, R., Frazier, W.A., et al. (2010). CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer. Nat. Immunol. 11, 155–161. Immunity The Function of Granulin on TLR9 Signaling 512 Immunity 34, 505–513, April 22, 2011 ª2011 Elsevier Inc. rTakaoka, A., Yanai, H., Kondo, S., Duncan, G., Negishi, H., Mizutani, T., Kano, S., Honda, K., Ohba, Y., Mak, T.W., and Taniguchi, T. (2005). Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434, 243–249
- E Latz
- A Schoenemeyer
- A Visintin
- K A Fitzgerald
- B G Monks
- C F Knetter
- E Lien
- N J Nilsen
- T Espevik
- D T Golenbock
Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K.A., Monks, B.G., Knetter,
C.F., Lien, E., Nilsen, N.J., Espevik, T., and Golenbock, D.T. (2004). TLR9
signals after translocating from the ER to CpG DNA in the lysosome. Nat.
Immunol. 5, 190–198.
TLR3 and TLR7 are targeted to the same intracellular compartments by distinct regulatory elements.
- Nishiya T.
- Kajita E.
- Miwa S.
- Defranco A.L.
Nishiya, T., Kajita, E., Miwa, S., and Defranco, A.L. (2005). TLR3 and TLR7 are
targeted to the same intracellular compartments by distinct regulatory
elements. J. Biol. Chem. 280, 37107–37117.
TLR3 and TLR7 are targeted to the same intracellular compartments by distinct regulatory elements
- Nishiya
Conversion of proepithelin to epithelins: Roles of SLPI and elastase in host defense and wound repair
- Zhu