[Show abstract][Hide abstract] ABSTRACT: Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia–reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the
Proceedings of the National Academy of Sciences 11/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is characterized by extracellular amyloid-β (Aβ) deposits and microglia-dominated inflammatory activation. Innate immune signaling controls microglial inflammatory activities and Aβ clearance. However, studies examining innate immunity in Aβ pathology and neuronal degeneration have produced conflicting results. In this study, we investigated the pathogenic role of innate immunity in AD by ablating a key signaling molecule, IKKβ, specifically in the myeloid cells of TgCRND8 APP-transgenic mice. Deficiency of IKKβ in myeloid cells, especially microglia, simultaneously reduced inflammatory activation and Aβ load in the brain and these effects were associated with reduction of cognitive deficits and preservation of synaptic structure proteins. IKKβ deficiency enhanced microglial recruitment to Aβ deposits and facilitated Aβ internalization, perhaps by inhibiting TGF-β-SMAD2/3 signaling, but did not affect Aβ production and efflux. Therefore, inhibition of IKKβ signaling in myeloid cells improves cognitive functions in AD mice by reducing inflammatory activation and enhancing Aβ clearance. These results contribute to a better understanding of AD pathogenesis and could offer a new therapeutic option for delaying AD progression.
The Journal of neuroscience : the official journal of the Society for Neuroscience. 09/2014; 34(39):12982-99.
[Show abstract][Hide abstract] ABSTRACT: Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1(-/-) mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER(T2)Ripk1(fl/fl) mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1(fl/fl) fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1(fl/fl) mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1(fl/fl) Ripk3(-/-) progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.
Proceedings of the National Academy of Sciences of the United States of America. 09/2014;
[Show abstract][Hide abstract] ABSTRACT: Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-κB signaling pathway has been implicated; however, the distinct roles of the individual NF-κB transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-κB subunits c-REL or RELA, which are both activated by the canonical NF-κB pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-κB pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-κB in GC lymphomagenesis.
[Show abstract][Hide abstract] ABSTRACT: Interferon Regulatory Factor 5 (IRF5) plays a major role in setting up an inflammatory macrophage phenotype, but the molecular basis of its transcriptional activity is not fully understood. In this study, we conduct a comprehensive genome-wide analysis of IRF5 recruitment in macrophages stimulated with bacterial lipopolysaccharide and discover that IRF5 binds to regulatory elements of highly transcribed genes. Analysis of protein:DNA microarrays demonstrates that IRF5 recognizes the canonical IRF-binding (interferon-stimulated response element [ISRE]) motif in vitro. However, IRF5 binding in vivo appears to rely on its interactions with other proteins. IRF5 binds to a noncanonical composite PU.1:ISRE motif, and its recruitment is aided by RelA. Global gene expression analysis in macrophages deficient in IRF5 and RelA highlights the direct role of the RelA:IRF5 cistrome in regulation of a subset of key inflammatory genes. We map the RelA:IRF5 interaction domain and suggest that interfering with it would offer selective targeting of macrophage inflammatory activities.
[Show abstract][Hide abstract] ABSTRACT: Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation. RIPK1 is implicated in inflammatory and cell death signalling and its kinase activity is believed to drive RIPK3-mediated necroptosis. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.
[Show abstract][Hide abstract] ABSTRACT: The serine/threonine kinase RIPK1 is recruited to TNFR1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. A RIPK1 deficiency results in perinatal lethality, impaired NFκB and MAPK signaling, and sensitivity to TNF-induced apoptosis. Chemical inhibitor and in vitro-reconstitution studies suggested that RIPK1 displays distinct kinase activity-dependent and -independent functions. To determine the contribution of RIPK1 kinase to inflammation in vivo, we generated knock-in mice endogenously expressing catalytically inactive RIPK1 D138N. Unlike Ripk1(-/-) mice, which die shortly after birth, Ripk1(D138N/D138N) mice are viable. Cells expressing RIPK1 D138N are resistant to TNF- and polyinosinic-polycytidylic acid-induced necroptosis in vitro, and Ripk1(D138N/D138N) mice are protected from TNF-induced shock in vivo. Moreover, Ripk1(D138N/D138N) mice fail to control vaccinia virus replication in vivo. This study provides genetic evidence that the kinase activity of RIPK1 is not required for survival but is essential for TNF-, TRIF-, and viral-initiated necroptosis.
Journal of immunology (Baltimore, Md. : 1950). 07/2014;
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO(LPC-KO) mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO(LPC-KO) mice. To address potential functional redundancies between death receptors we generated and analysed NEMO(LPC-KO) mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO(LPC-KO) mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO(LPC-KO) mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO(LPC-KO) mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.Cell Death and Differentiation advance online publication, 27 June 2014; doi:10.1038/cdd.2014.83.
Cell Death and Differentiation 06/2014; · 8.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nuclear factor kappa B (NF-κB) signalling pathway exhibits both tumour-promoting and tumour-suppressing functions in different tissues and models of carcinogenesis. In particular in epidermal keratinocytes, NF-κB signalling was reported to exert primarily growth inhibitory and tumour-suppressing functions. Here, we show that mice with keratinocyte-restricted p65/RelA deficiency were resistant to 7, 12-dimethylbenz(a)anthracene (DMBA)-/12-O-tetra decanoylphorbol-13 acetate (TPA)-induced skin carcinogenesis. p65 deficiency sensitized epidermal keratinocytes to DNA damage-induced death in vivo and in vitro, suggesting that inhibition of p65-dependent prosurvival functions prevented tumour initiation by facilitating the elimination of cells carrying damaged DNA. In addition, lack of p65 strongly inhibited TPA-induced epidermal hyperplasia and skin inflammation by suppressing the expression of proinflammatory cytokines and chemokines by epidermal keratinocytes. Therefore, p65-dependent NF-κB signalling in keratinocytes promotes DMBA-/TPA-induced skin carcinogenesis by protecting keratinocytes from DNA damage-induced death and facilitating the establishment of a tumour-nurturing proinflammatory microenvironment.
EMBO Molecular Medicine 06/2014; · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress.
Journal of Experimental Medicine 04/2014; · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immune responses in the skin are important for host defence against pathogenic microorganisms. However, dysregulated immune reactions can cause chronic inflammatory skin diseases. Extensive crosstalk between the different cellular and microbial components of the skin regulates local immune responses to ensure efficient host defence, to maintain and restore homeostasis, and to prevent chronic disease. In this Review, we discuss recent findings that highlight the complex regulatory networks that control skin immunity, and we provide new paradigms for the mechanisms that regulate skin immune responses in host defence and in chronic inflammation.
[Show abstract][Hide abstract] ABSTRACT: CYLD is a tumour suppressor gene mutated in familial cylindromatosis, a genetic disorder leading to the development of skin appendage tumours. It encodes a deubiquitinating enzyme that removes Lys63- or linear-linked ubiquitin chains. CYLD was shown to regulate cell proliferation, cell survival and inflammatory responses, through various signalling pathways. Here we show that CYLD localizes at centrosomes and basal bodies via interaction with the centrosomal protein CAP350 and demonstrate that CYLD must be both at the centrosome and catalytically active to promote ciliogenesis independently of NF-κB. In transgenic mice engineered to mimic the smallest truncation found in cylindromatosis patients, CYLD interaction with CAP350 is lost disrupting CYLD centrosome localization, which results in cilia formation defects due to impairment of basal body migration and docking. These results point to an undiscovered regulation of ciliogenesis by Lys63 ubiquitination and provide new perspectives regarding CYLD function that should be considered in the context of cylindromatosis.
[Show abstract][Hide abstract] ABSTRACT: Choroidal neovascularization (CNV) is aberrant angiogenesis associated with exudative age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Inflammation has been suggested as a risk factor for AMD. The IKK2/NF-κB pathway plays a key role in the inflammatory response through regulation of the transcription of cytokines, chemokines, growth factors and angiogenic factors. We investigated the functional role of IKK2 in development of the laser-induced CNV using either Ikk2 conditional knockout mice or an IKK2 inhibitor. The retinal neuronal tissue and RPE deletion of IKK2 was generated by breeding Ikk2(-/flox) mice with Nestin-Cre mice. Deletion of Ikk2 in the retina caused no obvious defect in retinal development or function, but resulted in a significant reduction in laser-induced CNV. In addition, intravitreal or retrobulbar injection of an IKK2 specific chemical inhibitor, TPCA-1, also showed similar inhibition of CNV. Furthermore, in vitro inhibition of IKK2 in ARPE-19 cells significantly reduced heat shock-induced expression of NFKBIA, IL1B, CCL2, VEGFA, PDGFA, HIF1A, and MMP-2, suggesting that IKK2 may regulate multiple molecular pathways involved in laser-induced CNV. The in vivo laser-induced expression of VEGFA, and HIF1A in RPE and choroidal tissue was also blocked by TPCA-1 treatment. Thus, IKK2/NF-κB signaling appears responsible for production of pro-inflammatory and pro-angiogenic factors in laser-induced CNV, suggesting that this intracellular pathway may serve as an important therapeutic target for aberrant angiogenesis in exudative AMD.
PLoS ONE 01/2014; 9(1):e87530. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Psoriasis is a common chronic inflammatory skin disease with a prevalence of about 2% in the Caucasian population. Tumor necrosis factor (TNF) plays an essential role in the pathogenesis of psoriasis, but its mechanism of action remains poorly understood. Here we report that the development of psoriasis-like skin inflammation in mice with epidermis-specific inhibition of the transcription factor NF-κB was triggered by TNF receptor 1 (TNFR1)-dependent upregulation of interleukin-24 (IL-24) and activation of signal transducer and activator of transcription 3 (STAT3) signaling in keratinocytes. IL-24 was strongly expressed in human psoriatic epidermis, and pharmacological inhibition of NF-κB increased IL-24 expression in TNF-stimulated human primary keratinocytes, suggesting that this mechanism is relevant for human psoriasis. Therefore, our results expand current views on psoriasis pathogenesis by revealing a new keratinocyte-intrinsic mechanism that links TNFR1, NF-κB, ERK, IL-24, IL-22R1, and STAT3 signaling to disease initiation.
[Show abstract][Hide abstract] ABSTRACT: COPD is an inflammatory lung disease largely associated with exposure to cigarette smoke (CS). The mechanism by which CS leads to the pathogenesis of COPD is currently unclear; it is known however that many of the inflammatory mediators present in the COPD lung can be produced via the actions of the transcription factor Nuclear Factor-kappaB (NF-κB) and its upstream signalling kinase, Inhibitor of κB kinase-2 (IKK-2). Therefore the NF-κB/IKK-2 signalling pathway may represent a therapeutic target to attenuate the inflammation associated with COPD.
To use a range of assays, genetically modified animals and pharmacological tools to determine the role of NF-κB in CS-induced airway inflammation.
NF-κB pathway activation was measured in pre-clinical models of CS-induced airway inflammation and in human lung tissue from COPD patients. This data was complemented by employing mice missing a functional NF-κB pathway in specific cell types (epithelial and myeloid cells) and with systemic inhibitors of IKK-2.
We showed in an airway inflammation model known to be NF-κB-dependent that the NF-κB pathway activity assays and modulators were functional in the mouse lung. Then, using the same methods, we demonstrated that the NF-κB pathway appears not to play an important role in the inflammation observed after exposure to CS. Furthermore, assaying human lung tissue revealed that in the clinical samples there was also no increase in NF-κB pathway activation in the COPD lung, suggesting that our pre-clinical data is translational to human disease.
In this study we present compelling evidence that the IKK-2/NF-κB signalling pathway does not play a prominent role in the inflammatory response to CS exposure and that this pathway may not be important in COPD pathogenesis.
PLoS ONE 10/2013; 8(1):e54128. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pneumocystis is an atypical fungal pathogen that causes severe, often fatal pneumonia in immunocompromised patients. Healthy humans and animals also encounter this pathogen, but they generate a protective CD4(+) T cell-dependent immune response that clears the pathogen with little evidence of disease. Pneumocystis organisms attach tightly to respiratory epithelial cells, and in vitro studies have demonstrated that this interaction triggers NF-κB-dependent epithelial cell responses. However, the contribution of respiratory epithelial cells to the normal host response to Pneumocystis remains unknown. IκB kinase 2 (IKK2) is the upstream kinase that is critical for inducible NF-κB activation. To determine whether IKK2-dependent lung epithelial cell (LEC) responses contribute to the anti-Pneumocystis immune response in vivo, transgenic mice with LEC-specific deletion of IKK2 (IKK2(ΔLEC)) were generated. Compared to wild-type mice, IKK2(ΔLEC) mice exhibited a delayed onset of Th17 and B cell responses in the lung and delayed fungal clearance. Importantly, delayed Pneumocystis clearance in IKK2(ΔLEC) mice was associated with an exacerbated immune response, impaired pulmonary function, and altered lung histology. These data demonstrate that IKK2-dependent LEC responses are important regulators of pulmonary adaptive immune responses and are required for optimal host defense against Pneumocystis infection. LECs likely set the threshold for initiation of the pulmonary immune response and serve to prevent exacerbated lung inflammation by promoting the rapid control of respiratory fungal infection.
The Journal of Immunology 09/2013; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell.
Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted.
Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice.
Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.
PLoS ONE 01/2013; 8(2):e55620. · 3.53 Impact Factor