[Show abstract][Hide abstract] ABSTRACT: Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network. DOI: http://dx.doi.org/10.7554/eLife.10561.001
[Show abstract][Hide abstract] ABSTRACT: The intestinal immune system remains unresponsive to beneficial microbes and dietary antigens while activating pro-inflammatory
responses against pathogens for host defence. In intestinal mucosa, abnormal activation of innate immunity, which directs
adaptive immune responses, causes the onset and/or progression of inflammatory bowel diseases. Thus, innate immunity is finely
regulated in the gut. Multiple innate immune cell subsets have been identified in both murine and human intestinal lamina
propria. Some innate immune cells play a key role in the maintenance of gut homeostasis by preventing inappropriate adaptive
immune responses while others are associated with the pathogenesis of intestinal inflammation through development of Th1 and
Th17 cells. In addition, intestinal microbiota and their metabolites contribute to the regulation of innate/adaptive immune
responses. Accordingly, perturbation of microbiota composition can trigger intestinal inflammation by driving inappropriate
No preview · Article · Nov 2015 · Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIβ, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.
No preview · Article · Aug 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Wnt5a, which regulates various cellular functions in Wnt signaling, is involved in inflammatory responses, however the mechanism is not well understood. We examined the role of Wnt5a signaling in intestinal immunity using conditional knockout mice for Wnt5a and its receptor Ror2. Removing Wnt5a or Ror2 in adult mice suppressed dextran sodium sulfate (DSS)-induced colitis. It also attenuated the DSS-dependent increase in inflammatory cytokine production and decreased interferon-γ (IFN-γ)-producing CD4(+) Th1 cell numbers in the colon. Wnt5a was highly expressed in stromal fibroblasts in ulcerative lesions in the DSS-treated mice and inflammatory bowel disease patients. Dendritic cells (DCs) isolated from the colon of Wnt5a and Ror2 deficient mice reduced the ability to differentiate naïve CD4(+) T cells to IFN-γ-producing CD4(+) Th1 cells. In vitro experiments demonstrated that the Wnt5a-Ror2 signaling axis augmented the DCs priming effect of IFN-γ, leading to enhanced lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression. Taken together, these results suggest that Wnt5a promotes IFN-γ signaling, leading to IL-12 expression in DCs, and thereby inducing Th1 differentiation in colitis.
[Show abstract][Hide abstract] ABSTRACT: Extracellular adenosine 5'-triphophate (ATP) mediates the immune response. Several ecto-enzymes hydrolyze ATP, including the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-nucleotide pyrophosphate/phosphodiesterase (E-NPP) protein families. Among these, E-NTPD1, E-NTPD7, and E-NPP3 have been shown to regulate the immune response. E-NTPD1 is expressed in lymphocytes and myeloid cells and modulates their function. E-NTPD7, which is selectively expressed in the epithelial cells of the small intestine, regulates Th17 responses in the small intestine by controlling ATP levels. E-NPP3 is rapidly induced on activated basophils and mast cells, and regulates ATP-dependent activation in basophils and mast cells to prevent chronic allergic inflammation. Thus, ATP-hydrolyzing ecto-enzymes modulate the immune response through ATP hydrolysis.
Preview · Article · Jan 2015 · Inflammation and Regeneration
[Show abstract][Hide abstract] ABSTRACT: The mammalian gastrointestinal tract, the site of nutrient digestion and absorption, harbors a dense microbial community. The intestinal immune system can distinguish between symbiotic bacteria and pathogens, and activates pro-inflammatory responses against pathogenic bacteria for host defense while remaining unresponsive to the beneficial microbes and dietary antigens. Abnormal activity of innate immunity, which directs the development of adaptive immunity, causes the onset and/or progression of several inflammatory diseases. Thus, activity of innate immunity is finely regulated in the gut. Inflammatory bowel disease is a chronic inflammatory disorder caused by alteration of several factors, such as host genetics, commensal bacteria and diet-derived compounds and metabolites. In intestinal mucosa, multiple innate immune cells have been identified and some populations play a crucial role in the maintenance of gut homeostasis by preventing inadequate adaptive immune responses while others are implicated in the pathogenesis of inflammatory bowel disease by driving Th1 and Th17 responses. In addition, recent studies demonstrated that dietary components and their metabolites produced by commensal bacteria contribute to the generation of a unique intestinal environment and further regulation of a variety of immune responses. Accordingly, alterations of intestinal microbial composition and perturbation of metabolites can trigger intestinal inflammation by inducing inadequate innate/adaptive immune responses.
Preview · Article · Jan 2015 · Inflammation and Regeneration
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii infection results in co-option and subversion of host cellular signaling pathways. This process involves discharge of T. gondii effector molecules from parasite secretory organelles such as rhoptries and dense granules. We report that the T. gondii polymorphic dense granule protein GRA6 regulates activation of the host transcription factor nuclear factor of activated T cells 4 (NFAT4). GRA6 overexpression robustly and selectively activated NFAT4 via calcium modulating ligand (CAMLG). Infection with wild-type (WT) but not GRA6-deficient parasites induced NFAT4 activation. Moreover, GRA6-deficient parasites failed to exhibit full virulence in local infection, and the treatment of WT mice with an NFAT inhibitor mitigated virulence of WT parasites. Notably, NFAT4-deficient mice displayed prolonged survival, decreased recruitment of CD11b(+) Ly6G(+) cells to the site of infection, and impaired expression of chemokines such as Cxcl2 and Ccl2. In addition, infection with type I parasites culminated in significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C terminus of GRA6. Collectively, our data suggest that GRA6-dependent NFAT4 activation is required for T. gondii manipulation of host immune responses to maximize the parasite virulence in a strain-dependent manner.
Preview · Article · Sep 2014 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: In a recent paper published in Cell Host & Microbe, Dasgupta et al. (2014) demonstrate that a component of the gut microbiota, Bacteroides fragilis, induces IL-10-producing regulatory T cells by driving TLR2-dependent plasmacytoid dendritic cell activation.
[Show abstract][Hide abstract] ABSTRACT: Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyer's patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyer's patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.
[Show abstract][Hide abstract] ABSTRACT: To maintain a quiescent gut microenvironment, proper regulation of immune responses initiated by pro-inflammatory immune subsets is required. Several types of regulatory T cells are reported to exert pivotal roles in achieving this. Among various types of regulatory T cells, the crucial role of Foxp3(+) Treg cells has been well documented. Furthermore, accumulating evidence demonstrates that both microbial and dietary factors influence the induction and suppressor functions of intestinal Foxp3(+) Treg cells. Foxp3(+) Treg cells are a highly activated T cell subset which responds rapidly to environmental and nutritional stimuli. Thus, sufficient nutrient supply is required to fuel the high energetic status of Foxp3(+) Treg cells for the regulation of intestinal immunity.
[Show abstract][Hide abstract] ABSTRACT: IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ-regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti-T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti-T. gondii cellular immunity by recruiting IFN-γ-inducible GTPases. IFN-γ-induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti-T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti-T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ-mediated anti-T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ-induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ-induced recruitment of the immunity-related GTPases to the intracellular pathogen.
No preview · Article · Feb 2014 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response.
As an example, 2'-O methylation of the 5′ cap of viral RNA subverts mammalian antiviral responses by evading restriction of
Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing
Ifit1 even though their genomic RNA has a 5′ cap lacking 2'-O methylation. We show that pathogenic alphaviruses use secondary structural
motifs within the 5′ untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5′-UTR affecting RNA structural elements enabled restriction by or antagonism
of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid
[Show abstract][Hide abstract] ABSTRACT: To clarify the function of osteoclast-like multinuclear cells differentiated from bone marrow-derived macrophages (BMMs) by a combination of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and to investigate the molecular mechanisms underlying the differentiation.
BMMs were stimulated by TNFα and/or IL-6. The cells were then compared with conventional osteoclasts differentiated in vitro by RANKL. An in vitro pit formation assay on dentine slices and an in vivo resorption assay of calvarial bones were performed. We also evaluated the activities and expression levels of NF-κB, c-Fos, and NF-ATc1, which are essential to the differentiation of conventional osteoclasts. Small interfering RNA was used to knock down c-Fos. The effects of genetic ablation of STAT-3 and pharmacologic inhibitors of NF-AT, JAK, and ERK were also studied.
Osteoclast-like cell differentiation depended on TNFα and IL-6 and was not inhibited by osteoprotegerin. These differentiated cells were associated with both in vitro and in vivo bone resorption activity. TNFα and IL-6 had a synergistic effect on the activity and expression of c-Fos. Knockdown of c-Fos inhibited the expression of NF-ATc1 and the differentiation of osteoclast-like cells. All of these inhibitors blocked differentiation of the cells in vitro, but surprisingly, the conditional knockout of STAT-3 did not. Tofacitinib also inhibited the bone destruction caused by TNFα and IL-6 in vivo.
Our results demonstrate that a combination of the inflammatory cytokines TNFα and IL-6 can induce osteoclast-like cells that have in vitro and in vivo bone-resorptive activity.
No preview · Article · Jan 2014 · Arthritis and Rheumatology
[Show abstract][Hide abstract] ABSTRACT: The intestinal immune system has an ability to distinguish between the microbiota and pathogenic bacteria, and then activate pro-inflammatory pathways against pathogens for host defense while remaining unresponsive to the microbiota and dietary antigens. In the intestine, abnormal activation of innate immunity causes development of several inflammatory disorders such as inflammatory bowel diseases (IBD). Thus, activity of innate immunity is finely regulated in the intestine. To date, multiple innate immune cells have been shown to maintain gut homeostasis by preventing inadequate adaptive immune responses in the murine intestine. Additionally, several innate immune subsets, which promote Th1 and Th17 responses and are implicated in the pathogenesis of IBD, have recently been identified in the human intestinal mucosa. The demonstration of both murine and human intestinal innate immune subsets contributing to regulation of adaptive immunity emphasizes the conserved innate immune functions across species and might promote development of the intestinal innate immunity-based clinical therapy.
[Show abstract][Hide abstract] ABSTRACT: Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers in vivo. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier in vivo.
[Show abstract][Hide abstract] ABSTRACT: & Aims: Abnormal activity of innate immune cells and T-helper (Th)17 cells has been implicated in the pathogenesis of autoimmune and inflammatory diseases, including Crohn's disease (CD). Intestinal innate immune (myeloid) cells have been found to induce development of Th17 cells in mice, but it is not clear if this occurs in humans, or in patients with CD. We investigated whether human intestinal lamina propria cells (LPCs) induce development of Th17 cells and whether these have a role in pathogenesis of CD.
Normal intestinal mucosa samples were collected from patients with colorectal cancer, and non-inflammed and inflammed regions of mucosa were collected from patients with CD. LPCs were isolated by enzymatic digestion and analyzed for expression of HLA-DR, lineage markers (Lin), CD14, and CD163 using flow cytometry.
Among HLA-DR(high) Lin(-) cells, we identified a subset of CD14(+) CD163(low) cells in intestinal LPCs; this subset expressed Toll-like receptor (TLR)2, TLR4, and TLR5 mRNAs and produced interleukin (IL)6, IL1β, and tumor necrosis factor (TNF)α in response to lipopolysaccharide. In vitro co-culture with naïve T cells revealed that CD14(+) CD163(low) cells induced development of Th17 cells. CD14(+) CD163(low) cells from inflamed regions of mucosa of patients with CD expressed high levels of IL6, IL23p19, and TNF mRNAs, and strongly induced Th17 cells. CD14(+) CD163(low) cells from the non-inflamed mucosa of patients with CD also had increased abilities to induce Th17 cells, compared with those from normal intestinal mucosa.
CD14(+) CD163(low) cells in intestinal LPCs from normal intestinal mucosa induce differentiation of naïve T cells into Th17 cells; this activity is increased in mucosal samples from patients with CD. These findings show how intestinal myeloid cell types could contribute to pathogenesis of CD and possibly other Th17-associated diseases.