Lack of Toll IL-1R8 exacerbates Th17 cell responses in fungal infection

TLRs contribute to the inflammatory response in fungal infections. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases. In this study, we tested the hypothesis that Toll IL-1R8 (TIR8)/single Ig IL-1-related receptor, a member of the IL-1R family acting as a negative regulator of TLR/IL-1R signaling, affects TLR responses in fungal infections. Genetically engineered Tir8(-/-) mice were assessed for inflammatory and adaptive Th cell responses to Candida albicans and Aspergillus fumigatus. Inflammatory pathology and susceptibility to infection were higher in Tir8(-/-) mice and were causally linked to the activation of the Th17 pathway. IL-1R signaling was involved in Th17 cell activation by IL-6 and TGF-beta in that limited inflammatory pathology and relative absence of Th17 cell activation were observed in IL-1RI(-/-) mice. These data demonstrate that TIR8 is required for host resistance to fungal infections and that it functions to negatively regulate IL-1-dependent activation of inflammatory Th17 responses. TIR8 may contribute toward fine-tuning the balance between protective immunity and immunopathology in infection.     

Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT1/2 inhibit RELMbeta induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELMbeta, a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELMbeta expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxo1 nuclear localization, and robust induction of RELMbeta expression in wild-type, but not IL-4Ralpha knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4Ralpha.     

Induction of endothelial nitric oxide synthase expression by IL-17 in human vascular endothelial cells: implications for vascular remodeling in transplant vasculopathy

IL-17 is a signature cytokine of Th17 cells, a recently described subset of effector CD4 T cells implicated in the development of several pathologies. We have examined the role of IL-17 in regulating endothelial NO synthase (eNOS) expression in human vascular endothelial cells (ECs) because of the key role of eNOS in determining the pathological outcome of immune-mediated vascular diseases. In cultured ECs, IL-17 increased expression of eNOS, eNOS phosphorylation at Ser(1177), and NO production. The induction of eNOS expression by IL-17 was prevented by the pharmacological inhibition of NF-κB, MEK, and JNK, as well as by small interfering RNA-mediated gene silencing of these signaling pathways. The expression of IL-17 was then examined by immunohistochemistry in human arteries affected by transplant vasculopathy (TV), a vascular condition that is a leading reflection of chronic heart transplant rejection. IL-17 was expressed by infiltrating leukocytes in the intima of arteries with TV, and the majority of IL-17-positive cells were T cells. The number of IL-17-positive cells was not correlated with the intima/media ratio, but was negatively correlated with the amount of luminal occlusion. There was also a significant positive correlation between the number of IL-17-positive cells and the density of eNOS-expressing luminal ECs in arteries with TV. Altogether, these findings show that IL-17 induces the expression of eNOS in human ECs and that this may facilitate outward expansion of arteries afflicted with TV.     

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes

Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.     

Th17 and Th17-stimulated CD8<Superscript>+</Superscript> T cells play a distinct role in Th17-induced preventive and therapeutic antitumor immunity

CD4⁺ Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4⁺ T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-β, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell–killing activity. However, Th17 cells were able to stimulate CD8⁺ CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4⁺, and CD8⁺ T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8⁺ CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8⁺ CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.     

The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway

Vulvovaginal candidiasis (VVC) caused by Candida albicans affects a significant number of women during their reproductive ages. Clinical observations revealed that a robust vaginal polymorphonuclear neutrophil (PMN) migration occurs in susceptible women, promoting pathological inflammation without affecting fungal burden. Evidence to date in the mouse model suggests that a similar acute PMN migration into the vagina is mediated by chemotactic S100A8 and S100A9 alarmins produced by vaginal epithelial cells in response to Candida. Based on the putative role for the Th17 response in mucosal candidiasis as well as S100 alarmin induction, this study aimed to determine whether the Th17 pathway plays a role in the S100 alarmin-mediated acute inflammation during VVC using the experimental mouse model. For this, IL-23p19^−/−, IL-17RA^−/− and IL-22^−/− mice were intravaginally inoculated with Candida, and vaginal lavage fluids were evaluated for fungal burden, PMN infiltration, the presence of S100 alarmins and inflammatory cytokines and chemokines. Compared to wild-type mice, the cytokine-deficient mice showed comparative levels of vaginal fungal burden and PMN infiltration following inoculation. Likewise, inoculated mice of all strains with substantial PMN infiltration exhibited elevated levels of vaginal S100 alarmins in both vaginal epithelia and secretions in the vaginal lumen. Finally, cytokine analyses of vaginal lavage fluid from inoculated mice revealed equivalent expression profiles irrespective of the Th17 cytokine status or PMN response. These data suggest that the vaginal S100 alarmin response to Candida does not require the cells or cytokines of the Th17 lineage, and therefore, the immunopathogenic inflammatory response during VVC occurs independently of the Th17-pathway.     

Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine

The requirements for in vivo steady state differentiation of IL-17-producing T-helper (Th17) cells, which are potent inflammation effectors, remain obscure. We report that Th17 cell differentiation in the lamina propria (LP) of the small intestine requires specific commensal microbiota and is inhibited by treating mice with selective antibiotics. Mice from different sources had marked differences in their Th17 cell numbers and animals lacking Th17 cells acquired them after introduction of bacteria from Th17 cell-sufficient mice. Differentiation of Th17 cells correlated with the presence of cytophaga-flavobacter-bacteroidetes (CFB) bacteria in the intestine and was independent of toll-like receptor, IL-21 or IL-23 signaling, but required appropriate TGF-beta activation. Absence of Th17 cell-inducing bacteria was accompanied by increase in Foxp3+ regulatory T cells (Treg) in the LP. Our results suggest that composition of intestinal microbiota regulates the Th17:Treg balance in the LP and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases.     

Type I IFN promotes IL-10 production from T cells to suppress Th17 cells and Th17-associated autoimmune inflammation

Whereas the immune system is essential for host defense against pathogen infection or endogenous danger signals, dysregulated innate and adaptive immune cells may facilitate harmful inflammatory or autoimmune responses. In the CNS, chronic inflammation plays an important role in the pathogenesis of neurodegenerative diseases such as multiple sclerosis (MS). Our previous study has demonstrated a critical role for the type I IFN induction and signaling pathways in constraining Th17-mediated experimental autoimmune encephalomyelitis (EAE), an animal model of human MS. However, it remains unknown if self-reactive Th17 cells can be reprogrammed to have less encephalitogenic activities or even have regulatory effects through modulation of innate pathways. In this study, we investigated the direct effects of type I IFN on Th17 cells. Our data show that IFNβ treatment of T cells cultured under Th17 polarizing conditions resulted in reduced production of IL-17, but increased production of IL-10. We also found that IFNβ induced IL-10 production by antigen specific T cells derived from immunized mice. Furthermore, IFNβ treatment could suppress the encephalitogenic activity of myelin-specific T cells, and ameliorate clinical symptoms of EAE in an adoptive transfer model. Together, results from this study suggest that IFNβ may induce antigen-specific T cells to produce IL-10, which in turn negatively regulate Th17-mediate inflammatory and autoimmune response.     

IL-17 expression in human herpetic stromal keratitis: modulatory effects on chemokine production by corneal fibroblasts

Herpetic stromal keratitis (HSK) is an immunopathologic disease triggered by infection of the cornea with HSV. Key events in HSK involve the interaction between cornea-infiltrating inflammatory cells and resident cells. This interaction, in which macrophages, producing IL-1 and TNF-alpha, and IFN-gamma-producing Th1 cells play a crucial role, results in the local secretion of immune-modulatory factors and a major influx of neutrophils causing corneal lesions and blindness. The Th1-derived cytokine IL-17 has been shown to play an important role in several inflammatory diseases characterized by a massive infiltration of neutrophils into inflamed tissue. Here we show that IL-17 is expressed in corneas from patients with HSK and that the IL-17R is constitutively expressed by human corneal fibroblasts (HCF). IL-17 exhibited a strong synergistic effect with TNF-alpha on the induction of IL-6 and IL-8 secretion by cultured HCF. Secreted IL-8 in these cultures had a strong chemotactic effect on neutrophils. IL-17 also enhanced TNF-alpha- and IFN-gamma-induced secretion of macrophage-inflammatory proteins 1alpha and 3alpha, while inhibiting the induced secretion of RANTES. Furthermore, considerable levels of IFN-gamma-inducible protein 10 and matrix metalloproteinase 1 were measured in stimulated HCF cultures, while the constitutive secretion of monocyte chemotactic protein 1 remained unaffected. The data presented suggest that IL-17 may play an important role in the induction and/or perpetuation of the immunopathologic processes in human HSK by modulating the secretion of proinflammatory and neutrophil chemotactic factors by corneal resident fibroblasts.     

Type 17 T-helper cells might be a promising therapeutic target for osteoporosis

Osteoporosis, a disease characterized by low bone mass and deterioration of bone tissue, is a pressing public health problem. Recent studies have suggested a possible role of T-helper (Th) cells in the pathogenesis of bone loss which occurs in systemic inflammatory diseases. However, there are contradictions in the published literature regarding the functional role of Th1/Th2 cells in the regulation of the differentiation of osteoclasts. These paradoxes have now been clarified by the recent discovery of Th17 cells, a novel subset of Th cells that selectively secrete several proinflammatory cytokines, mainly IL-17. It has been confirmed that Th17 cells have stimulatory effects on osteoclastogenesis and accelerate bone loss in animal models with inflammatory disorders. Targeting Th17 cells or IL-17 may inhibit the bone resorption with RA. Thus, we are led to suppose that Th17 cells might be promising therapeutic targets in osteoporosis.     

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.     

Intranasal Administration of Recombinant Mycobacterium smegmatis Inducing IL-17A Autoantibody Attenuates Airway Inflammation in a Murine Model of Allergic Asthma

Asthma is a chronic inflammatory disorder, previous studies have shown that IL-17A contributes to the development of asthma, and there is a positive correlation between the level of IL-17A and the severity of disease. Here, we constructed recombinant Mycobacterium smegmatis expressing fusion protein Ag85A-IL-17A (rMS-Ag85a-IL-17a) and evaluated whether it could attenuate allergic airway inflammation, and further investigated the underlying mechanism. In this work, the murine model of asthma was established with ovalbumin, and mice were intranasally vaccinated with rMS-Ag85a-IL-17a. Autoantibody of IL-17A in sera was detected, and the airway inflammatory cells infiltration, the local cytokines and chemokines production and the histopathological changes of lung tissue were investigated. We found that the administration of rMS-Ag85a-IL-17a induced the autoantibody of IL-17A in sera. The vaccination of rMS-Ag85a-IL-17a remarkably reduced the infiltration of inflammatory cells and the secretion of mucus in lung tissue and significantly decreased the numbers of the total cells, eosinophils and neutrophils in BALF. Th1 cells count in spleen, Th1 cytokine levels in BALF and supernatant of splenocytes and mediastinal lymph nodes, and T-bet mRNA in lung tissue were significantly increased with rMS-Ag85a-IL-17a administration. Meanwhile, rMS-Ag85a-IL-17a vaccination markedly decreased Th2 cells count, Th2 cytokine and Th17 cytokine levels in BALF and supernatant of splenocytes and mediastinal lymph nodes, and chemokines mRNA expression in lung tissue. These data confirmed that recombinant Mycobacterium smegmatis in vivo could induce autoantibody of IL-17A, which attenuated asthmatic airway inflammation.     

Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Background

Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.

Objective

To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.

Methods

Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.

Results

OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.

Conclusions

Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0164-4) contains supplementary material, which is available to authorized users.     

A knowledgebase resource for interleukin-17 family mediated signaling

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.     

Imbalanced Frequencies of Th17 and Treg Cells in Acute Coronary Syndromes Are Mediated by IL-6-STAT3 Signaling

Aims

Extensive evidence suggests inflammatory components participate in the pathogenic processes of acute coronary syndromes (ACS). In this study, we aimed to elucidate the role and mechanism underlying the imbalance of Th17 and Treg cell peripheral populations in the pathogenesis of ACS.

Methods and Results

Using a flow cytometric analysis, we observed a significantly increased frequency of Th17 cells and a concurrently decreased CD4⁺CD25⁺Foxp3⁺ Treg cells in patients with ACS. To elucidate the mechanism of Th17/Treg imbalance in ACS, 22 inflammatory cytokines were measured using multiplexed immunobead-based assays. Of six elevated cytokines in ACS patients, only IL-6 was positively correlated with a higher Th17 cell level (r = 0.39, P<0.01). Relying on IL-6 stimulating and neutralizing studies, we demonstrated a direct role for IL-6 in sera from ACS patients with an increased frequency of Th17 cells. IL-6 induces the differentiation of Th17 cells from naïve CD4⁺ T cells through STAT3 activation and RORγt induction. However, we observed that high levels of TGF-β1 inhibited IL-6-dependent Th17 cell differentiation, indicating a complex interplay between the two cytokines in the control of Th17 and Treg cell populations.

Conclusions

Our results demonstrate the role of IL-6-STAT3 signaling in ACS through increased Th17 cell differentiation. These findings indicate that IL-6 neutralizing strategies could present novel therapeutic avenues in the treatment of ACS.     

Human Th17 cells

The discovery in mice of a new lineage of CD4⁺ effector T helper (Th) cells that selectively produce IL-17 has provided exciting new insights into immune regulation, host defence, and the pathogenesis of autoimmune and other chronic inflammatory disorders. This population of CD4⁺ Th cells, which has been termed 'Th17', indeed plays an apparently critical role in the pathogenesis of some murine models of autoimmunity. Interestingly, murine Th17 cells share a common origin with Foxp3⁺ T regulatory cells, because both populations are produced in response to transforming growth factor-β, but they develop into Th17 cells only when IL-6 is simultaneously produced. Initial studies in humans have confirmed the existence of Th17 cells, but they have shown that the origin of these cells in humans differs from that in mice, with IL-1β and IL-23 being the major cytokines responsible for their development. Moreover, the presence in the circulation and in various tissues of Th cells that can produce both IL-17 and interferon-γ, as well as the flexibility of human Th17 clones to produce interferon-γ in addition to IL-17 in response to IL-12, suggests that there may be a developmental relationship between Th17 and Th1 cells, at least in humans. Resolving this issue has great implications in tems of establishing the respective pathogenic roles of Th1 and Th17 cells in autoimmune disorders. In contrast, it is unlikely that Th17 cells contribute to the pathogenesis of human allergic IgE-mediated disorders, because IL-4 and IL-25 (a powerful inducer of IL-4) are both potent inhibitors of Th17 cell development.