STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4(+) T cells and stimulating the proliferation of memory CD4(+) T cells. We investigated the pathogenic role of IL-23 in CD4(+) T cells in mice lacking the IL-1R antagonist (IL-1Ra(-/-)), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra(-/-) mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1beta further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4(+) T cells of IL-1Ra(-/-) mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4(+) T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-kappaB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra(-/-) model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.     

IL-23 and IL-17 in tuberculosis

Tuberculosis is a chronic disease requiring the constant expression of cellular immunity to limit bacterial growth. The constant expression of immunity also results in chronic inflammation, which requires regulation. While IFN-gamma-producing CD4+ T helper cells (Th1) are required for control of bacterial growth they also initiate and maintain a mononuclear inflammatory response. Other T cell subsets are induced by Mycobacterium tuberculosis (Mtb) infection including those able to produce IL-17 (Th17). IL-17 is a potent inflammatory cytokine capable of inducing chemokine expression and recruitment of cells to parenchymal tissue. Both the IL-17 and the Th17 response to Mtb are largely dependent upon IL-23. Although both Th17 and Th1 cells are induced following primary infection with Mtb, the protective response is significantly altered in the absence of Th1 cells but not in the absence of Th17. In contrast, in vaccinated animals the absence of memory Th17 cells results in loss of both the accelerated memory Th1 response and protection. Th1 and Th17 responses cross-regulate each other during mycobacterial infection and this may be important for immunopathologic consequences not only in tuberculosis but also other mycobacterial infections.     

Development of experimental cerebral malaria is independent of IL-23 and IL-17

Cerebral malaria (CM) is the most severe complication of Plasmodium infection. Although inappropriate immune responses to Plasmodium falciparum are reported as the major causes of CM, the precise mechanisms for development remain unclear. IL-23 and IL-17 have critical roles in the onset of autoimmunity and inflammatory diseases triggered by microbial infections. Thus, we investigated the influence of IL-23 and IL-17 on experimental CM (ECM) using Plasmodium berghei ANKA infection of C57BL/6 mice. Both IL-23 deficient mice and wild-type (WT) mice developed ECM. IL-17 deficient mice also developed ECM, while IL-17 producing cells other than CD4⁺ T cells (Th17) were increased in WT mice that developed ECM. In conclusion, this study showed that IL-23 and IL-17 are not involved in ECM development.     

IL-6 and IL-10 induction from dendritic cells in response to Mycobacterium tuberculosis is predominantly dependent on TLR2-mediated recognition

The initial TLR-mediated interaction between Mycobacterium tuberculosis and dendritic cells is critical, since the cytokine production that ensues can greatly influence the class of adaptive immunity that is generated to the pathogen. In this study, we therefore determined the dependency on TLR2 and TLR4 for M. tuberculosis-induced cytokine production by murine dendritic cells. A key new finding of this study is that production of IL-6 and IL-10 from dendritic cells in response to M. tuberculosis is principally dependent on TLR2. The study also indicates that M. tuberculosis can induce IL-12 production in the absence of either TLR2 or TLR4, suggesting redundancy or possibly involvement of other receptors in IL-12 production. In addition, the data also reveal that lack of TLR2 or TLR4 does not impact on dendritic cell maturation or on their ability to influence the polarity of differentiating naive T cells. Collectively, data presented here provide a mechanistic insight for the contribution of TLR2 and TLR4 to tuberculosis disease progression and offer strategies for regulating IL-6 and IL-10 production in dendritic cell-based vaccine strategies.     

IL-17A is involved in diabetic inflammatory pathogenesis by its receptor IL-17RA

Interleukin (IL)-17A, a proinflammatory cytokine produced by T-helper (Th)17 cells, has been associated with autoimmune diseases. Type 1 diabetes (T1D) is caused either due to mutation of insulin gene or developed as an autoimmune disease. Studies have shown that IL-17A expression is upregulated in the pancreas in T1D patients and animal models. However, role or importance of IL-17A in T1D pathogenesis needs elucidation. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells through activating IL-17 receptor A (IL-17RA) is lacking. Ins2^Akita (Akita) mouse, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis, was crossed with IL-17A-knockout mouse and male IL-17A-deficient Akita mice were used. Streptozotocin, a pancreatic β-cell-specific cytotoxin, was employed to induce a diabetic model in MIN6 cells, a mouse insulinoma cell line. IL-17A expression in the pancreas was upregulated in both Akita and streptozotocin-induced diabetic mice. IL-17A-knockout Akita mice manifested reduced blood glucose concentration and raised serum insulin level. IL-17A deficiency also decreased production of the proinflammatory cytokines tumor necrosis factor (TNF)-α, IL-1β, and interferon (IFN)-γ in Akita mice. IL-17RA expression in MIN6 cells was upregulated by IL-17A. IL-17A enhanced expression of TNF-α, IL-1β, IFN-γ, and inducible nitric oxide synthase (iNOS) and further increased streptozotocin-induced expression of the inflammatory factors in MIN6 cells. IL-17A exacerbated streptozotocin-induced MIN6 cell apoptosis and insulin secretion impairment. Blocking IL-17RA with anti-IL-17RA-neutralizing antibody reduced all these deleterious effects of IL-17A on MIN6 cells. Collectively, IL-17A deficiency alleviated hyperglycemia, hypoinsulinemia, and inflammatory response in Akita mice that are characteristic for T1D. IL-17A exerted an alone and synergistic destruction with streptozotocin to pancreatic β cells through IL-17RA pathway. Thus, the data suggest that targeting IL-17A and/or IL-17RA is likely to preserve remaining β-cell function and treat T1D.Impact statementThe participation of interleukin (IL)-17A in diabetic pathogenesis is suggested in animal models of autoimmune diabetes and in patients with type 1 diabetes (T1D), but with some contradictory results. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells is lacking. We showed that IL-17A deficiency alleviated diabetic signs including hyperglycemia, hypoinsulinemia, and inflammatory response in Ins2^Akita (Akita) mice, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis. IL-17A enhanced inflammatory reaction, oxidative stress, and cell apoptosis but attenuated insulin level in mouse insulin-producing MIN6 cells. IL-17A had also a synergistic destruction to MIN6 cells with streptozotocin (STZ), a pancreatic β-cell-specific cytotoxin. Blocking IL-17 receptor A (IL-17RA) reduced all these deleterious effects of IL-17A on MIN6 cells. The results demonstrate the role and the importance of IL-17A in T1D pathogenesis and suggest a potential therapeutic strategy for T1D targeting IL-17A and/or IL-17RA.     

Evidence for anti-inflammatory effects of C5a on the innate IL-17A/IL-23 axis

There is growing evidence that the complement activation product C5a positively or negatively regulates inflammatory functions. The studies presented here report that C5a exerts anti-inflammatory effects by altering production of the cytokines IL-17A and IL-23 during endotoxic shock in young adult male C57BL/6J mice and has similar effects on macrophages from the same mice. IL-17A and IL-23 both appeared in plasma during endotoxemia, and their neutralization improved survival. The relevant sources of IL-17A during endotoxemia were not CD4(+) cells, γδ T cells, or NK cells but CD11b(+)F4/80(+) macrophages. The addition in vitro of C5a to lipopolysaccharide-activated peritoneal macrophages dose dependently antagonized the production of IL-17A (IC(50), 50-100 nM C5a) and IL-23 (IC(50), 10 nM C5a). This suppression required the receptor C5aR, but was independent of the second C5a receptor, C5L2. Genetic absence of C5aR was associated with much higher levels of IL-17A and IL-23 during endotoxic shock. Mechanistically, C5a mediated its effects on the IL-17A/IL-23 axis in a 2-step process. C5a caused activation of the PI3K-Akt and MEK1/2-ERK1/2 pathways, resulting in induction of IL-10, which powerfully inhibited production of IL-17A and IL-23. These data identify previously unknown mechanisms by which the anaphylatoxin C5a limits acute inflammation and antagonizes the IL-17A/IL-23 axis.     

IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis

Background

Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.

Methods

The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.

Results

We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt⁺ γδ T cells and to a lesser extent by CD4αβ⁺ T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.

Conclusions

Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.     

High-fat-diet-associated intestinal microbiota exacerbates psoriasis-like inflammation by enhancing systemic γδ T cell IL-17 production

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IL-23/IL-17轴在脓毒症中的表达及意义

目的:探讨IL-23/IL-17轴在脓毒症患者中的表达及意义.方法:符合诊断标准的脓毒症患者40例,以28天预后为终点,将患者分为存活组(n=21)和病死组(n=19),分析各组病人的急性生理和慢性健康评分(APACHE)Ⅱ和序贯器官衰竭估计(SOFA)评分,同时在入ICU第1天采取外周静脉血做IL-23和IL-17检测,并对病死率和IL-23、IL-17、APACHEⅡ、SOFA做相关性分析.结果:与存活组比较,病死组患者拥有较高的APACHEⅡ和SOFA评分(P<0.01),且外周血的IL-23和IL-17蛋白含量均明显升高(P<0.05).APACHEⅡ和SOFA评分、IL-17和IL-23含量与28天预后有明显的相关性(P<0.05).结论:脓毒症Th17细胞分泌的IL-23/IL-17增加,加重患者病情,在脓毒症发病机制中可能扮演重要角色.     

IL-23 is required for the development of severe egg-induced immunopathology in schistosomiasis and for lesional expression of IL-17

In infection with the trematode helminth Schistosoma mansoni, the severity of CD4 T cell-mediated hepatic granulomatous and fibrosing inflammation against parasite eggs varies considerably in humans and among mouse strains. In mice, either the natural high pathology, or high pathology induced by concomitant immunization with schistosome egg Ags (SEA) in CFA (SEA/CFA), results from a failure to contain a net proinflammatory cytokine environment. We previously demonstrated that the induction of severe immunopathology was dependent on the IL-12/IL-23 common p40 subunit, and correlated with an increase in IL-17, thus implying IL-23 in the pathogenesis. We now show that mice lacking the IL-23-specific subunit p19 are impaired in developing severe immunopathology following immunization with SEA/CFA, which is associated with a marked drop of IL-17 in the granulomas, but not in the draining mesenteric lymph nodes, and with a markedly suppressed SEA-specific IFN-gamma response regulated by a striking increase in IL-10. The granulomas are characterized by a significant reduction in Gr-1(+) cell recruitment and by alternative macrophage activation. Taken together, these results demonstrate that IL-23 per se is not necessary for the generation of IL-17-producing T cells, but is essential for the development of severe schistosome egg-induced immunopathology, and its absence cannot be overcome with other possible compensatory mechanisms.     

IL-17-mediated oxidative stress is an important stimulator of AT1-AA and hypertension during pregnancy

Preeclampsia is associated with autoimmune cells T(H)17, secreting interleukin-17, autoantibodies activating the angiotensin II type I receptor (AT1-AA), and placental oxidative stress (ROS). The objective of our study was to determine whether chronic IL-17 increases blood pressure by stimulating ROS and AT1-AAs during pregnancy. To answer this question four groups of rats were examined: normal pregnant (NP, n = 20), NP+IL-17 (n = 12), NP+tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (n = 7) (a superoxide dismutase mimetic that scavenges ROS), and NP+IL-17+tempol (n = 11). IL-17 (150 pg/day) was infused into NP rats while tempol was administered via the drinking water ad libitum. On day 19 blood pressure (MAP) was recorded, and plasma, urine, and tissue were collected for isolation of ROS detected by chemilluminescent technique. Urinary isoprostane was measured by ELISA. AT1-AAs were determined via cardiomyocyte assay and expressed as beats per minute. MAP increased from 98 ± 3 mmHg in NP to 123 ± 3 mmHg in IL-17-infused NP rats. Urinary isoprostane increased from 1,029 ± 1 in NP to 3,526 ± 2 pg·mg(-1)·day(-1) in IL-17-infused rats (P < 0.05). Placental ROS was 436 ± 4 RLU·ml(-1)·min(-1) (n = 4) in NP and 702 ± 5 (n = 5) RLU·ml(-1)·min(-1) in IL-17-treated rats. Importantly, AT1-AA increased from 0.41 ± 0.05 beats/min in NP rats (n = 8) to 18.4 ± 1 beats/min in IL-17 rats (n = 12). Administration of tempol attenuated the hypertension (101 ± 3 mmHg) ROS (459 ± 5 RLU·ml(-1)·min(-1)) and blunted AT1-AAs (7.3 ± 0.6 beats/min) in NP+IL-17+tempol-treated rats. Additionally, AT1 receptor blockade inhibited IL-17-induced hypertension and placental oxidative stress. MAP was 105 ± 5 mmHg and ROS was 418 ± 5 RLU·ml(-1)·min(-1) in NP+IL 17-treated with losartan. These data indicate that IL-17 causes placental oxidative stress, which serves as stimulus modulating AT1-AAs that may play an important role in mediating IL-17-induced hypertension during pregnancy.     

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.     

Robust antigen specific th17 T cell response to group A Streptococcus is dependent on IL-6 and intranasal route of infection

Group A streptococcus (GAS, Streptococcus pyogenes) is the cause of a variety of clinical conditions, ranging from pharyngitis to autoimmune disease. Peptide-major histocompatibility complex class II (pMHCII) tetramers have recently emerged as a highly sensitive means to quantify pMHCII-specific CD4+ helper T cells and evaluate their contribution to both protective immunity and autoimmune complications induced by specific bacterial pathogens. In lieu of identifying an immunodominant peptide expressed by GAS, a surrogate peptide (2W) was fused to the highly expressed M1 protein on the surface of GAS to allow in-depth analysis of the CD4+ helper T cell response in C57BL/6 mice that express the I-A(b) MHCII molecule. Following intranasal inoculation with GAS-2W, antigen-experienced 2W:I-A(b)-specific CD4+ T cells were identified in the nasal-associated lymphoid tissue (NALT) that produced IL-17A or IL-17A and IFN-γ if infection was recurrent. The dominant Th17 response was also dependent on the intranasal route of inoculation; intravenous or subcutaneous inoculations produced primarily IFN-γ+ 2W:I-A(b+) CD4+ T cells. The acquisition of IL-17A production by 2W:I-A(b)-specific T cells and the capacity of mice to survive infection depended on the innate cytokine IL-6. IL-6-deficient mice that survived infection became long-term carriers despite the presence of abundant IFN-γ-producing 2W:I-A(b)-specific CD4+ T cells. Our results suggest that an imbalance between IL-17- and IFN-γ-producing CD4+ T cells could contribute to GAS carriage in humans.     

Molecular mechanisms of cytokine and chemokine release from eosinophils activated by IL-17A, IL-17F, and IL-23: implication for Th17 lymphocytes-mediated allergic inflammation

IL-17A and IL-17F are members of the IL-17 family that play crucial roles in allergic inflammation. Recent studies reported that IL-17A and IL-17F production from a distinct Th lymphocyte subset, Th17, was specifically induced by IL-23, which was produced by dendritic cells and macrophages in response to microbial stimuli. The IL-23-IL-17 axis might therefore provide a link between infections and allergic diseases. In the present study, we investigated the effects of IL-17A, IL-17F, and IL-23, alone or in combination, on cytokine and chemokine release from eosinophils and the underlying intracellular mechanisms. Human eosinophils were found to constitutively express receptors for IL-17A, IL-17F, and IL-23 at the protein level. IL-17A, IL-17F, and IL-23 could induce the release of chemokines GRO-alpha/CXCL1, IL-8/CXCL8, and MIP-1beta/CCL4 from eosinophils, while IL-17F and IL-23 could also increase the production of proinflammatory cytokines IL-1beta and IL-6. Synergistic effects were observed in the combined treatment of IL-17F and IL-23 on the release of proinflammatory cytokines, and the effects were dose-dependently enhanced by IL-23, but not IL-17F. Further investigations showed that IL-17A, IL-17F, and IL-23 differentially activated the ERK, p38 MAPK, and NF-kappaB pathways. Moreover, inhibition of these pathways using selective inhibitors could significantly abolish the chemokine release induced by IL-17A, IL-17F, and IL-23 and the synergistic increases on IL-1beta and IL-6 production mediated by combined treatment of IL-17F and IL-23. Taken together, our findings provide insight for the Th17 lymphocyte-mediated activation of eosinophils via differential intracellular signaling cascades in allergic inflammation.     

Accessory protein-like is essential for IL-18-mediated signaling

IL-18 is an essential cytokine for both innate and adaptive immunity. Signaling by IL-18 requires IL-18Ralpha, which binds specifically to the ligand and contains sequence homology to IL-1R and TLRs. It is well established that IL-1R signaling requires an accessory cell surface protein, AcP. Other accessory proteins also exist with roles in regulating TLR signaling, but some have inhibitory functions. An AcP-like molecule (AcPL) has been identified with the ability to cooperate with IL-18Ralpha in vitro; however, the physiological function of AcPL remains unknown. In this study, we demonstrate that IL-18 signals are abolished in AcPL-deficient mice and cells. Splenocytes from mutant mice fail to respond to IL-18-induced proliferation and IFN-gamma production. In particular, Th1 cells lacking AcPL fail to produce IFN-gamma in response to IL-18. AcPL-deficient neutrophils also fail to respond to IL-18-induced activation and cytokine production. Furthermore, AcPL is required for NK-mediated cytotoxicity induced by in vivo IL-18 stimulation. However, AcPL is dispensable for the activation or inhibition of IL-1R and the various TLR signals that we have examined. These results suggest that AcPL is a critical and specific cell surface receptor that is required for IL-18 signaling.     

Allergen-induced airway hyperresponsiveness mediated by cyclooxygenase inhibition is not dependent on 5-lipoxygenase or IL-5, but is IL-13 dependent

Cyclooxygenase (COX) inhibition during allergic sensitization and allergen airway challenge results in augmented allergic inflammation. We hypothesized that this increase in allergic inflammation was dependent on increased generation of leukotrienes that results from COX inhibition, as leukotrienes are important proinflammatory mediators of allergic disease. To test this hypothesis, we allergically sensitized and challenged mice deficient in 5-lipoxygenase (5-LO). We found that 5-LO knockout mice that were treated with a COX inhibitor during allergic sensitization and challenge had significantly increased airway hyperresponsiveness (AHR) (p < 0.01) and airway eosinophilia (p < 0.01) compared with 5-LO knockout mice that were treated with vehicle. The proinflammatory cytokines have also been hypothesized to be critical regulators of airway inflammation and AHR. We found that the increase in airway eosinophilia seen with COX inhibition is dependent on IL-5, whereas the increase in AHR is not dependent on this cytokine. In contrast, the COX inhibition-mediated increase in AHR is dependent on IL-13, but airway eosinophilia is not. These results elucidate the pathways by which COX inhibition exerts a critical effect of the pulmonary allergen-induced inflammatory response and confirm that COX products are important regulators of allergic inflammation.     

Basement membrane formation during wound healing is dependent on epidermal transplants

The purpose of the study was to compare directly the effect of healing and the formation of the basement membrane during wound healing from two autologous primary keratinocyte cultures in the liquid environment in full-thickness wounds in pigs. Wounds were either transplanted with cultured epidermal autografts (n = 26) or autologous keratinocyte suspensions (n = 24) or treated with saline alone (n = 40) and covered with a chamber. All wounds transplanted with cultured epidermal autografts and keratinocyte cell suspensions had positive "take" after transplantation. Healing times were significantly shorter for wounds treated with either cultured epidermal autografts or keratinocyte suspensions (p = 0.0001) compared with saline-treated wounds but were not different from each other (p = 0.1835). There were no differences in cytokeratin and laminin expression; however, staining with monoclonal antibody against collagen type VII showed a lower signal for cultured epidermal autografts only on days 8 and 16 compared with keratinocyte suspensions. Electron microscope evaluation showed a higher incidence of anchoring fibrils and a more mature dermal-epidermal junction in wounds treated with keratinocyte cell suspensions at day 8. These findings may be due to the single, noncontact-inhibited cells and the early formation of an in vivo neodermis to the wet wound environment. These data suggest that wounds transplanted with autologous keratinocyte suspensions in a wet environment may be an alternative method in the treatment of wounds.