HBsAg Inhibits IFN-α Production in Plasmacytoid Dendritic Cells through TNF-α and IL-10 Induction in Monocytes

Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-α-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-α production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-α production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-α (IFN-α) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-α and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-α, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-α production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-α by pDCs through the induction of monocytes that secreted TNF-α and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections.     

Activation of Toll-Like Receptor 2 Prevents Suppression of T-Cell Interferon γ Production by Modulating p38/Extracellular Signal-Regulated Kinase Pathways following Alcohol and Burn Injury

Recent studies indicate that toll-like receptors (TLRs) are expressed on T cells and that these receptors directly or indirectly activate the adaptive immune system. We have shown previously that acute alcohol/ethanol (EtOH) intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell interleukin-2 (IL-2) and interferon γ (IFN-γ) production. We examined whether direct stimulation of T cells with TLR2, 4, 5 and 7 agonists modulates CD3-mediated T-cell IL-2/IFN-γ release following EtOH and burn injury. Male mice were gavaged with EtOH (2.9 gm/kg) 4 h prior to receiving an ~12.5% total body surface area sham or full-thickness burn injury. Animals were killed on d 1 after injury and T cells were purified from MLN and spleens. T cells were cultured with plate-bound anti-CD3 in the presence or absence of various TLR ligands. Although TLR2, 4 and 5 agonists potentiate anti-CD3–dependent IFN-γ by T cells, the TLR2 agonist alone induced IFN-γ production independent of CD3 stimulation. Furthermore, T cells were treated with inhibitors of myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), p38 and/or extracellular signal-regulated kinase (ERK) to determine the mechanism by which TLR2 mediates IL-2/IFN-γ production. IL-2 was not influenced by TLR agonists. MyD88 and TIRAP inhibitory peptides dose-dependently diminished the ability of T cells to release IFN-γ. p38 and ERK inhibitors also abolished TLR2-mediated T-cell IFN-γ. Together, our findings suggest that TLR2 directly modulates T-cell IFN-γ production following EtOH and burn injury, independent of antigen-presenting cells. Furthermore, we demonstrated that MyD88/TIRAP-dependent p38/ERK activation is critical to TLR2-mediated T-cell IFN-γ release following EtOH and burn injury.     

DNase Sda1 Allows Invasive M1T1 Group A Streptococcus to Prevent TLR9-Dependent Recognition

Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.     

Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium tuberculosis–Induced Apoptosis in Mice

Background

Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive.

Methodology/Principal Findings

We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-α, IL-1β, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration.

Conclusions/Significance

These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.     

Intranasal Treatment with Poly(I·C) Protects Aged Mice from Lethal Respiratory Virus Infections

In the 2002-2003 severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, no patients under 24 years of age died, while mortality was greater than 50% in those over 65 years. Greater than 90% of all deaths from influenza A virus (IAV) occur in the elderly (>65 years of age). To address this age-related susceptibility to SARS-CoV and IAV, we infected C57BL/6 (B6) mice with mouse-adapted SARS-CoV (MA15) or IAV (PR8), both of which cause severe disease in aged mice. Intranasal pretreatment of aged mice with poly(I·C) (a TLR3 agonist) and, to a lesser extent, CpG, R848, or lipopolysaccharide (TLR9, TLR7/8, or TLR4 agonists), provided a high level of protection [90% to 100% survival rate after poly(I·C) treatment] against lethal MA15 or IAV challenge and reduced pathological changes and virus loads in the lungs at early times after infection. Poly(I·C) pretreatment upregulated beta interferon (IFN-β), IFN-γ, IL-1β, and tumor necrosis factor (TNF) gene expression in the lungs. Intranasal pretreatment with IFN-β or IFN-γ but not IL-1β or TNF also protected aged mice, consistent with the notion that poly(I·C) pretreatment functioned, at least in part, by inducing IFN-β and IFN-γ. We also identified a potential cellular target for poly(I·C) by showing that treatment inhibited virus replication in primary human airway epithelial cells. These results suggest that intranasal poly(I·C) should be evaluated as a prophylactic agent in aged individuals at high risk for contracting SARS-CoV or IAV infections.     

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4^lps-d), mice deficient in TLR9 (TLR9^−/−) and TLR4/9 double mutant mice (TLR4^lps-d/TLR9^−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4^lps-d/TLR9^−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4^lps-d/TLR9^−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.     

Key Role of Splenic Myeloid DCs in the IFN-αβ Response to Adenoviruses In Vivo

The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR^−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.     

IFN-β Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

Purpose

It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-β on its expression.

Methods

EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161–180 (IRBP_161–180). IFN-β was administered subcutaneously to IRBP_161–180 immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-β or PBS were stimulated with anti-CD3/CD28 or IRBP_161–180 for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-β for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-β for 3 days. IFN-β-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA.

Results

IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-β in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-β suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-β also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-β-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells.

Conclusion

IL-9 expression is increased during EAU which could be suppressed by IFN-β.     

Bruton's Tyrosine Kinase and Protein Kinase C μ Are Required for TLR7/9-Induced IKKα and IRF-1 Activation and Interferon-β Production in Conventional Dendritic Cells

Stimulation of TLR7/9 by their respective ligands leads to the activation of IκB kinase α (IKKα) and Interferon Regulatory Factor 1 (IRF-1) and results in interferon (IFN)-β production in conventional dendritic cells (cDC). However, which other signaling molecules are involved in IKKα and IRF-1 activation during TLR7/9 signaling pathway are not known. We and others have shown that Bruton''s Tyrosine Kinase (BTK) played a part in TLR9-mediated cytokine production in B cells and macrophages. However, it is unclear if BTK participates in TLR7/9-induced IFN-β production in cDC. In this study, we show that BTK is required for IFN-β synthesis in cDC upon TLR7/9 stimulation and that stimulated BTK-deficient cDC are defective in the induction of IKKα/β phosphorylation and IRF-1 activation. In addition, we demonstrate that Protein Kinase C µ (PKCµ) is also required for TLR7/9-induced IRF-1 activation and IFN-β upregulation in cDC and acts downstream of BTK. Taken together, we have uncovered two new molecules, BTK and PKCµ, that are involved in TLR7/9-triggered IFN-β production in cDC.     

Blocking TLR7- and TLR9-mediated IFN-α Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection

Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.     

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for β-Cell Dysfunction Induced by Inflammatory Cytokines

Pathology driving β-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with β-cell dysfunction. Acute in vitro exposure of islets and β-cells to an inflammatory cytokine cocktail (IL-1β/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse β-cell lines and primary mouse islets. Cytokine cooperation is required for β-cell apoptosis with the most potent combinations including IL-1β. Single cytokine exposure did not induce β-cell apoptosis. Expression of endogenous interleukin-12 in β-cells correlated with inflammatory cytokine combinations that induced β-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to β-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced β-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/β-cell viability in established diabetes.     

Activation of the NLRP3 Inflammasome Complex is Not Required for Stress-Induced Death of Pancreatic Islets

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H₂O_2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.     

TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns

Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway could function as an adjuvant to improve maternal-neonatal innate immunity.     

Favorable Alteration of Tumor Microenvironment by Immunomodulatory Cytokines for Efficient T-Cell Therapy in Solid Tumors

Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16.OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-α2, IFN-γ, TNF-α, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-α2, IFN-γ and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-α2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-α2, IFN-γ and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience.     

The Host-Protective Effect of Arabinosylated Lipoarabinomannan against Leishmania donovani Infection Is Associated with Restoration of IFN-γ Responsiveness

Visceral leishmaniasis (VL), which is endemic as a major infectious disease in the tropical and subtropical countries, is caused by a protozoan parasite Leishmania donovani. At present, restricted treatment options and lack of vaccines intensify the problem of controlling VL. Therefore, finding a novel immunoprophylactic or therapeutic principle is a pressing need. Here, we report that arabinosylated lipoarabinomannan (Ara-LAM), a TLR2-ligand isolated from Mycobacterium smegmatis, exhibits a strong immunomodulatory property that conferred protection against L. donovani infection. Although, Ara-LAM modulates TLR2 and MAPK signaling, it is not known whether Ara-LAM involves IFN-γ signaling for effective parasite clearance. Because, it is reported that IFN-γ signaling, a principle mediator of NO generation and macrophage and Tcell activation, is hampered during leishmanial pathogenesis. Ara-LAM increases IFN-γ receptor expression and potentiates IFN-γ receptor signaling through JAK-STAT pathway. Moreover, Ara-LAM reciprocally modulates IRF4 and IRF8 expression and reinstates anti-leishmanial Th1 response that eventuates in significantly reduced parasite load in spleen and liver of L. donovani-infected BALB/c mice. IFN-γRα silencing resulted in the suppression of these host-protective mechanisms affected by Ara-LAM. Thus, Ara-LAM-mediated restoration of IFN-γ responsiveness is a novel immuno-modulatory principle for protection against L. donovani susceptible host.     

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.     

Type I Interferons Function as Autocrine and Paracrine Factors to Induce Autotaxin in Response to TLR Activation

Lysophosphatidic acid (LPA) is an important phospholipid mediator in inflammation and immunity. However, the mechanism of LPA regulation during inflammatory response is largely unknown. Autotaxin (ATX) is the key enzyme to produce extracellular LPA from lysophosphatidylcholine (LPC). In this study, we found that ATX was induced in monocytic THP-1 cells by TLR4 ligand lipopolysaccharide (LPS), TLR9 ligand CpG oligonucleotide, and TLR3 ligand poly(I:C), respectively. The ATX induction by TLR ligand was abolished by the neutralizing antibody against IFN-β or the knockdown of IFNAR1, indicating that type I IFN autocrine loop is responsible for the ATX induction upon TLR activation. Both IFN-β and IFN-α were able to induce ATX expression via the JAK-STAT and PI3K-AKT pathways but with different time-dependent manners. The ATX induction by IFN-β was dramatically enhanced by IFN-γ, which had no significant effect on ATX expression alone, suggesting a synergy effect between type I and type II IFNs in ATX induction. Extracellular LPA levels were significantly increased when THP-1 cells were treated with IFN-α/β or TLR ligands. In addition, the type I IFN-mediated ATX induction was identified in human monocyte-derived dendritic cells (moDCs) stimulated with LPS or poly(I:C), and IFN-α/β could induce ATX expression in human peripheral blood mononuclear cells (PBMCs) and monocytes isolated form blood samples. These results suggest that, in response to TLR activation, ATX is induced through a type I INF autocrine-paracrine loop to enhance LPA generation.