Kensuke Shibata

Resident Vδ1+ γδ T Cells Control Early Infiltration of Neutrophils after Escherichia coli Infection via IL-17 Production

Neutrophils infiltrate the site of infection and play critical roles in host defense, especially against extracellular bacteria. In the present study, we found a rapid and transient production of IL-17 after i.p. infection with Escherichia coli, preceding the influx of neutrophils. Neutralization of IL-17 resulted in a reduced infiltration of neutrophils and an impaired bacterial clearance. Ex vivo intracellular cytokine flow cytometric analysis revealed that γδ T cell population was the major source of IL-17. Mice depleted of γδ T cells by mAb treatment or mice genetically lacking Vδ1 showed diminished IL-17 production and reduced neutrophil infiltration after E. coli infection, indicating an importance of Vδ1+ γδ T cells as the source of IL-17. It was further revealed that γδ T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23, which was induced rapidly after E. coli infection in a TLR4 signaling-dependent manner. Thus, although γδ T cells are generally regarded as a part of early induced immune responses, which bridge innate and adaptive immune responses, our study demonstrated a novel role of γδ T cells as a first line of host defense controlling neutrophil-mediated innate immune responses.

Identification of CD25+ γδ T Cells As Fetal Thymus-Derived Naturally Occurring IL-17 Producers

We previously reported that resident γδ T cells in the peritoneal cavity rapidly produced IL-17 in response to Escherichia coli infection to mobilize neutrophils. We found in this study that the IL-17-producing γδ T cells did not produce IFN-γ or IL-4, similar to Th17 cells. IL-17-producing γδ T cells specifically express CD25 but not CD122, whereas CD122+ γδ T cells produced IFN-γ. IL-17-producing γδ T cells were decreased but still present in IL-2- or CD25-deficient mice, suggesting a role of IL-2 for their maintenance. IFN-γ-producing CD122+ γδ T cells were selectively decreased in IL-15-deficient mice. Surprisingly, IL-17-producing γδ T cells were already detected in the thymus, although CD25 was not expressed on the intrathymic IL-17-producing γδ T cells. The number of thymic IL-17-producing γδ T cells was peaked at perinatal period and decreased thereafter, coincided with the developmental kinetics of Vγ6+Vδ1+ γδ T cells. The number of IL-17-producing γδ T cells was decreased in fetal thymus of Vδ1-deficient mice, whereas Vγ5+ fetal thymocytes in normal mice did not produce IL-17. Thus, it was revealed that the fetal thymus-derived Vγ6+Vδ1+ T cells functionally differentiate to produce IL-17 within thymus and thereafter express CD25 to be maintained in the periphery.

Notch-Hes1 pathway is required for the development of IL-17–producing γδ T cells

Unlike conventional T cells, which are exported from the thymus as naive cells and acquire effector functions upon antigen encounter in the periphery, a subset of γδ T cells differentiates into effectors that produce IL-17 within the fetal thymus. We demonstrate here that intrathymic development of the naturally occurring IL-17-producing γδ T cells is independent of STAT3 and partly dependent on RORγt. Comparative gene-expression analysis identified Hes1, one of the basic helix-loop-helix proteins involved in Notch signaling, as a factor specifically expressed in IL-17-producing γδ T cells. Hes1 is critically involved in the development of IL-17-producing γδ T cells, as evidenced by their severe decrease in the thymi of Hes1-deficient fetal mice. Delta-like 4 (Dll4)-expressing stromal cells support the development of IL-17-producing γδ T cells in vitro. In addition, conditional Hes1 ablation in peripheral γδ T cells decreases their IL-17 production but not their IFN-γ production. These results reveal a unique differentiation pathway of IL-17-producing γδ T cells.

Tyk2-Signaling Plays an Important Role in Host Defense against Escherichia coli through IL-23-Induced IL-17 Production by γδ T Cells

Tyrosine kinase 2 (Tyk2), a member of the JAK-signal transducer family, is involved in intracellular signaling triggered by various cytokines, including IL-23. We have recently reported that resident γδ T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23. In this study, we examined importance of Tyk2-mediated signaling in the IL-17 production by γδ T cells using Tyk2 deficient (−/−) mice. γδ T cells in the peritoneal cavity of Tyk2−/− mice displayed effecter/memory phenotypes and TCR V repertoire similar to those in Tyk2+/+ mice and produced comparable level of IL-17 to those in Tyk2+/+ mice in response to PMA and ionomycin, indicating normal differentiation to IL-17-producing effectors in the absence of Tyk2-signaling. However, γδ T cells in Tyk2−/− mice produced less amount of IL-17 in response to IL-23 in vitro than those in Tyk2+/+ mice. Similarly, γδ T cells in the peritoneal cavity of Tyk2−/− mice showed severely impaired IL-17 production after an i.p. infection with E. coli despite comparable level of IL-23 production to Tyk2+/+ mice. As a consequence, Tyk2−/− mice showed a reduced infiltration of neutrophils and severely impaired bacterial clearance after Escherichia coli infection. These results indicate that Tyk2-signaling is critical for IL-23-induced IL-17 production by γδ T cells, which is involved in the first line of host defense by controlling neutrophil-mediated immune responses.

IL-17 production by γδ T cells is important for the antitumor effect of Mycobacterium bovis bacillus Calmette-Guérin treatment against bladder cancer.

Intravesical inoculation of Mycobacterium bovis bacillus Calmette‐Guérin (BCG) has been used for the treatment of bladder cancer. Recent studies implied the requirement of neutrophil infiltration for the antitumor effect. In this study, we found that IL‐17 was produced in the bladder after BCG treatment, preceding the infiltration of neutrophils. Neutrophils in the bladder after BCG treatment were reduced in IL‐17‐deficient mice, in which BCG‐induced antitumor effect against intravesically inoculated bladder cancer was abolished. Notably, the level of IL‐17 production and the number of neutrophils in BCG‐treated bladder was reduced in γδ T‐cell‐deficient mice but not in CD4‐depleted mice. Survival of bladder cancer‐inoculated γδ T‐cell‐deficient mice was not improved by BCG treatment. These results suggest that IL‐17‐producing γδ T cells play a key role in the BCG‐induced recruitment of neutrophils to the bladder, which is essential for the antitumor activity against bladder cancer.

Protective Role of Naturally Occurring Interleukin-17A-Producing γδ T Cells in the Lung at the Early Stage of Systemic Candidiasis in Mice

ABSTRACT Interleukin-17A (IL-17A)-producing γδ T cells differentiate in the fetal thymus and reside in the peripheral tissues, such as the lungs of naïve adult mice. We show here that naturally occurring γδ T cells play a protective role in the lung at a very early stage after systemic infection with Candida albicans. Selective depletion of neutrophils by in vivo administration of anti-Ly6G monoclonal antibody (MAb) impaired fungal clearance more prominently in the lung than in the kidney 24 h after intravenous infection with C. albicans. Rapid and transient production of IL-23 was detected in the lung at 12 h, preceding IL-17A production and the influx of neutrophils, which reached a peak at 24 h after infection. IL-17A knockout (KO) mice showed reduced infiltration of neutrophils concurrently with impaired fungal clearance in the lung after infection. The major source of IL-17A was the γδ T cell population in the lung, and Cδ KO mice showed little IL-17A production and reduced neutrophil infiltration after infection. Early IL-23 production in a TLR2/MyD88-dependent manner and IL-23-triggered tyrosine kinase 2 (Tyk2) signaling were essential for IL-17A production by γδ T cells. Thus, our study demonstrated a novel role of naturally occurring IL-17A-producing γδ T cells in the first line of host defense against C. albicans infection.

Type I interferon limits influenza virus-induced acute lung injury by regulation of excessive inflammation in mice

Antiviral immune responses play as a double edged sword in resolution of infection and pathogenesis of acute lung injury caused by infection with highly pathogenic influenza A viruses. Here we show that type I interferons (IFNs) are important in protection against acute influenza A virus infection not only via their antiviral activity but also via their anti-inflammatory activity. IFN α receptor (IFNAR) knock-out (KO) mice exhibited increased mortality and morbidity with higher viral load after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) compared with wild-type (WT) mice, though the viruses were finally eliminated in both groups. The levels of proinflammatory cytokines in the lungs were significantly higher, while the level of IL-10 in the lungs was significantly lower in IFNAR KO mice than in WT mice during the course of infection. Restoration of IL-10 during an ongoing virus infection significantly reduced the levels of proinflammatory cytokines and improved mortality of IFNAR KO mice. These results suggest that type I IFNs are responsible not only for direct resolution of viral load but also for suppression of immunopathology caused by influenza A virus through IL-10 production.

A Critical Role of CD30 Ligand/CD30 in Controlling Inflammatory Bowel Diseases in Mice

BACKGROUND & AIMS A CD30-ligand (CD30L) is a 40-kilodalton, type II membrane-associated glycoprotein belonging to the tumor necrosis factor family. Serum levels of soluble CD30 increased in inflammatory bowel diseases (IBD), suggesting that CD30L/CD30 signaling is involved in the pathogenesis of IBD. In this study, we investigated the role of CD30L in oxazolone (OXA)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis in CD30L knockout (KO) mice. METHODS Colitis was induced by OXA or TNBS in CD30LKO mice with BALB/c or C57BL/6 background, respectively, and diverse clinical signs of the disease were evaluated. Cytokine production from lamina propria T cells of the colon was assessed by enzyme-linked immunosorbent assay. Anti-interleukin (IL)-4 monoclonal antibody (mAb) or agonistic anti-CD30 mAb was inoculated in mice with colitis induced by OXA or TNBS. RESULTS CD30LKO mice were susceptible to OXA-induced colitis but resistant to TNBS-induced acute colitis. The levels of T helper cell 2 type cytokines such as IL-4 and IL-13 in the LP T cells were significantly higher, but the levels of interferon gamma were lower in OXA- or TNBS-treated CD30LKO mice than in wild-type mice. In vivo administration of agonistic anti-CD30 mAb ameliorated OXA-induced colitis but aggravated TNBS-induced colitis in CD30LKO mice. CONCLUSIONS These results suggest that CD30L/CD30 signaling is involved in development of both OXA- and TNBS-induced colitis. Modulation of CD30L/CD30 signaling by mAb could be a novel biologic therapy for IBD.

Interleukin-15 Is Critical in the Pathogenesis of Influenza A Virus-Induced Acute Lung Injury

ABSTRACT Highly pathogenic influenza A viruses cause acute severe pneumonia to which the occurrence of “cytokine storm” has been proposed to contribute. Here we show that interleukin-15 (IL-15) knockout (KO) mice exhibited reduced mortality after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) albeit the viral titers of these mice showed no difference from those of control mice. There were significantly fewer antigen-specific CD44+ CD8+ T cells in the lungs of infected IL-15 KO mice, and adoptive transfer of the CD8+ T cells caused reduced survival of IL-15 KO mice following influenza virus infection. Mice deficient in β2-microglobulin by gene targeting and those depleted of CD8+ T cells by in vivo administration of anti-CD8 monoclonal antibody displayed a reduced mortality rate after infection. These results indicate that IL-15-dependent CD8+ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus.

Efficacy of Recombinant Bacille Calmette-Guérin Vaccine Secreting Interleukin-15/Antigen 85B Fusion Protein in Providing Protection against Mycobacterium tuberculosis

Protection against Mycobacterium tuberculosis not only depends on CD4+ T helper type 1 (Th1) cells but, also, on CD8+ T cells. Interleukin (IL)-15 has an important function in the maintenance of memory CD8+ T cells. In the present study, we examined the efficacy of recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) secreting fusion protein antigen (Ag) 85B murine IL-15 (rBCG-Ag85B-IL15) in providing protection against M. tuberculosis infection. The levels of major histocompatibility (MHC) class Ib (H2-M3)-binding TB2- or MHC class Ia (H-2Db)-binding MPT64-specific CD8+ T cells producing interferon (IFN)-gamma were significantly higher after immunization with rBCG-Ag85B-IL15 than after immunization with rBCG secreting Ag85B (rBCG-Ag85B). The levels of purified protein derivative- or Ag85B-specific CD4+ T cells producing IFN-gamma were also higher in mice immunized with rBCG-Ag85B-IL15 than in mice immunized with rBCG-Ag85B. Mice immunized with rBCG-Ag85B-IL15 exhibited CD8+ and CD4+ T cells responses that were stronger than those in mice immunized with rBCG-Ag85B, as well as robust protection in the lung against intratracheal challenge of M. tuberculosis. Thus, rBCG-Ag85B-IL15 vaccination capable of inducing efficient cell-mediated immunity might be used as an effective vaccine for tuberculosis.