Ji Hyung Kim

Natural Killer T (NKT) Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis by Producing Interferon-γ

Pulmonary fibrosis is a progressive illness characterized by interstitial fibrosis. Although the precise mechanism for pulmonary fibrosis is not completely understood, an immune response involving interferon (IFN)-γ appears to play a role. Therefore, we examined the functional roles of natural killer T (NKT) cells, which produce IFN-γ and interleukin-4 on activation, in bleomycin-induced pulmonary fibrosis. In NKT cell-deficient mice, pulmonary fibrosis was worse in terms of histology, hydroxyproline levels, and mortality than in control mice. The transforming growth factor (TGF)-β1 levels were higher in the lung after injecting bleomycin, and blockade of TGF-β1 by neutralizing monoclonal antibody attenuated the pulmonary fibrosis in CD1d−/− mice. In contrast, the production of IFN-γ was reduced in lungs from CD1d−/− mice. Moreover, the adoptive transfer of NKT cells into CD1d−/− mice increased IFN-γ and reduced TGF-β1 production, attenuating pulmonary fibrosis. An in vitro assay demonstrated that IFN-γ was involved in suppressing TGF-β1 production in cells collected from bronchoalveolar lavage. The adoptive transfer of NKT cells from IFN-γ−/− mice did not reverse pulmonary fibrosis or TGF-β1 production in lungs of CD1d−/− mice whereas NKT cells from B6 control mice attenuated fibrosis and reduced TGF-β1 production. In conclusion, IFN-γ-producing NKT cells play a novel anti-fibrotic role in pulmonary fibrosis by regulating TGF-β1 production.

Donor bone marrow type II (non-Valpha14Jalpha18 CD1d-restricted) NKT cells suppress graft-versus-host disease by producing IFN-gamma and IL-4.

NKT cells in donor bone marrow (BM) have been demonstrated to protect against graft-vs-host disease (GVHD) following BM transplantation. Murine NKT cells are divided into two distinct subsets based on the invariant Vα14Jα18 TCR expression. However, details of the subset and mechanisms of the BM NKT cells involved in suppressing GVHD have not been clarified. Irradiated BALB/c or C3H/HeN mice administered B6 or Jα18−/− BM cells show attenuation of GVHD, whereas recipients given CD1d−/− BM cells did not show attenuation. Moreover, coinjection of BM non-Vα14Jα18 CD1d-restricted (type II) NKT cells and CD1d−/− BM cells suppressed GVHD, whereas coinjection of BM Vα14Jα18 TCR (type I) NKT cells did not. These protective effects on GVHD depended upon IFN-γ-producing type II NKT cells, which induced the apoptosis of donor T cells. The splenocytes of mice administered BM cells from B6.IL-4−/− or Jα18−/−IL-4−/− mice produced lower levels of IL-4 and IL-10 than the splenocytes of mice transplanted with BM cells from B6, B6.IFN-γ−/−, Jα18−/−, or Jα18−/−IFN-γ−/− mice. Taken together, our results show that IFN-γ-producing BM type II NKT cells suppress GVHD by inducing the apoptosis of donor T cells, while IL-4-producing BM type II NKT cells protect against GVHD by deviating the immune system toward a Th2-type response.

Direct engagement of TLR4 in invariant NKT cells regulates immune diseases by differential IL-4 and IFN-γ production in mice.

During interaction with APCs, invariant (i) NKT cells are thought to be indirectly activated by TLR4-dependently activated APCs. However, whether TLR4 directly activates iNKT cells is unknown. Therefore, the expression and function of TLR4 in iNKT cells were investigated. Flow cytometric and confocal microscopic analysis revealed TLR4 expression on the surface and in the endosome of iNKT cells. Upon LPS stimulation, iNKT cells enhanced IFN-γ production, but reduced IL-4 production, in the presence of TCR signals, depending on TLR4, MyD88, TRIF, and the endosome. However, enhanced TLR4-mediated IFN-γ production by iNKT cells did not affect IL-12 production or CD1d expression by DCs. Adoptive transfer of WT, but not TLR4-deficient, iNKT cells promoted antibody-induced arthritis in CD1d−/− mice, suggesting that endogenous TLR4 ligands modulate iNKT cell function in arthritis. Furthermore, LPS-pretreated WT, but not TLR4-deficient, iNKT cells suppressed pulmonary fibrosis, but worsened hypersensitivity pneumonitis more than untreated WT iNKT cells, indicating that exogenous TLR4 ligands regulate iNKT cell functions in pulmonary diseases. Taken together, we propose a novel direct activation pathway of iNKT cells in the presence of TCR signals via endogenous or exogenous ligand-mediated engagement of TLR4 in iNKT cells, which regulates immune diseases by altering IFN-γ and IL-4 production.

NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis

Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, whether NOD2 regulates C5a generation during sepsis remains to be determined. To address this issue, cecal ligation & puncture (CLP)-induced sepsis was compared in wild type and Nod2−/− mice. Nod2−/− mice showed lower levels of C5a, IL-10, and IL-1β in serum and peritoneum, but higher survival rate during CLP-induced sepsis compared to wild type mice. Injection of recombinant C5a decreased survival rates of Nod2−/− mice rate during sepsis, whereas it did not alter those in wild type mice. These findings suggest a novel provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis. SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis. Therefore, we propose a novel NOD2-mediated complement cascade regulatory pathway in sepsis, which may be a useful therapeutic target.

CD1d-Restricted IFN-γ–Secreting NKT Cells Promote Immune Complex-Induced Acute Lung Injury by Regulating Macrophage-Inflammatory Protein-1α Production and Activation of Macrophages and Dendritic Cells

Immune complex-induced acute lung injury (IC-ALI) has been implicated in various pulmonary disease states. However, the role of NKT cells in IC-ALI remains unknown. Therefore, we explored NKT cell functions in IC-ALI using chicken egg albumin and anti-chicken egg albumin IgG. The bronchoalveolar lavage fluid of CD1d−/− and Jα18−/− mice contained few Ly6G+CD11b+ granulocytes, whereas levels in B6 mice were greater and were increased further by α-galactosyl ceramide. IFN-γ and MIP-1α production in the lungs was greater in B6 than CD1d−/− mice. Adoptive transfer of wild type (WT) but not IFN-γ–, MIP-1α–, or FcγR-deficient NKT cells into CD1d−/− mice caused recruitment of inflammatory cells to the lungs. Moreover, adoptive transfer of IFN-γR–deficient NKT cells enhanced MIP-1α production and cell recruitment in the lungs of CD1d−/− or CD1d−/−IFN-γ−/− mice, but to a lesser extent than WT NKT cells. This suggests that IFN-γ–producing NKT cells enhance MIP-1α production in both an autocrine and a paracrine manner. IFN-γ–deficient NKT cells induced less IL-1β and TNF-α production by alveolar macrophages and dendritic cells in CD1d−/− mice than did WT NKT cells. Taken together, these data suggest that CD1d-restricted IFN-γ–producing NKT cells promote IC-ALI by producing MIP-1α and enhancing proinflammatory cytokine production by alveolar macrophages and dendritic cells.

FTY720, a sphingosine 1-phosphate receptor modulator, inhibits CD1d-restricted NKT cells by suppressing cytokine production but not migration.

FTY720, a sphingosine 1-phosphate (S1P) receptor modulator, suppresses immune responses by inhibiting T-cell migration into target tissues; however, it does not alter T-cell functions. In this study, we investigated the biological effects of FTY720 on NKT cells. Unlike T cells, FTY720 suppressed the production of IL-4, IFN-γ, IL-10, and IL-13 by NKT cells through the S1P1 receptor (S1P1). Moreover, FTY720 also inhibited the expression of T-bet and GATA-3 of NKT cells in the presence of TCR engagement. However, it did not inhibit NKT cell migration in vitro or in vivo. In a K/BxN serum transfer arthritis model, FTY720 suppressed arthritis in B6, but not in CD1d−/− mice. Moreover, the adoptive transfer of control NKT cells restored arthritis in CD1d−/− mice, whereas FTY720-pretreated NKT cells did not. The number of NKT cells in the joints of B6 mice given FTY720 was similar to that in the joints of untreated B6 mice, whereas the production of IL-4 and IFN-γ was reduced in the FTY720-treated B6 mice. Taken together, these data show that FTY720 suppresses cytokine production in NKT cells through S1P1, but not NKT cell migration. Thus, FTY720 may be useful in the treatment of NKT cell-promoted immune diseases.

IFN-γ-producing NKT cells exacerbate sepsis by enhancing C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils.

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT‐cell‐deficient mice of C57BL/6 background during cecal ligation and puncture‐induced sepsis. The levels of C5a, IFN‐γ, and IL‐10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d−/− mice, while the mortality rate was lower in CD1d−/− mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d−/− mice. As assessed by intracellular staining, NKT cells expressed IFN‐γ, while neutrophils expressed IL‐10. Upon coculture, IL‐10‐deficient NKT cells enhanced IL‐10 production by WT, but not IFN‐γR‐deficient, neutrophils. Meanwhile, CD1d−/− mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL‐10 administration into CD1d−/− mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN‐γ‐deficient, NKT cells into CD1d−/− mice suppressed CD55 expression on neutrophils, but increased IL‐10 and C5a levels. Taken together, IFN‐γ‐producing NKT cells enhance C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.

Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells

Invariant natural killer T cells are a distinct subset of T cells that exert Janus‐like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4‐specific gene ablation of Notch 1 and Notch 2 (N1N2−/−) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N2−/− mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.1−CD44+ to the NK1.1+CD44+ stage, resulting in accumulation of NK1.1−CD44+ invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N2−/− mice produced lower cytokine levels and increased apoptosis versus wild‐type invariant natural killer T cells. Furthermore, Notch 1/Notch 2‐deficient, but not wild type, invariant natural killer T cells failed to promote antibody‐induced arthritis in CD1d−/− mice. Unlike N1N2−/− mice, RBP‐jlox/lox CD4‐Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild‐type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP‐j in the thymus.

Role of non-classical T cells in skin immunity

HIGHLIGHTSThe non‐classical MHC pathways are important in skin immunity.CD1 proteins are involved in skin inflammatory diseases.&ggr;&dgr; T cells are key players in skin immunity and tissue homeostasis.Other non‐classical MHC restricted T cells may contribute to skin immunity.Targeting non‐classical T cells has potential in inflammatory skin disease therapy. ABSTRACT The immune network controls homeostasis and inflammation of the skin. Immune cells use their antigen receptors to respond to a wide range of insults originating from microbes and allergens. T cells, which are key effector cells in the immune system, engage their T cell receptors (TCRs) to recognize self and foreign antigens in the context of classical major histocompatibility complex (MHC) molecules, MHC‐like CD1 proteins, or MHC class I‐related molecules. Recently, increasing evidence has demonstrated that T cells activated by non‐canonical antigens are important in skin diseases. This review focuses on recent studies examining the roles of non‐classical antigen‐presenting molecules and their reactive T cells in the skin immune system. Additionally, we describe the types of ligands that activate these unconventional T cells through the non‐classical MHC pathway. Finally, we highlight recent advances in the understanding of the physiological functions of non‐classical T cells in the skin. Further investigation may result in the development of new therapeutic strategies for treating immune‐related skin diseases.

Lipid-Reactive T Cells in Immunological Disorders of the Lung

Regulation of T cell-mediated immunity in the lungs is critical for prevention of immune-related lung disorders and for host protection from pathogens. While the prevalent view of pulmonary T cell responses is based on peptide recognition by antigen receptors, called T cell receptors (TCR), on the T cell surface in the context of classical major histocompatibility complex (MHC) molecules, novel pathways involving the presentation of lipid antigens by cluster of differentiation 1 (CD1) molecules to lipid-reactive T cells are emerging as key players in pulmonary immune system. Whereas, genetic conservation of group II CD1 (CD1d) in mouse and human genomes facilitated numerous in vivo studies of CD1d-restricted invariant natural killer T (iNKT) cells in lung diseases, the recent development of human CD1-transgenic mice has made it possible to examine the physiological roles of group I CD1 (CD1a-c) molecules in lung immunity. Here, we discuss current understanding of the biology of CD1-reactive T cells with a specific focus on their roles in several pulmonary disorders.