Sae Jin Oh

Invariant NKT Cells Producing IL-4 or IL-10, But Not IFN-γ, Inhibit the Th1 Response in Experimental Autoimmune Encephalomyelitis, Whereas None of These Cells Inhibits the Th17 Response

Experimental autoimmune encephalomyelitis (EAE) is mediated by Th1 and Th17 cells. Invariant NKT (iNKT) cells prevent EAE in an IL-4–, IL-10–, and IFN-γ–dependent manner. However, which of the iNKT cell-produced cytokines regulates the Th1 or Th17 response in EAE remains unclear. Wild-type B6 and Jα18−/− mice were immunized with MOG35–55 peptide to address this issue. Clinical scores for EAE, IL-17, and IFN-γ transcript levels, and IL-17– or IFN-γ–expressing CD4+ T cell percentages in the CNS and draining lymph nodes were higher in Jα18−/− than in B6 mice, but all of these parameters in the CNS were reduced by the adoptive transfer of wild-type or IFN-γ–deficient iNKT cells into the Jα18−/− mice before immunization. In contrast, adoptive transfer of IL-4– or IL-10–deficient iNKT cells into Jα18−/− mice decreased IL-17 transcript levels and the percentage of IL-17–expressing CD4+ T cells in the CNS but did not affect clinical scores, IFN-γ transcript levels, or the percentage of IFN-γ–expressing CD4+ T cells in the CNS. Taken together, IL-4– and IL-10–producing iNKT cells inhibit the Th1 cell response, but not the Th17 cell response, although wild-type iNKT cells suppress both the Th1 and Th17 responses in the CNS during EAE. Moreover, IFN-γ–producing iNKT cells have a minimal role in the regulation of the Th1 and Th17 responses in EAE.

CD4+CD25+ regulatory T cells attenuate hypersensitivity pneumonitis by suppressing IFN-γ production by CD4+ and CD8+ T cells

HP results from the repeated inhalation of environmental antigens; however, the roles of CD4+CD25+ Treg cells in HP are unknown. Therefore, we investigated the functions of CD4+CD25+ Treg cells in SR‐induced murine HP. More severe HP was observed in CD4+CD25+ Treg cell‐depleted mice than in control mice in terms of histological alterations, inflammatory cell numbers in BALF, and the serum level of SR‐specific IgG, which were restored by the adoptive transfer of CD4+CD25+ Treg cells. The CD4+CD25+ Treg cell‐depleted mice also showed elevated levels of IFN‐γ, TGF‐β, and reduced IL‐4 production in the lungs. Moreover, IL‐10 production of CD4+CD25+ Treg cells and direct contact between CD4+CD25+ Treg cells and CD4+ or CD8+ T cells in BALF resulted in reduced IFN‐γ production. Taken together, CD4+CD25+ Treg cells play a protective role in SR‐induced HP by suppressing IFN‐γ production by T cells.

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.

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.

Comparison of Invariant NKT Cells with Conventional T Cells by Using Gene Set Enrichment Analysis (GSEA)

Background Invariant Natural killer T (iNKT) cells, a distinct subset of CD1d-restricted T cells with invariant Vαβ TCR, functionally bridge innate and adaptive immunity. While iNKT cells share features with conventional T cells in some functional aspects, they simultaneously produce large amount of Th1 and Th2 cytokines upon T-cell receptor (TCR) ligation. However, gene expression pattern in two types of cells has not been well characterized. Methods we performed comparative microarray analyses of gene expression in murine iNKT cells and conventional CD4+CD25-γδTCR- T cells by using Gene Set Enrichment Analysis (GSEA) method. Results Here, we describe profound differences in gene expression pattern between iNKT cells and conventional CD4+CD25-γδTCR- T cells. Conclusion Our results provide new insights into the functional competence of iNKT cells and a better understanding of their various roles during immune responses.

GM-CSF and IL-4 produced by NKT cells inversely regulate IL-1β production by macrophages

Natural Killer T (NKT) cells are distinct T cell subset that link innate and adaptive immune responses. IL-1β, produced by various immune cells, plays a key role in the regulation of innate immunity in vivo. However, it is unclear whether NKT cells regulate IL-1β production by macrophages. To address this, we co-cultured NKT cells and peritoneal macrophages in the presence of TCR stimulation and inflammasome activators. Among cytokines secreted from NKT cells, GM-CSF enhanced IL-1β production by macrophages via regulating LPS-mediated pro-IL-1β expression and NLRP3-dependent inflammasome activation, whereas IL-4 enhanced M2-differentiation of macrophages and decreased IL-1β production. Together, our findings suggest the NKT cells have double-sided effects on IL-1β-mediated innate immune responses by producing IL-4 and GM-CSF. These findings may be helpful for a comprehensive understanding of NKT cell-mediated regulatory mechanisms of the pro-inflammatory effects of IL-1β in inflammatory diseases in vivo.

CD4+FOXP3+ Regulatory T Cells Exhibit Impaired Ability to Suppress Effector T Cell Proliferation in Patients with Turner Syndrome

Objective We investigated whether the frequency, phenotype, and suppressive function of CD4+FOXP3+ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls. Design and Methods Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4–35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (–) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4+CD25bright were co-cultured with autologous CD4+CD25− target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function. Results Despite a lower frequency of CD4+ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3+ Tregs among CD4+ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3+, and CCR4+CCR6+ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4+CD25− T cells was significantly impaired in the TS (–) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (–) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group. Conclusions The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4+ T cells.