Activation and IL-10 production of specific CD4+ T cells are regulated by IL-27 during chronic infection with Plasmodium chabaudi

IL-27, a regulatory cytokine, plays critical roles in the prevention of immunopathology during Plasmodium infection. We examined these roles in the immune responses against Plasmodium chabaudi infection using the Il-27ra^−/− mice. While IL-27 was expressed at high levels during the early phase of the infection, enhanced CD4⁺ T cell function and reduction in parasitemia were observed mainly during the chronic phase in the mutant mice. In mice infected with P. chabaudi and cured with drug, CD4⁺ T cells in the Il-27ra^−/− mice exhibited enhanced CD4⁺ T-cell responses, indicating the inhibitory role of IL-27 on the protective immune responses. To determine the role of IL-27 in detail, we performed CD4⁺ T-cell transfer experiments. The Il-27ra^−/− and Il27p28^−/− mice were first infected with P. chabaudi and then cured using drug treatment. Plasmodium-antigen primed CD4⁺ T cells were prepared from these mice and transferred into the recipient mice, followed by infection with the heterologous parasite P. berghei ANKA. Il-27ra^−/− CD4⁺ T cells in the infected recipient mice did not produce IL-10, indicating that IL-10 production by primed CD4⁺ T cells is IL-27 dependent. Il27p28^−/− CD4⁺ T cells that were primed in the absence of IL-27 exhibited enhanced recall responses during the challenge infection with P. berghei ANKA, implying that IL-27 receptor signaling during the primary infection affects recall responses in the long-term via the regulation of the memory CD4⁺ T cell generation. These features highlighted direct and time-transcending roles of IL-27 in the regulation of immune responses against chronic infection with Plasmodium parasites.     

B Cells Enhance Antigen-Specific CD4 T Cell Priming and Prevent Bacteria Dissemination following Chlamydia muridarum Genital Tract Infection

B cells can contribute to acquired immunity against intracellular bacteria, but do not usually participate in primary clearance. Here, we examined the endogenous CD4 T cell response to genital infection with Chlamydia muridarum using MHC class-II tetramers. Chlamydia-specific CD4 T cells expanded rapidly and persisted as a stable memory pool for several months after infection. While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract. Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites. However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection. Together, these data reveal heterogeneity in pathogen-specific CD4 T cell responses within the genital tract and an unexpected requirement for B cells in regulating local T cell activation and bacterial dissemination during genital infection.     

Anti-Chlamydial Th17 Responses Are Controlled by the Inducible Costimulator Partially through Phosphoinositide 3-Kinase Signaling

We previously showed that mice deficient in the Inducible Costimulator ligand (ICOSL-KO) develop more severe disease and lung pathology with delayed bacterial clearance upon respiratory infection of Chlamydia muridarum. Importantly, the exacerbation of disease in ICOSL-KO mice was seen despite heightened IFN-γ/Th1 responses, the major defense mechanisms against Chlamydia. To gain insight into the mechanism of ICOS function in this model, we presently analyzed anti-Chlamydia immune responses in mice lacking the entire ICOS (ICOS-KO) versus knock-in mice expressing a mutant ICOS (ICOS-Y181F) that has selectively lost the ability to activate phosphoinositide 3-kinase (PI3K). Like ICOSL-KO mice, ICOS-KO mice showed worse disease with elevated IFN-γ/Th1 responses compared to wild-type (WT) mice. ICOS-Y181F mice developed much milder disease compared to ICOS-KO mice, yet they were still not fully protected to the WT level. This partial protection in ICOS-Y181F mice could not be explained by the magnitude of IFN-γ/Th1 responses since these mice developed a similar level of IFN-γ response compared to WT mice. It was rather IL-17/Th17 responses that reflected disease severity: IL-17/Th17 response was partially impaired in ICOS-Y181F mice compared to WT, but was substantially stronger than that of ICOS-KO mice. Consistently, we found that both polarization and expansion of Th17 cells were partially impaired in ICOS-Y181F CD4 T cells, and was further reduced in ICOS-KO CD4 T cells in vitro. Our results indicate that once the IFN-γ/Th1 response is above a threshold level, the IL-17/Th17 response becomes a limiting factor in controlling Chlamydia lung infection, and that ICOS plays an important role in promoting Th17 responses in part through the activation of PI3K.     

SND1 promotes Th1/17 immunity against chlamydial lung infection through enhancing dendritic cell function

To date, no reports have linked the multifunctional protein, staphylococcal nuclease domain-containing protein 1 (SND1), to host defense against intracellular infections. In this study, we investigated the role and mechanisms of SND1, by using SND1 knockout (SND1^-/-) mice, in host defense against the lung infection of Chlamydia muridarum, an obligate intracellular bacterium. Our data showed that SND1^-/- mice exhibited significantly greater body weight loss, higher organism growth, and more severe pathological changes compared with wild-type mice following the infection. Further analysis showed significantly reduced Chlamydia-specific Th1/17 immune responses in SND1^-/- mice after infection. Interestingly, the dendritic cells (DCs) isolated from SND1^-/- mice showed lower costimulatory molecules expression and IL-12 production, but higher IL-10 production compared with those from wild-type control mice. In the DC-T cell co-culture system, DCs isolated from SND1^-/- infected mice showed significantly reduced ability to promote Chlamydia-specific IFN-γ producing Th1 cells but enhanced capacity to induce CD4⁺T cells into Foxp3⁺ Treg cells. Adoptive transfer of DCs isolated from SND1^-/- mice, unlike those from wild-type control mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that SND1 plays an important role in host defense against intracellular bacterial infection, and suggest that SND1 can promote Th1/17 immunity and inhibit the expansion of Treg cells through modulation of the function of DCs.     

Interruption of CXCL13-CXCR5 Axis Increases Upper Genital Tract Pathology and Activation of NKT Cells following Chlamydial Genital Infection

Background

Regulation of immune responses is critical for controlling inflammation and disruption of this process can lead to tissue damage. We reported that CXCL13 was induced in fallopian tube tissue following C. trachomatis infection. Here, we examined the influence of the CXCL13-CXCR5 axis in chlamydial genital infection.

Methodology and Principal Findings

Disruption of the CXCL13-CXCR5 axis by injecting anti-CXCL13 Ab to BALB/c mice or using Cxcr5−/− mice increased chronic inflammation in the upper genital tract (UGT; uterine horns and oviducts) after Chlamydia muridarum genital infection (GT). Further studies in Cxcr5−/− mice showed an elevation in bacterial burden in the GT and increased numbers of neutrophils, activated DCs and activated NKT cells early after infection. After resolution, we noted increased fibrosis and the accumulation of a variety of T cells subsets (CD4-IFNγ, CD4-IL-17, CD4-IL-10 & CD8-TNFα) in the oviducts. NKT cell depletion in vitro reduced IL-17α and various cytokines and chemokines, suggesting that activated NKT cells modulate neutrophils and DCs through cytokine/chemokine secretion. Further, chlamydial glycolipids directly activated two distinct types of NKT cell hybridomas in a cell-free CD1d presentation assay and genital infection of Cd1d−/− mice showed reduced oviduct inflammation compared to WT mice. CXCR5 involvement in pathology was also noted using single-nucleotide polymorphism analysis in C. trachomatis infected women attending a sub-fertility clinic. Women who developed tubal pathology after a C. trachomatis infection had a decrease in the frequency of CXCR5 SNP +10950 T>C (rs3922).

Conclusions/Significance

These experiments indicate that disruption of the CXCL13-CXCR5 axis permits increased activation of NKT cells by type I and type II glycolipids of Chlamydia muridarum and results in UGT pathology potentially through increased numbers of neutrophils and T cell subsets associated with UGT pathology. In addition, CXCR5 appears to contribute to inter-individual differences in human tubal pathology following C. trachomatis infection.     

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19⁺CD1d^highCD5⁺ B cells (CD1d^highCD5⁺ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1d^highCD5⁺ Breg cells as compared to age-matched WT control mice. CD1d^highCD5⁺ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4⁺ T cells. Interestingly, CD1d^highCD5⁺ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.     

The immunoregulatory role of type I and type II NKT cells in cancer and other diseases

NKT cells are CD1d-restricted T cells that recognize lipid antigens. They also have been shown to play critical roles in the regulation of immune responses. In the immune responses against tumors, two subsets of NKT cells, type I and type II, play opposing roles and cross-regulate each other. As members of both the innate and adaptive immune systems, which form a network of multiple components, they also interact with other immune components. Here, we discuss the function of NKT cells in tumor immunity and their interaction with other regulatory cells, especially CD4⁺CD25⁺Foxp3⁺ regulatory T cells.     

Natural killer T cells promote collagen-induced arthritis in DBA/1 mice

The role of NKT cells in the pathogenesis of collagen-induced arthritis (CIA) remains unclear since most studies have used C57BL/6 (B6) mice, which are less susceptible to CIA than mice with a DBA/1 background. To clarify the immunological functions of NKT cells in CIA, it is necessary to analyze in detail the effects of NKT cell deficiency on CIA development in DBA/1 mice. The incidence and severity of CIA were significantly exacerbated in DBA/1CD1d^+/− mice as compared to DBA/1CD1d^−/− mice. In DBA/1CD1d^+/− mice, antigen-specific responses of B and T cells against CII were remarkably increased and inflammatory cytokine levels were also increased in vivo and in vitro. The number of IL-17-producing NKT cells significantly increased in DBA/1CD1d^+/− mice as the disease progressed. Our results clearly show that NKT cells are involved not only in accelerating the severity and incidence of CIA but also in perpetuating the disease progression.     

Improving Mycobacterium bovis Bacillus Calmette-Guèrin as a Vaccine Delivery Vector for Viral Antigens by Incorporation of Glycolipid Activators of NKT Cells

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8⁺ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8⁺ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8⁺ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8⁺ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8⁺ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.     

Mice Lacking Natural Killer T Cells Are More Susceptible to Metabolic Alterations following High Fat Diet Feeding

Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD). Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT) cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d^−/− mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD) feeding. Compared with their WT counterparts, CD1d^−/− mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d^−/− mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.     

Regulation of IL-21 signaling by suppressor of cytokine signaling-1 (SOCS1) in CD8(+) T lymphocytes

Mice lacking the gene for suppressor of cytokine signaling 1 (SOCS1) show defective homeostasis of T lymphocytes due to accumulation of CD8⁺ T cells, resulting at least partly from dysregulated IL-15 signaling. IL-15 alone does not stimulate proliferation of naïve CD8 T cells, but can synergize with IL-21 to induce proliferation, suggesting a potential role for IL-21 in the defective homeostasis of CD8⁺ T lymphocytes in SOCS1^−/− mice. Since IL-21 strongly induced SOCS1 mRNA in CD8⁺ T cells, we investigated whether SOCS1 regulates their response to IL-21. CD8⁺ T cells isolated from SOCS1-deficient mice proliferated vigorously in response to IL-21 + IL-15. In CD8⁺ T lymphocytes expressing transgenic TCR, IL-21 + IL-7 provided a stronger stimulus to naïve cells whereas IL-15 + IL-21 potently stimulated memory cells. Compared to truly naïve or memory cells, SOCS1^−/− H-Y TCR⁺ CD8⁺ T cells displayed CD44^loLy6C^hiCD122^intCD127^lo partial memory phenotype and exhibited stronger response to IL-15 + IL-21 than truly naïve cells. In SOCS1^−/− CD8⁺ T cells, IL-21 caused greater reduction in IL-15 threshold for activation in a dose-dependent manner. SOCS1 deficiency did not modulate IL-21Rα expression or sensitivity to IL-21, but delayed the loss of IL-21-induced phospho-STAT3 signal. These results show that SOCS1 is a critical regulator of IL-21 signaling in CD8⁺ T cells, and support the notion that sustained IL-21 signaling might also contribute to the aberrant T cell homeostasis in SOCS1-deficient mice.     

Resistance to Intranasal Infection with Balamuthia mandrillaris Amebae is T‐Cell Dependent

T and B cell‐deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild‐type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4⁺ T cells remained susceptible. When adult WT mice were depleted of both CD4⁺ and CD8⁺ T cells or of CD4⁺ T cells alone, these mice also became susceptible to BAE. Depletion of CD8⁺ T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain.     

Regulatory role of CD1d in neurotropic virus infection

The GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) causes an acute fatal polioencephalomyelitis in mice. Infection of susceptible mice with the DA strain of TMEV results in an acute polioencephalomyelitis followed by chronic immune-mediated demyelination with virus persistence in the central nervous system (CNS); DA virus infection is used as an animal model for multiple sclerosis. CD1d-restricted natural killer T (NKT) cells can contribute to viral clearance and regulation of autoimmune responses. To investigate the role of CD1d in TMEV infection, we first infected CD1d-deficient mice (CD1^−/−) and wild-type BALB/c mice with GDVII virus. Wild-type mice were more resistant to virus than CD1^−/− mice (50% lethal dose titers: wild-type mice, 10 PFU; CD1^−/− mice, 1.6 PFU). Wild-type mice had fewer viral antigen-positive cells with greater inflammation in the CNS than CD1^−/− mice. Second, an analysis of DA virus infection in CD1^−/− mice was conducted. Although both wild-type and CD1^−/− mice had similar clinical signs during the first 2 weeks after infection, CD1^−/− mice had an increase in neurological deficits over those observed in wild-type mice at 3 to 5 weeks after infection. Although wild-type mice had no demyelination, 20 and 60% of CD1^−/− mice developed demyelination at 3 and 5 weeks after infection, respectively. TMEV-specific lymphoproliferative responses, interleukin-4 (IL-4) production, and IL-4/gamma interferon ratios were higher in CD1^−/− mice than in wild-type mice. Thus, CD1d-restricted NKT cells may play a protective role in TMEV-induced neurological disease by alteration of the cytokine profile and virus-specific immune responses.     

Innate Invariant NKT Cells Recognize Mycobacterium tuberculosis–Infected Macrophages,Produce Interferon-γ, and Kill Intracellular Bacteria

Cellular immunity to Mycobacterium tuberculosis (Mtb) requires a coordinated response between the innate and adaptive arms of the immune system, resulting in a type 1 cytokine response, which is associated with control of infection. The contribution of innate lymphocytes to immunity against Mtb remains controversial. We established an in vitro system to study this question. Interferon-γ is produced when splenocytes from uninfected mice are cultured with Mtb-infected macrophages, and, under these conditions, bacterial replication is suppressed. This innate control of bacterial replication is dependent on CD1d-restricted invariant NKT (iNKT) cells, and their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. We show that iNKT cells, even in limiting quantities, are sufficient to restrict Mtb replication. To determine whether iNKT cells contribute to host defense against tuberculosis in vivo, we adoptively transferred iNKT cells into mice. Primary splenic iNKT cells obtained from uninfected mice significantly reduce the bacterial burden in the lungs of mice infected with virulent Mtb by the aerosol route. Thus, iNKT cells have a direct bactericidal effect, even in the absence of synthetic ligands such as α-galactosylceramide. Our finding that iNKT cells protect mice against aerosol Mtb infection is the first evidence that CD1d-restricted NKT cells mediate protection against Mtb in vivo.     

Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ

Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant Vα14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NKT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGFβ. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from Vα14 TCR Tg mice to naïve CD4⁺ T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFNγ neutralization, we showed that this reduction was mediated by IFNγ. Furthermore, the main source of IFNγ during iTreg differentiation was NK1.1⁻CD4⁺Foxp3⁻ T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFNγ.     

NKT cells mediate organ-specific resistance against Leishmania major infection

Whereas the acquired T cell-mediated protection against intracellular pathogens such as Leishmania major has been well studied in the past, the cells and mechanisms involved in their innate control are still poorly understood. Here, we investigated the role of natural killer T (NKT) cells in a high dose L. major mouse infection model. In vitro, L. major only weakly stimulated NKT cells and antagonized their response to the prototypic NKT cell ligand alpha-galactosylceramide, indicating that L. major partially escapes the activation of NKT cells. NKT cell deficiency as analyzed by subcutaneous infection of Jalpha281-/- mice (lacking invariant CD1d-restricted NKT cells) and CD1-/- mice (lacking all CD1d-restricted NKT cells) led to a transient increase in skin lesions, but did not impair the clinical cure of the infection, NK cell cytotoxicity, the production of IFN-gamma, the expression of inducible nitric oxide synthase, and the control of the parasites in the lymph node. In the spleen, however, NKT cells were required for NK cell cytotoxicity and early IFN-gamma production, they lowered the parasite burden, and exerted bystander effects on Leishmania antigen-specific T cell responses, most notably after systemic infection. Thus, NKT cells fulfill organ-specific protective functions during infection with L. major, but are not essential for parasite control.     

Protective immunity and delayed type hypersensitivity reaction are uncoupled in experimental Leishmania major infection of CCR6-negative mice

The chemokine receptor CCR6 is expressed on naïve B cells, dendritic cell and T-cell subpopulations and is involved in cell navigation during organogenesis and recruitment in response to inflammatory stimuli. Gene-deficient C57BL/6 CCR6^−/− mice infected with the protozoan parasite Leishmania (L.) major were able to mount a protective immune response and survived the infection. Whereas macrophage production of nitric oxide (NO), the key leishmanicidal effector molecule during the immune response to L. major, did not require CCR6, the migration of CD4⁺ T cells to the site of infection was reduced in CCR6^−/− mice. Furthermore, the induction of a T-cell-dependent delayed-type-hypersensitivity (DTH) reaction was defective in CCR6^−/− mice, whereas resistance to re-infection was maintained in the absence of CCR6. We conclude that CCR6 contributes to the recruitment of T cells to the site of infection, but is largely dispensable for the control of L. major parasites during primary or secondary infection.     

WSX-1 Signalling Inhibits CD4+ T Cell Migration to the Liver during Malaria Infection by Repressing Chemokine-Independent Pathways

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4⁺ T cells in the liver of infected IL27R^−/− (WSX-1^−/−) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4⁺ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4⁺ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1^−/− mice than infected WT mice, and hepatic CD4⁺ T cells from WSX-1^−/− mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4⁺ T cells to the liver of WSX-1^−/− mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4⁺ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4⁺ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.