Type 2 diabetes mellitus is associated with altered CD8+ T and natural killer cell function in pulmonary tuberculosis

Type 2 diabetes mellitus (DM) is associated with expanded frequencies of mycobacterial antigen-specific CD4⁺ T helper type 1 (Th1) and Th17 cells in individuals with active pulmonary tuberculosis (TB). No data are available on the role of CD8⁺ T and natural killer (NK) cells in TB with coincident DM. To identify the role of CD8⁺ T and NK cells in pulmonary TB with diabetes, we examined mycobacteria-specific immune responses in the whole blood of individuals with TB and DM (TB-DM) and compared them with those without DM (TB-NDM). We found that TB-DM is characterized by elevated frequencies of mycobacterial antigen-stimulated CD8⁺ T cells expressing type 1 [interferon-γ and interleukin-2 (IL-2)] and type 17 (IL-17F) cytokines. We also found that TB-DM is characterized by expanded frequencies of TB antigen-stimulated NK cells expressing type 1 (tumour necrosis factor-α) and type 17 (IL-17A and IL-17F) cytokines. In contrast, CD8⁺ T cells were associated with significantly diminished expression of the cytotoxic markers perforin, granzyme B and CD107a both at baseline and following antigen or anti-CD3 stimulation, while NK cells were associated with significantly decreased antigen-stimulated expression of CD107a only. This was not associated with alterations in CD8⁺ T-cell or NK cell numbers or subset distribution. Therefore, our data suggest that pulmonary TB complicated with type 2 DM is associated with an altered repertoire of cytokine-producing and cytotoxic molecule-expressing CD8⁺ T and NK cells, possibly contributing to increased pathology.     

IL-36 cytokines imprint a colitogenic phenotype on CD4+ T helper cells

IL-36 cytokines are emerging as potent orchestrators of intestinal inflammation and are being implicated in the pathogenesis of inflammatory bowel diseases (IBD). However, the mechanisms through which these cytokines mediate these effects remain to be fully uncovered. Here, we report specifically elevated expression of IL-36α, and not IL-36β or IL-36γ in the serum of newly diagnosed, treatment naïve, paediatric IBD patients and identify T cells as primary cellular mediators of IL-36 responses in the inflamed gut. IL-36R expression on CD4⁺ T cells was found to promote intestinal pathology in a murine model of colitis. Consistent with these effects, IL-36R can act as a potent instructor of CD4⁺ T cell differentiation in vivo, enhancing Th1 responses, while inhibiting the generation of Tregs. In addition, loss of IL-36 responsiveness significantly reduced the migration of pathogenic CD4⁺ T cells towards intestinal tissues and IL-36 was found to act, uniquely among IL-1 family members, to induce the expression of gut homing receptors in proinflammatory murine and human CD4⁺ T cells. These data reveal an important role for IL-36 cytokines in driving the colitogenic potential of CD4⁺ T cells and identify a new mechanism through which they may contribute to disease pathogenesis.     

Host-dependent control of early regulatory and effector T-cell differentiation underlies the genetic susceptibility of RAG2-deficient mouse strains to transfer colitis

De novo differentiation of CD4⁺Foxp3⁺ regulatory T cells (induced (i) Tregs) occurs preferentially in the gut-associated lymphoid tissues (GALT). We addressed the contribution of background genetic factors in affecting the balance of iTreg, T helper type 1 (Th1), and Th17 cell differentiation in GALT in vivo following the transfer of naive CD4⁺CD45RB^high T cells to strains of RAG2-deficient mice with differential susceptibility to inflammatory colitis. iTregs represented up to 5% of CD4⁺ T cells in mesenteric lymph nodes of less-susceptible C57BL/6 RAG2^−/− mice compared with <1% in highly susceptible C57BL/10 RAG2^−/− mice 2 weeks following T-cell transfer before the onset of colitis. Early Treg induction was correlated inversely with effector cell expansion and the severity of colitis development, was controlled primarily by host and not T-cell-dependent factors, and was strongly associated with interleukin-12 (IL-12)/23 production by host CD11c⁺CD103⁺ dendritic cells. These data highlight the importance of genetic factors regulating IL-12/23 production in controlling the balance between iTreg differentiation and effector-pathogenic CD4⁺ T-cell expansion in lymphopenic mice and indicate a direct role for iTregs in the regulation of colonic inflammation in vivo.     

CD101 inhibits the expansion of colitogenic T cells

CD101 exerts negative-costimulatory effects in vitro, but its function in vivo remains poorly defined. CD101 is abundantly expressed on lymphoid and myeloid cells in intestinal tissues, but absent from naïve splenic T cells. Here, we assessed the impact of CD101 on the course of inflammatory bowel disease (IBD). Using a T-cell transfer model of chronic colitis, we found that in recipients of naïve T cells from CD101^+/+ donors up to 30% of the recovered lymphocytes expressed CD101, correlating with an increased interleukin (IL)-2-mediated FoxP3 expression. Transfer of CD101^−/− T cells caused more severe colitis and was associated with an expansion of IL-17-producing T cells and an enhanced expression of IL-2Rα/β independently of FoxP3. The co-transfer of naïve and regulatory T cells (Treg) protected most effectively from colitis, when both donor and recipient mice expressed CD101. Although the expression of CD101 on T cells was sufficient for Treg-function and the inhibition of T-cell proliferation, sustained IL-10 production required additional CD101 expression by myeloid cells. Finally, in patients with IBD a reduced CD101 expression on peripheral and intestinal monocytes and CD4⁺ T cells correlated with enhanced IL-17 production and disease activity. Thus, CD101 deficiency is a novel marker for progressive colitis and potential target for therapeutic intervention.     

Bach2 in CD4+ T cells from SLE patients modulates B-cell differentiation and IgG production

T and B cells participate in the development of systemic lupus erythematosus (SLE). BTB and CNC homology 2 (Bach2) is an irreplaceable regulator in the T and B lineages that helps to maintain immune homeostasis. However, the function of Bach2 in the pathogenesis of SLE has not been studied in depth. Flow cytometry and qRT‒PCR were used to assess Bach2 levels, bisulfite sequencing PCR was used to measure the methylation level, and silencing by electroporation and stimulation with a cytokine concentration gradient were used to investigate the effect of Bach2 on T cells. Bach2 expression was elevated in the helper T-cell subsets (T follicular helper, Th1, Th2, Th17, and Treg cells) of SLE patients and negatively correlated with disease severity and autoantibody levels. CD4⁺ T cells from SLE patients had decreased methylation levels in the Bach2 promoter region. Silencing Bach2 in CD4⁺ T cells induced increases in the CD19⁺ B-cell count, plasmablasts, and secretion of IgG by prompting the secretion of cytokines. The activation signals CD3/CD28, IL-6, and IL-21 upregulated Bach2 expression in CD4⁺ T cells. The regulation of Bach2 by cytokines and T-cell activation signals in CD4⁺ T cells was shown to act on B cells and play a protective role against SLE.     

BTLA associates with increased Foxp3 expression in CD4+ T cells in dextran sulfate sodium-induced colitis

Ulcerative colitis (UC) is an inflammatory bowel disease, and its pathogenesis involves a variety of genetic, environmental, and immunological factors such as T helper cells and their secreted cytokines. B and T lymphocyte attenuator (BTLA) is an immunoregulatory receptor that has a strong suppressive effect on T-cell function. However the role of BTLA in UC remains poorly understood. Here we demonstrated that the frequency of BTLA-expressing CD3⁺ T cells, especially CD4⁺ T cells, increased in blood and mucosa in mice with DSS-induced colitis. The frequency of Foxp3-expressing cells in BTLA+ CD4⁺ T cell from lamina propria mononuclear cells (LPMCs) was much higher in DSS-treated mice than that in controls. Similarly, the proportion of IL-17+ cells in BTLA+ CD4⁺ T cells from LPMCs in DSS-treated mice is much higher than that in controls, while no perceptible difference for the proportion of IFN-γ+ cells in BTLA+ CD4⁺ T cells was noted between DSS-treated mice and controls. Treatment of mesalazine, an anti-ulcerative colitis drug, down-regulated Foxp3 and IL-17 expression in BTLA positive T cells along with attenuated severity for colitis. Our findings indicate that BTLA may be involved in the control of inflammatory responses through increasing Foxp3 expression, rather than attenuating IL-17 production, in DSS-induced colitis.     

T helper cell IL-4 drives intestinal Th2 priming to oral peanut antigen,under the control of OX40L and independent of innate-like lymphocytes

Intestinal T helper type 2 (Th2) immunity in food allergy results in IgG1 and IgE production, and antigen re-exposure elicits responses such as anaphylaxis and eosinophilic inflammation. Although interleukin-4 (IL-4) is critically required for allergic sensitization, the source and control of IL-4 during the initiation of Th2 immunity in vivo remains unclear. Non-intestinal and non-food allergy systems have suggested that natural killer-like T (NKT) or γδ T-cell innate lymphocytes can supply the IL-4 required to induce Th2 polarization. Group 2 innate lymphoid cells (ILCs) are a novel IL-4-competent population, but their contribution to initiating adaptive Th2 immunity is unclear. There are also reports of IL-4-independent Th2 responses. Here, we show that IL-4-dependent peanut allergic Th2 responses are completely intact in NKT-deficient, γδ T-deficient or ILC-deficient mice, including antigen-specific IgG1/IgE production, anaphylaxis, and cytokine production. Instead, IL-4 solely from CD4⁺ Th cells induces full Th2 immunity. Further, CD4⁺ Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule on dendritic cells (DCs) required for intestinal allergic priming. However, both Th2 cells and ILCs orchestrated IL-13-dependent eosinophilic inflammation. Thus, intestinal Th2 priming is initiated by an autocrine/paracrine acting CD4⁺ Th cell-intrinsic IL-4 program that is controlled by DC OX40L, and not by NKT, γδ T, or ILC cells.     

Expression of selectin-binding epitopes and cytokines by CD4+ T cells repopulating scid mice with colitis

Recruitment into the gut of CD4⁺ T cells and their activation in the colonic lamina propria (LP) are key events in the development of colitis in scid mice reconstituted with CD4⁺ T cells from immunocompetent, congenic donor mice. This study investigated the expression of cytokines and selectin-binding epitopes by CD4⁺ T cells repopulating different tissues of the adoptive scid host. Cells from the inflamed colonic LP of transplanted scid mice produced high amounts of IL-12, IFN-γ and TNF-α but only low amounts IL-4 and IL-10. Intracellular cytokine staining confirmed the presence of large numbers of IFN-γ- and TNF-α-producing effector CD4⁺ T cells in the colonic LP of scid mice with colitis but also in non-inflamed tissues [spleen (S), peritoneal cavity (PC) and mesenteric lymph nodes (mLN)] of the adoptive host. Cells from these tissues furthermore produced large amounts of IL-12. Ligands for endothelial selectins are involved in recruiting T cells into inflamed tissues. We have analyzed the expression of selectin-binding epitopes on CD4⁺ T cells repopulating different tissues of the adoptive scid host. We found that a large fraction of CD4⁺ T cells from inflamed colonic LP and from non-inflamed PC, mLN and S expressed high levels of P- and E-selectin-binding epitopes (P-L^hi) in transplanted scid mice, but not in congenic, immunocompetent control mice. Although P-L^hi CD4⁺ T cells were enriched in IFN-γ-producing subsets from most (but not all) tissues, we also found large numbers of in vivo generated P-L^lo CD4⁺ T cells producing pro-inflammatory cytokines. This was in contrast to in vitro generated Th1 CD4⁺ T blasts that were almost exclusively P-L^hi. In this mouse model, production of Th1-type pro-inflammatory cytokines and expression of surface epitopes binding endothelial selectins are hence strikingly up-regulated in CD4⁺ T cells residing in inflamed and non-inflamed tissues during the development of colitis.     

Protein kinase 2 (CK2) controls CD4+ T cell effector function in the pathogenesis of colitis

Crohn's disease (CD), one of the major forms of inflammatory bowel disease (IBD), is characterized by chronic inflammation of the gastrointestinal tract and associated with aberrant CD4⁺ T-helper type 1 (Th1) and Th17 responses. Protein kinase 2 (CK2) is a conserved serine–threonine kinase involved in signal transduction pathways, which regulate immune responses. CK2 promotes Th17 cell differentiation and suppresses the generation of Foxp3⁺ regulatory T cells. The function of CK2 in CD4⁺ T cells during the pathogenesis of CD is unknown. We utilized the T cell-induced colitis model, transferring CD45RB^hi-naive CD4⁺ T cells from CK2α^fl/fl controls and CK2α^fl/fldLck-Cre mice into Rag1^−/− mice. CD4⁺ T cells from CK2α^fl/fldLck-Cre mice failed to induce wasting disease and significant intestinal inflammation, which was associated with decreased interleukin-17A-positive (IL-17A⁺), interferon-γ-positive (IFN-γ⁺), and double-positive IL-17A⁺IFN-γ⁺ CD4⁺ T cells in the spleen and colon. We determined that CK2α regulates CD4⁺ T cell proliferation through a cell-intrinsic manner. CK2α is also important in controlling CD4⁺ T cell responses by regulating NFAT2, which is vital for T cell activation and proliferation. Our findings indicate that CK2α contributes to the pathogenesis of colitis by promoting CD4⁺ T cell proliferation and Th1 and Th17 responses, and that targeting CK2 may be a novel therapeutic treatment for patients with CD.     

IL-27, targeting antigen-presenting cells,promotes Th17 differentiation and colitis in mice

T helper type 17 (Th17) cells have been implicated in autoimmunity and inflammatory bowel disease (IBD). Antigen-presenting cell (APC) -derived cytokines such as interleukin (IL)-1β and IL-6 are key mediators supporting Th17 differentiation, yet how these factors are induced in vivo remains unclear. Here, we show that IL-27 acting on APCs enhances IL-6 and IL-1β production and Th17 differentiation. IL-27Rα−/− T-cell receptor (TCR)β−/− recipients fail to develop gut inflammation following naive CD4 T-cell transfer, whereas IL-27Rα+/+ TCRβ−/− recipients develop severe colitis. Investigation of T-cell responses exhibits that IL-27Rα−/− TCRβ−/− mice do not support Th17 differentiation with significantly decreased levels of IL-6 and IL-1β by APCs. Our study has identified a novel proinflammatory role for IL-27 in vivo that promotes Th17 differentiation by inducing Th17-supporting cytokines in APCs.     

T1/ST2 promotes T helper 2 cell activation and polyfunctionality in bronchopulmonary mycosis

Interleukin (IL)-33 enhances T helper (Th)2 immunity via its receptor T1/ST2. Infection with the yeast-like pathogen Cryptococcus neoformans is usually controlled by a Th1-mediated immune response. The mechanisms responsible for nonprotective Th2 immunity leading to allergic inflammation in pulmonary cryptococcosis are still not fully understood. Using a murine pulmonary model of C. neoformans infection, we report that T1/ST2 expression correlates with the intensity of Th2 activation, as demonstrated by the expression of CD25 and CD44 and downregulation of CD62L. Antigen-specific T1/ST2⁺ Th cells are the primary source of the Th2 cytokines IL-5 and IL-13 as compared with wild-type T1/ST2⁻ Th cells or Th cells from T1/ST2^−/− mice. In addition, T1/ST2⁺ Th cells almost exclusively contain bi- and trifunctional Th2 cytokine-producing Th cells compared with T1/ST2⁻ Th cells or Th cells from T1/ST2^−/− mice. Finally, T1/ST2-driven Th2 development resulted in defective pulmonary fungal control. These data demonstrate that T1/ST2 directs Th2 cell activation and polyfunctionality in allergic bronchopulmonary mycosis.     

Increased CD45RO CD62L CD4 T-cell subpopulation responsible for Th2 response in Kimura’s disease

Kimura’s disease is characterized by subcutaneous masses, eosinophilia, and markedly elevated serum immunoglobulin E, suggesting that T helper (Th)2 cells may play a role in the pathogenesis. We investigated Th2 cytokine synthesis by mononuclear cells and possible Th1/Th2 subpopulations in Kimura’s disease. Peripheral blood samples were obtained from seven patients with Kimura’s disease and CD4⁺ T-cell subpopulations separated by CD45RO and CD62L were isolated. Purified cells were stimulated with PHA or anti-CD3 mAb, and the cytokine levels were measured by Cytometric Bead Array kit. Peripheral blood mononuclear cells in the majority of the patients produced Th2 cytokines such as interleukin (IL)-3, IL-4, IL-5, IL-13 or GM-CSF higher than those of controls. The ratio of CD45RO⁺ CD62L⁺ cells in CD4⁺ T cells was increased in six out of seven patients compared to age-matched controls. Especially, patient 1 had remarkably increased levels of CD45RO⁺ CD62L⁺ population in CD4⁺ T cells. In addition, IL-4 production levels by CD45RO⁺ CD62L⁺ CD4⁺ T cells of patients 1 and 2 were higher than those of their CD45RO⁺ CD62L⁻ CD4⁺ T cells, in the same manner as those by a normal control. Taken together, the synthesis of Th2 cytokines and CD62L-positive subpopulation in CD45RO⁺ CD4⁺ T cells, which may represent characteristics of Th2, are increased in patients with Kimura’s disease, suggesting that deviation to Th2 may involve in pathogenesis of the disease.     

Contraction of intestinal effector T cells by retinoic acid-induced purinergic receptor P2X7

The intestinal environment harbors a large number of activated T cells, which are potentially inflammatory. To prevent inflammatory responses, intestinal T cells are controlled by various tolerogenic mechanisms, including T-cell apoptosis. We investigated the expression mechanism and function of the purinergic receptor P2X7 in contraction of intestinal CD4⁺ effector T cells. We found that P2X7 upregulation on CD4⁺ effector T cells is induced by retinoic acid through retinoic acid receptor α binding to an intragenic enhancer region of the P2rx7 gene. P2X7 is highly expressed by most intestinal αβ and γδ T cells, including T-helper type 1 (Th1) and Th17 cells. The intestinal effector T cells are effectively deleted by P2X7 activation-dependent apoptosis. Moreover, P2X7 activation suppressed T-cell-induced colitis in Rag1^−/− mice. The data from vitamin A-deficient and P2rx7^−/− mice indicate that the retinoic acid-P2X7 pathway is important in preventing aberrant buildup of activated T cells. We conclude that retinoic acid controls intestinal effector T-cell populations by inducing P2X7 expression. These findings have important ramifications in preventing inflammatory diseases in the intestine.     

Microbial Amyloids Induce Interleukin 17A (IL-17A) and IL-22 Responses via Toll-Like Receptor 2 Activation in the Intestinal Mucosa

The Toll-like receptor 2 (TLR2)/TLR1 receptor complex responds to amyloid fibrils, a common component of biofilm material produced by members of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. To determine whether this TLR2/TLR1 ligand stimulates inflammatory responses when bacteria enter intestinal tissue, we investigated whether expression of curli amyloid fibrils by the invasive enteric pathogen Salmonella enterica serotype Typhimurium contributes to T helper 1 and T helper 17 responses by measuring cytokine production in the mouse colitis model. A csgBA mutant, deficient in curli production, elicited decreased expression of interleukin 17A (IL-17A) and IL-22 in the cecal mucosa compared to the S. Typhimurium wild type. In TLR2-deficient mice, IL-17A and IL-22 expression was blunted during S. Typhimurium infection, suggesting that activation of the TLR2 signaling pathway contributes to the expression of these cytokines. T cells incubated with supernatants from bone marrow-derived dendritic cells (BMDCs) treated with curli fibrils released IL-17A in a TLR2-dependent manner in vitro. Lower levels of IL-6 and IL-23 production were detected in the supernatants of the TLR2-deficient BMDCs treated with curli fibrils. Consistent with this, three distinct T-cell populations—CD4⁺ T helper cells, cytotoxic CD8⁺ T cells, and γδ T cells—produced IL-17A in response to curli fibrils in the intestinal mucosa during S. Typhimurium infection. Notably, decreased IL-6 expression by the dendritic cells and decreased IL-23 expression by the dendritic cells and macrophages were observed in the cecal mucosa of mice infected with the curli mutant. We conclude that TLR2 recognition of bacterial amyloid fibrils in the intestinal mucosa represents a novel mechanism of immunoregulation, which contributes to the generation of inflammatory responses, including production of IL-17A and IL-22, in response to bacterial entry into the intestinal mucosa.