Interleukin-9 as a T helper type 17 cytokine

The production of interleukin‐9 (IL‐9) by CD4 T cells has gathered renewed interest as the result of the observation that its expression is broader than originally thought. This includes the production of IL‐9 by a recently characterized subset of CD4 helper T (Th) cells that are termed Th9 as well as production by additional T‐cell subsets including Th17 cells. There is an incomplete understanding as to which IL‐9‐producing T‐cell subsets develop under physiological conditions. We describe the conditions used to generate IL‐9 in Th17 cells in vitro. We also summarize conditions where both IL‐9 and IL‐17 are found in vivo and propose that Th17 cells producing IL‐9 may co‐exist and interact with Th9 cells during conditions of autoimmunity, allergy and infection.     

MicroRNAs regulate T‐cell production of interleukin‐9 and identify hypoxia‐inducible factor‐2α as an important regulator of T helper 9 and regulatory T‐cell differentiation

MicroRNAs (miRNAs) regulate many aspects of helper T cell (Th) development and function. Here we found that they are required for the suppression of interleukin‐9 (IL‐9) expression in Th9 cells and other Th subsets. Two highly related miRNAs (miR‐15b and miR‐16) that we previously found to play an important role in regulatory T (Treg) cell differentiation were capable of suppressing IL‐9 expression when they were over‐expressed in Th9 cells. We used these miRNAs as tools to identify novel regulators of IL‐9 expression and found that they could regulate the expression of Epas1, which encodes hypoxia‐inducible factor (HIF)‐2α. HIF proteins regulate metabolic pathway usage that is important in determining appropriate Th differentiation. The related protein, HIF‐1α enhances Th17 differentiation and inhibits Treg cell differentiation. Here we found that HIF‐2α was required for IL‐9 expression in Th9 cells, but its expression was not sufficient in other Th subsets. Furthermore, HIF‐2α suppressed Treg cell differentiation like HIF‐1α, demonstrating both similar and distinct roles of the HIF proteins in Th differentiation and adding a further dimension to their function. Ironically, even though miR‐15b and miR‐16 suppressed HIF‐2α expression in Treg cells, inhibiting their function in Treg cells did not lead to an increase in IL‐9 expression. Therefore, the physiologically relevant miRNAs that regulate IL‐9 expression in Treg cells and other subsets remain unknown. Nevertheless, the analysis of miR‐15b and miR‐16 function led to the discovery of the importance of HIF‐2α so this work demonstrated the utility of studying miRNA function to identify novel regulatory pathways in helper T‐cell development.     

MicroRNA‐155 may be involved in the pathogenesis of atopic dermatitis by modulating the differentiation and function of T helper type 17 (Th17) cells

Our aims were to identify the differential expression of microRNA (miR)‐155, as well as to explore the possible regulatory effects of miR‐155 on the differentiation and function of T helper type 17 (Th17) cells in atopic dermatitis (AD). The Th17 cell percentage and expression levels of miR‐155, retinoic acid‐related orphan receptor (ROR)γt, interleukin (IL)‐17 and suppressor of cytokine signalling‐1 (SOCS1) in peripheral CD4⁺ T cells, plasma and skin specimens were detected and compared in AD patients and healthy subjects. A miR‐155 mimic and an inhibitor were transfected separately into AD CD4⁺ T cells to confirm the in‐vivo data. The Th17 cell percentage, miR‐155 expression, RORγt mRNA expression, IL‐17 mRNA expression and plasma concentration were increased significantly in AD patients compared with healthy subjects. Conversely, SOCS1 mRNA expression and plasma concentration were decreased significantly. Similar results were detected in cultured CD4⁺ T cells transfected with the miR‐155 mimic compared with a miR‐155 inhibitor or a negative control. Additionally, there was a sequential decrease in miR‐155 expression, as well as RORγt and IL‐17 mRNA expression, but an increase in SOCS1 mRNA expression, from AD lesional skin and perilesional skin to normal skin. Positive correlations were found between miR‐155 expression and AD severity, Th17 cell percentage, RORγt mRNA expression and IL‐17 mRNA expression and plasma concentration, while negative correlations were observed between miR‐155 expression and SOCS1 mRNA expression and plasma concentration in AD peripheral circulation and skin lesions. In conclusion, miR‐155 is over‐expressed and may be involved in AD pathogenesis by modulating the differentiation and function of Th17 cells.     

Serum cytokine pattern in young children with screening detected coeliac disease

Coeliac disease is an autoimmune disease characterized by inflammation localized to the small bowel, but less is known about systemic signs of inflammation. The aim was to measure cytokines of the T helper 1 (Th1) and T helper 2 (Th2) cell patterns in children with screening‐detected coeliac disease before and after treatment with a gluten‐free diet. Serum samples selected before and after the start of a gluten‐free diet from 26 3‐year‐old children diagnosed with biopsy‐proven coeliac disease and from 52 matched controls were assayed in an multiplex enzyme‐linked immunosorbent assay (ELISA) for the 10 cytokines: interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐8, IL‐10, IL‐12p70, IL‐13 and tumour necrosis factor (TNF)‐α. Among Th1 cytokines, IFN‐γ and IL‐12p70 were elevated significantly in children with coeliac disease compared to controls (P < 0·001 and P = 0·001, respectively). Similar findings were demonstrated for the Th2 cytokines IL‐5 (P < 0·001), IL‐10 (P = 0·001) and IL‐13 (P = 0·002). No difference in cytokine levels between the two groups was found for TNF‐α, IL‐1β, IL‐2, IL‐4 and IL‐8. After gluten‐free diet, levels of IL‐5, IL‐12 and IL‐10 decreased significantly (P < 0·001, P = 0·002 and P = 0·007) and IFN‐γ levels were reduced (P = 0·059). Young children with coeliac disease detected by screening demonstrate elevated levels of serum cytokines at time of diagnosis. A prolonged systemic inflammation may, in turn, contribute to long‐term complications known to be associated with untreated coeliac disease.     

Interleukin‐9 and T helper type 9 cells in rheumatic diseases

Interleukin (IL)‐9 is a 28‐30 kDa monomeric glycosylated polypeptide belonging to the IL‐7/IL‐9 family of proteins that bind to a composite receptor consisting of the private receptor IL‐9R and the IL‐2 receptor, gamma (IL‐2RG), a common gamma subunit shared by the receptors of many different cytokines. The IL‐9R is expressed widely and IL‐9 impacts a number of effector cells, such as effector T cells, B cells, innate lymphoid cells, mast cells, polymorphonuclear cells, epithelial cells and smooth muscle cells, playing an important role in regulating inflammatory immunity. The critical role of IL‐9 in promoting cellular and humoral immune responses makes it an important focus of potential therapeutic interventions. Recently, a defined subset of T helper type cells, Th9 cells, has been identified by the potent production of IL‐9. The involvement of the Th9 cell subset has been described in many types of inflammatory diseases, namely atopic diseases, helminth infections, experimental autoimmune encephalomyelitis and ulcerative colitis. In this review, we summarize the IL‐9 biological activities, highlighting roles for IL‐9 and Th9 cells in rheumatoid and psoriatic arthritis, systemic vasculitis, systemic lupus erythematosus and systemic sclerosis.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

Galectin‐9 expands immunosuppressive macrophages to ameliorate T‐cell‐mediated lung inflammation

Galectin‐9 (Gal‐9) plays pivotal roles in the modulation of innate and adaptive immunity to suppress T‐cell‐mediated autoimmune models. However, it remains unclear if Gal‐9 plays a suppressive role for T‐cell function in non‐autoimmune disease models. We assessed the effects of Gal‐9 on experimental hypersensitivity pneumonitis induced by Trichosporon asahii. When Gal‐9 was given subcutaneously to C57BL/6 mice at the time of challenge with T. asahii, it significantly suppressed T. asahii‐induced lung inflammation, as the levels of IL‐1, IL‐6, IFN‐γ, and IL‐17 were significantly reduced in the BALF of Gal‐9‐treated mice. Moreover, co‐culture of anti‐CD3‐stimulated CD4 T cells with BALF cells harvested from Gal‐9‐treated mice on day 1 resulted in diminished CD4 T‐cell proliferation and decreased levels of IFN‐γ and IL‐17. CD11b⁺Ly‐6C^highF4/80⁺ BALF M? expanded by Gal‐9 were responsible for the suppression. We further found in vitro that Gal‐9, only in the presence of T. asahii, expands CD11b⁺Ly‐6C^highF4/80⁺ cells from BM cells, and the cells suppress T‐cell proliferation and IFN‐γ and IL‐17 production. The present results indicate that Gal‐9 expands immunosuppressive CD11b⁺Ly‐6C^high M? to ameliorate Th1/Th17 cell‐mediated hypersensitivity pneumonitis.     

Indirect co‐cultures of healthy mesenchymal stem cells restore the physiological phenotypical profile of psoriatic mesenchymal stem cells

Psoriasis microenvironment, characterized by an imbalance between T helper type 1 (Th1)/Th17 and Th2 cytokines and also influences the mesenchymal stem cells (MSCs) phenotypical profile. MSCs from healthy donors (H‐MSCs) can exert a strong paracrine effect by secreting active soluble factors, able to modulate the inflammation in the microenvironment. To evaluate the influence of H‐MSCs on MSCs from psoriatic patients (PsO‐MSCs), H‐MSCs and PsO‐MSCs were isolated and characterized. Indirect co‐culture of H‐MSCs with PsO‐MSCs was performed; effects on proliferation and expression of cytokines linked to Th1/Th17 and Th2 pathways were assayed before and after co‐culture. The results show that before co‐culture, proliferation of PsO‐MSCs was significantly higher than H‐MSCs (P < 0·05) and the levels of secreted cytokines confirmed the imbalance of Th1/Th17 versus the Th2 axis. After co‐culture of H‐MSCs with PsO‐MSCs, healthy MSCs seem to exert a ‘positive’ influence on PsO‐MSCs, driving the inflammatory phenotypical profile of PsO‐MSCs towards a physiological pattern. The proliferation rate decreased towards values nearer to those observed in H‐MSCs and the secretion of the cytokines that mostly identified the inflammatory microenvironment that characterized psoriasis, such as interleukin (IL)‐6, IL‐12, IL‐13, IL‐17A, tumour necrosis factor (TNF)‐α and granulocyte–macrophage colony‐stimulating factor (G‐CSF), is significantly lower in co‐cultured PsO‐MSCs than in individually cultured PSO‐MSCs (P at least < 0·05). In conclusion, our preliminary results seem to provide an intriguing molecular explanation for the ever‐increasing evidence of therapeutic efficacy of allogeneic MSCs infusion in psoriatic patients.     

Decreased Plasma IL‐22 Levels and Correlations with IL‐22‐Producing T Helper Cells in Patients with New‐Onset Systemic Lupus Erythematosus

Interleukin‐22 (IL‐22) and IL‐22‐producing T helper (Th) cells are involved in the pathogenesis of autoimmune diseases. However, the roles of IL‐22 and IL‐22‐producing T helper cells in systemic lupus erythematosus (SLE) remain unclear. Plasma levels of IL‐22 were measured in 41 patients with SLE (19 new‐onset and 22 relapsing patients) and 20 healthy controls by enzyme‐linked immunosorbent assay (ELISA). Meanwhile, the percentages of CD4⁺IFN‐γ⁺ (Th1), CD4⁺IL‐17⁺ (Th17) and CD4⁺IFN‐γ^?IL‐17^? IL‐22⁺ (Th22) cells in peripheral lymphocytes were determined by flow cytometry, and plasma IL‐22 autoantibodies were detected by ELISA in 19 new‐onset SLE patients and 20 healthy controls. Plasma IL‐22 levels in new‐onset SLE patients were significantly decreased compared with relapsing SLE patients and healthy controls. After treatment with prednisone and hydroxychloroquine, the levels of plasma IL‐22 in new‐onset SLE patients were obviously increased but still lower than healthy controls. There was a positive correlation between plasma IL‐22 levels and the percentages of Th22 cells, but not Th1 and Th17 cells. Moreover, plasma IL‐22 levels as well as peripheral Th17 and Th22 cells correlated with SLE disease activity index (SLEDAI) scores and erythrocyte sedimentation rate (ESR). High frequencies of plasma IL‐22 autoantibodies were detected in new‐onset SLE patients. However, IL‐22 levels did not correlate with IL‐22 autoantibody. Decreased plasma IL‐22 levels and correlation with Th22 cells may be distinct features in new‐onset SLE. Moreover, IL‐22 and Th22 cell correlated with SLE disease activity.     

The influence of regulatory T cells and diurnal hormone rhythms on T helper cell activity

Symptoms of diseases such as rheumatoid arthritis, which is T helper 1 (Th1) dependent, and asthma, which is T helper 2 (Th2) dependent, are influenced by diurnal rhythms and natural regulatory T cells (nTreg). However, the mechanisms responsible for the diurnal rhythm of disease activity have not been identified and it is unclear whether nTreg activity is diurnal rhythm‐dependent. We therefore investigated whether a 24‐hr diurnal cycle affected the ability of various helper T‐cell populations to generate immunomodulatory and pro‐inflammatory cytokines, as well as its suppression by nTreg cells. Using a within‐subject crossover design, sleep versus continuous wakefulness was compared over a 24‐hr period in healthy young volunteers under defined environmental conditions. Venous blood was drawn periodically every 5 hr and the function of T cells was explored in vitro. We demonstrated that interleukin (IL)‐2, interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and IL‐10 secretion by naïve CD4⁺ T cells follows a diurnal rhythm. Furthermore, multiple regression analysis, as well as subsequent in vitro experiments, suggested that serum levels of cortisol and prolactin are part of the underlying mechanism. Additionally, we observed that nTreg suppressed the secretion of IFN‐γ, IL‐2 and TNF‐α, but not the secretion of IL‐4, IL‐6, IL‐10 and IL‐17A. However, the abrogation of IL‐2 release was reversed upon inhibiting CD25 on nTreg. Highly purified nTreg secreted IL‐6, IL‐10 and IL‐17A, but not IL‐2, IL‐4, IFN‐γ or TNF‐α. Taken together, our results demonstrate that hormones and nTreg modulate the diurnal rhythm of T helper cell activity.     

Th9 cytokines curb cervical cancer progression and immune evasion

Cervical cancer is one of the most common cancers among women in developing countries. Persistent infection with high-risk human papillomavirus (HPV) is the major determinant for the development of cervical cancer. Role of newly discovered T helper 9 (Th9) cells in cervical cancer pathogenesis is yet unfolded. In this study, we observed a huge infiltration of PU.1⁺ cells and overrepresentation of IL-9R in tissue biopsy specimens of CIN patients in cervical cancer cases. Treatment with Th9 signatory cytokines, IL-9 and IL-21, suppressed proliferation, enhanced apoptosis and stimulated the expression of MHC I and e-cadherin on HeLa cell lines. Th9 thus seems enhance antitumor immune response through T cell cytotoxicity and play crucial role in a controlling malignant cell transformation. Therefore, this study helps in firmer understanding of relevance of Th9 in cervical cancer immunity.     

The proportion of different interleukin‐17‐producing T‐cell subsets is associated with liver fibrosis in chronic hepatitis C

Studies have suggested the pivotal role of T helper type 1 (Th1) ‐related cytokines on the outcome of hepatitis C virus (HCV) infection. Nevertheless, the role of different interleukin‐17 (IL‐17) ‐secreting T cells on chronic hepatitis C (CHC) is less clear. Here, the in vivo IL‐1β, IL‐6, and IL‐17 levels were positively correlated with both alanine transaminase (ALT) levels and hepatic lesions. When compared with the control group, CHC patients showed a lower proportion of IL‐17‐secreting (CD4⁺ and CD8⁺) T cells capable of simultaneously producing IL‐21. Moreover, the percentage of IL‐10‐secreting Th17 cells was also lower in CHC patients. Notably, advanced liver lesions were observed among those patients with lower percentage levels of IL‐17‐producing T cells positive for IL‐21, interferon‐γ (IFN‐γ) and IL‐10. In contrast, the severity of hepatic damage was associated with peripheral single IL‐17⁺ T cells. The percentage of IL‐17⁺ IL‐21^– IFN‐γ⁺ (CD4⁺ and CD8⁺) T‐cell phenotypes was positively associated with plasma CD14 levels. Finally, elevated levels of circulating CD14 were detected among CHC patients with extensive liver damage. In summary, although preliminary, our results suggest that a balance between different IL‐17‐producing T cells, associated with peripheral levels of CD14, may be a progress marker for liver disease in chronically HCV‐infected patients.     

The microRNA‐9/B‐lymphocyte‐induced maturation protein‐1/IL‐2 axis is differentially regulated in progressive HIV infection

The fine control of T‐cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B‐lymphocyte‐induced maturation protein‐1 (Blimp‐1/Prdm1) is highly expressed in CD4⁺ T cells from chronically HIV‐infected (CHI) patients compared to cells from long‐term nonprogressors or healthy controls. Stimulation through the T‐cell receptor in the presence ofIL‐2 induces Blimp‐1 protein expression. We show here that Blimp‐1 levels are translationally regulated by microRNA‐9 (miR‐9). Overexpression of miR‐9 induces Blimp‐1 repression, restoring IL‐2 secretion in CD4⁺ T cells via reduction in the binding of Blimp‐1 to the il‐2 promoter. In CHI patients where IL‐2 expression is reduced and there is generalized T‐cell dysfunction, we show differential expression of both miR‐9 and Blimp‐1 in CD4⁺ cells compared with levels in long‐term nonprogressors. These data identify a novel miR‐9/Blimp‐1/IL‐2 axis that is dysregulated in progressive HIV infection.     

Functional TLR9 modulates bone marrow B cells from rheumatoid arthritis patients

TLR9 recognizes unmethylated CpG‐rich, pathogen‐derived DNA sequences and represents the component of the innate immune system that heavily influences adaptive immunity and may contribute to the immunological disturbances in rheumatoid arthritis (RA). Accumulating data indicate that BM of RA patients participates in the pathogenesis of this disease as a site of proinflammatory cytokines overproduction and lymphocytes activation. Here, we investigated the functionality of TLR9 and its role in the modulation of RA BM B‐cell functions. We report that BM B cells isolated from RA patients express TLR9 at the mRNA and protein levels acquired at the stage of preB/immature B‐cell maturation. Stimulation of BM CD20⁺ B cells by CpG‐containing oligodeoxynucleotide‐enhanced expression of activation markers (CD86 and CD54) triggered IL‐6 and TNF‐α secretion and cell proliferation. Significantly higher levels of eubacterial DNA encoding 16S‐rRNA were found in BM samples from RA than osteoarthritis patients. Moreover, RA BM B cells exerted higher expression of CD86 than their osteoarthritis counterparts, suggesting their in situ activation via TLR9. Thus, our data indicate that TLR9 may participate in direct activation and proliferation of B cells in BM, and therefore could play a role in the pathogenesis of RA.