Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4⁺ T cells toward the Th17 phenotype was examined. CD4⁺ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.     

Periodontitis‐associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14+ monocytes leading to enhanced Th17/IL‐17 responses

The Th17/IL‐17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL‐17 response in vitro, and studied IL‐17⁺ CD4⁺ T‐cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4⁺ T‐cell co‐cultures promoted a Th17/IL‐17 response in vitro in a dose‐ and time‐dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA‐DR expression, and enhanced TNF‐α, IL‐1β, IL‐6 and IL‐23 production. Mechanistically, IL‐17 production in Pg‐stimulated co‐cultures was partially dependent on IL‐1β, IL‐23 and TLR2/TLR4 signalling. Increased frequencies of IL‐17⁺ cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL‐17⁺ CD4⁺ T‐cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL‐17 production in anti‐CD3 mAb‐stimulated monocyte/CD4⁺ T‐cell co‐cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL‐17 production by human CD4⁺ T cells, a process that appears enhanced in patients with PD.     

Notch signaling suppresses CD14+ monocytes cells activity in patients with chronic hepatitis C

Hepatitis C virus (HCV) infection always leads to chronic hepatitis via dysregulation of host immunity. Notch signaling also modulates the response of monocytes/macrophages. Thus, we aimed to investigate the regulatory role of Notch signaling to CD14⁺ monocytes. Forty patients with chronic hepatitis C and twenty normal controls (NC) were enrolled. CD14⁺ monocytes and CD4⁺ T cells were purified from peripheral bloods. Notch receptors' mRNA expression in CD14⁺ monocytes was semi‐quantified by real‐time PCR. Cytokine production by CD14⁺ monocytes in response to γ‐secretase inhibitor (GSI) was investigated by ELISA. GSI‐induced CD14⁺ monocytes activity to HCV clearance in Huh7.5 cells and to CD4⁺ T cell differentiation was also assessed in direct and indirect contact co‐culture system. Notch1 mRNA relative level was approximately 10‐fold elevated in CD14⁺ monocytes from chronic hepatitis C patients when compared with NC. GSI stimulation resulted in enhanced cytokines production by CD14⁺ monocytes from chronic hepatitis C patients. GSI‐stimulated CD14⁺ monocytes from chronic hepatitis C patients induced suppression of HCV RNA replication in both direct and indirect contact co‐culture system of CD14⁺ monocytes and HCVcc‐infected Huh7.5 cells, and this process was accompanied by elevation of interferon‐γ production but not increased target cell death. Moreover, GSI stimulation also enhanced CD14⁺ monocytes‐induced Th1 and Th17 cells activation, and this process required direct cell‐to‐cell contact. Effective antiviral therapy down‐regulated Notch1 mRNA expression and promoted cytokine production by CD14⁺ monocytes from chronic hepatitis C. Current data revealed an important immunoregulatory property of Notch signaling to CD14⁺ monocytes in chronic HCV infection.     

Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers

‘Circulating’ T follicular helper cells (Tfh), characterized by their surface phenotypes CD4⁺chemokine receptor 5 (CXCR5)⁺ inducible co‐stimulatory molecule (ICOS)⁺, have been identified as the CD4⁺ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast‐like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA‐FLS co‐cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co‐cultured RA‐FLS with or without anti‐CD3/CD28‐stimulated PBMC. The results showed that RA‐FLS co‐cultured with stimulated PBMC could increase the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells of RA PBMC possibly via the production of interleukin (IL)‐6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co‐culture system of RA‐FLS and PBMC. The percentage of CD4⁺CXCR5⁺ICOS⁺ T cells was decreased when ROS production was inhibited by N‐acetyl‐L‐cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)‐α and IL‐1β in the co‐culture system and the blocking of TNF receptor 2 (TNF‐R2) and IL‐1β receptor (IL‐1βR) both decreased the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA‐FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA.     

CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis

CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17‐associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL‐17 production by human CD4⁺ T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4⁺ T cells to secrete IL‐17 that co‐expressed IFN‐γ and IL‐22, and differentiated naïve CD4⁺ T cells to become Th17‐cytokine producing cells. In a co‐culture system of human CD4⁺ T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL‐17 production upon triggering by superantigen. Moreover, when monocyte‐derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL‐17, IFN‐γ, and proliferation by CD4⁺ T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL‐17 and IL‐22 levels. A similar response to CXCL4 of enhanced IL‐17 production by CD4⁺ T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro‐inflammatory cytokine production especially IL‐17 by human CD4⁺ T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology.     

Boswellic acids reduce Th17 differentiation via blockade of IL‐1β‐mediated IRAK1 signaling

Interferon‐gamma producing CD4⁺ T (Th1) cells and IL‐17‐producing CD4⁺ T (Th17) cells are involved in the pathogenesis of several autoimmune diseases including multiple sclerosis. Therefore, the development of treatment strategies controlling the generation and expansion of these effector cells is of high interest. Frankincense, the resin from trees of the genus Boswellia, and particularly its prominent bioactive compound acetyl‐11‐keto‐β‐boswellic acid (AKBA), have potent anti‐inflammatory properties. Here, we demonstrate that AKBA is able to reduce the differentiation of human CD4⁺ T cells to Th17 cells, while slightly increasing Th2‐ and Treg‐cell differentiation. Furthermore, AKBA reduces the IL‐1β‐triggered IL‐17A release of memory Th17 cells. AKBA may affect IL‐1β signaling by preventing IL‐1 receptor‐associated kinase 1 phosphorylation and subsequently decreasing STAT3 phosphorylation at Ser727, which is required for Th17‐cell differentiation. The effects of AKBA on Th17 differentiation and IL‐17A release make the compound a good candidate for potential treatment of Th17‐driven diseases.     

Interleukin (IL)‐17‐producing pathogenic T lymphocytes co‐express CD20 and are depleted by rituximab in primary Sjögren's syndrome: a pilot study

Compelling evidence suggests that interleukin (IL)‐17 and IL‐17‐producing cells play a pivotal role in the pathogenesis of primary Sjögren's syndrome (pSS). We investigated phenotypical and functional effects of the anti‐CD20 antibody rituximab (RTX) on circulating and glandular IL‐17‐producing T cells in pSS. RTX is able to deplete glandular IL‐17⁺ CD3⁺CD4^–CD8^– double‐negative (DN) and CD4⁺ Th17 cells as well as circulating IL‐17⁺ DN T cells. A fraction of glandular and circulating IL‐17⁺ DN cells and CD4⁺ T helper type 17 (Th17) cells co‐expresses CD20 on the cell surface explaining, at least in part, such depletive capacity of RTX. The exposure to RTX does not rescue the in‐vitro corticosteroid resistance of IL‐17⁺ DN T cells. Our results support further the therapeutic role in pSS of RTX that, despite its B cell specificity, appears able to also hamper IL‐17‐producing T cells in this disease.     

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3⁺ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.     

Anti‐tumour necrosis factor treatment increases circulating T helper type 17 cells similarly in different types of inflammatory arthritis

We investigated changes in circulating T helper type 17 (Th17) cells following anti‐tumour necrosis factor (TNF) in rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) patients. Peripheral blood mononuclear cells (PBMC) were isolated from 25 RA, 15 AS and eight PsA patients at baseline 4 and 12 weeks after treatment, and Th17 cell frequencies were analysed using interleukin (IL)‐17 enzyme‐linked immunospot (ELISPOT) and flow cytometry. A significant increase in IL‐17‐producing cells was observed by ELISPOT in RA and AS patients at 12 weeks. Flow cytometry confirmed significant increases in CD4⁺IL‐17⁺ cells at 12 weeks in RA and AS and 4 weeks in PsA patients. Anti‐TNF treatment increases circulating Th17 cells in three different diseases.     

Fetal BM‐derived mesenchymal stem cells promote the expansion of human Th17 cells,but inhibit the production of Th1 cells

Th type 17 (Th17) cells have been identified as a proinflammatory T‐cell subset. Here, we investigated the regulation of human Th17 cells by fetal BM‐derived mesenchymal stem cells (FBM‐MSC). We cocultured FBM‐MSC with human PBMC or CD4⁺ T cells from healthy donors. FBM‐MSC significantly suppressed the proliferation of CD4⁺ T cells stimulated by PHA and recombinant IL‐2. Significantly higher levels of IL‐17 were observed in FBM‐MSC cocultured with either PBMC or CD4⁺ T cells than that in PBMC cultured alone or CD4⁺ T cells cultured alone. Flow cytometry analysis showed that the percentage of Th17 cells in coculture of FBM‐MSC and CD4⁺ T cells was significantly higher than that in CD4⁺ T‐cell cultured alone. FBM‐MSC did not express IL‐17 protein. Consistent with the augmentation of Th17 cells, significantly higher levels of IL‐6 and IL‐1 were observed in coculture of FBM‐MSC and CD4⁺ T cells than that in CD4⁺ T‐cell culture, while the levels of IL‐23 were similar between FBM‐MSC + PBMC coculture and PBMC alone, or FBM‐MSC + CD4⁺ T‐cell and CD4⁺ T‐cell alone. The presence of FBM‐MSC decreased the percentage of Th1 cells, but minimally affected the expansion of CD4⁺CD25⁺ T cells. In conclusion, our data demonstrate for the first time that FBM‐MSC promote the expansion of Th17 cells and decrease IFN‐γ‐producing Th1 cells. These data suggest that IL‐6 and IL‐1, instead of IL‐23, may be partly involved in the expansion of Th17 cells.     

Prediction of disease severity in neuromyelitis optica by the levels of interleukin (IL)‐6 produced during remission phase

T helper type 17 (Th17) cytokines have been implicated in the pathogenesis of neuromyelitis optica (NMO). As humanized anti‐interleukin (IL)‐6R (tocilizumab) immunoglobulin (Ig)G has been used as disease‐modifying therapy for NMO, the objective of our study was to investigate the role of endogenous IL‐6 on NMO‐derived CD4⁺ T cell behaviour. High production of IL‐6, IL‐17 and IL‐21 by CD4⁺ T‐cells was detected in NMO patients. Further, IL‐21 and IL‐6 levels were related directly to the level of neurological disabilities. The addition of anti‐IL‐6R IgG not only reduced directly the production of these cytokines, but also almost abolished the ability of activated autologous monocytes in enhancing IL‐6, IL‐17 and IL‐21 release by CD4⁺ T cells. In contrast, the production of IL‐10 was amplified in those cell cultures. Further, anti‐IL‐6R monoclonal antibodies (mAb) also potentiated the ability of glucocorticoid in reducing Th17 cytokines. Finally, the in‐vivo and in‐vitro IL‐6 levels were significantly higher among those patients who experienced clinical relapse during 2‐year follow‐up. In summary, our results suggest a deleterious role of IL‐6 in NMO by favouring, at least in part, the expansion of corticoid‐resistant Th17 cells.     

HMGB1 Promotes the Differentiation of Th17 via Up‐Regulating TLR2 and IL‐23 of CD14+ Monocytes from Patients with Rheumatoid Arthritis

High‐mobility group box 1 (HMGB1) is a non‐histone nuclear protein that is released extracellulary and has been implicated in autoimmune disease. Toll‐like receptor 2 (TLR2) signalling is thought to be essential for the inflammatory response and for immune disorders. In recent studies, enhanced HMGB1 and TLR2 expressions have been found in rheumatoid arthritis (RA), respectively. The aim of this study is to explore whether HMGB1 stimulation can up‐regulate the expression of TLR2 on CD14⁺ monocytes from patients with RA and to clarify the subsequent events involving Th17 cells and Th17 cell‐associated cytokine changes. Our results showed that the frequency of CD14⁺ cells in peripheral blood mononuclear cell (PBMC) was obviously increased, and enhanced expression of TLR2 on CD14⁺ monocytes was also found in patients with RA, compared with healthy controls with statistical significance (P < 0.001). In addition, the levels of IL‐17, IL‐23 and IL‐6 in supernatants from cultured monocytes from patients and in patient’s plasma were increased, and NF‐κB, the downstream target of TLR2, also showed a marked elevation after monocytes were stimulated by HMGB1. This implies that the enhanced TLR2 pathway and Th17 cell polarization may be due to HMGB1 stimulation in rheumatoid arthritis.     

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

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.     

Elevated circulating Th17 and follicular helper CD4+ T cells in patients with rheumatoid arthritis

It remains not fully elucidated the potential functions of Th17 cells and follicular helper T (Tfh) cells and secreting cytokines in the pathogenesis of rheumatoid arthritis (RA) and their association with disease activity. In this study, the frequencies of Th17 and Tfh cells were determined by flow cytometry, and the levels of interleukin (IL)‐17, IL‐21, and IL‐22 were measured by ELISA in RA patients with different disease activities. The dynamic changes of cell subsets were also detected in response to disease‐modify antirheumatic drugs (DMARDs) therapy. The percentages of CD3⁺CD4⁺IL‐17A⁺ (Th17) cells and CD3⁺CD4⁺CXCR5⁺ICOS^high (Tfh) cells, as well as the concentrations of IL‐17, IL‐21, and IL‐22 were significantly elevated in RA patients than those in healthy individuals. Furthermore, Tfh cells, IL‐21, and IL‐22 in the serum was positively correlated with the values of disease activity score. Concentrations of IL‐21 and IL‐22 in the serum were remarkably reduced following the DMARDs therapies. Our data suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL‐21 and IL‐22 were associated with the disease activity of RA patients, and might be potential therapeutic targets for treatment of RA.     

Relationship of CD146 expression to secretion of interleukin (IL)‐17, IL‐22 and interferon‐γ by CD4+ T cells in patients with inflammatory arthritis

Expression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4⁺ T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146⁺CD4⁺ and interleukin (IL)‐17⁺CD4⁺ T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146⁺CD4⁺ T cells were enriched for secretion of IL‐17 [alone or with IL‐22 or interferon (IFN)‐γ] and for some putative Th17‐associated surface markers (CD161 and CCR6), but not others (CD26 and IL‐23 receptor). CD4⁺ T cells producing IL‐22 or IFN‐γ without IL‐17 were also present in the CD146⁺ subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4⁺ helper T cells.