TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17⁺cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c⁺/langerin⁺ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.     

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.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

TGF‐β indirectly favors the development of human Th17 cells by inhibiting Th1 cells

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti‐proliferative effect of TGF‐β than Th1 and Th2 clones or circulating Th1‐oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl‐2 expression and reduced apoptosis in the presence of TGF‐β, in comparison with Th1 cells. Umbilical cord blood naïve CD161⁺CD4⁺ T cells, which contain the precursors of human Th17 cells, differentiated into IL‐17A‐producing cells only in response to IL‐1β plus IL‐23, even in serum‐free cultures. TGF‐β had no effect on constitutive RORγt expression by umbilical cord blood CD161⁺ T cells but it increased the relative proportions of CD161⁺ T cells differentiating into Th17 cells in response to IL‐1β plus IL‐23, whereas under the same conditions it inhibited both T‐bet expression and Th1 development. These data suggest that TGF‐β is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.     

Loss of regulatory characteristics in CD4+CD25+/hi T cells induced by impaired transforming growth factor beta secretion in pneumoconiosis

Pneumoconiosis is caused by the accumulation of airborne dust in the lung, which stimulates a progressive inflammatory response that ultimately results in lung fibrosis and respiratory failure. It is possible that regulatory cells in the immune system could function to suppress inflammation and possibly slow or reverse disease progression. However, results in this study suggest that in pneumoconiosis patients, the regulatory T cells (Tregs) and B cells are functionally impaired. First, we found that pneumoconiosis patients presented an upregulation of CD4⁺CD25⁺ T cells compared to controls, whereas the CD4⁺CD25⁺ and CD4⁺CD25^hi T cells were enriched with Th1‐ and Th17‐like cells but not Foxp3‐expressing Treg cells and evidenced by significantly higher T‐bet, interferon (IFN)‐γ, and interleukin (IL)‐17 expression but lower Foxp3 and transforming growth factor (TGF)‐β expression. Regarding the CD4⁺CD25^hi T‐cell subset, the frequency of this cell type in pneumoconiosis patients was significantly reduced compared to controls, together with a reduction in Foxp3 and TGF‐β and an enrichment in T‐bet, RORγt, IFN‐γ, and IL‐17. This skewing toward Th1 and Th17 types of inflammation could be driven by monocytes and B cells, since after depleting CD14⁺ monocytes and CD19⁺ B cells, the levels of IFN‐γ and IL‐17 were significantly decreased. Whole peripheral blood mononuclear cells and isolated monocytes and B cells in pneumoconiosis patients also presented reduced capacity of TGF‐β secretion. Furthermore, monocytes and B cells from pneumoconiosis patients presented reduced capacity in inducing Foxp3 upregulation, a function that could be rescued by exogenous TGF‐β. Together, these data indicated a potential pathway for the progression of pneumoconiosis through a loss of Foxp3⁺ Treg cells associated with impaired TGF‐β secretion.     

Role of CD4 T cell helper subsets in immune response and deviation of CD8 T cells in mice*

The ability of different CD4⁺ T cell subsets to help CD8⁺ T‐cell response is not fully understood. Here, we found using the murine system that Th17 cells induced by IL‐1β, unlike Th1, were not effective helpers for antiviral CD8 responses as measured by IFNγ‐producing cells or protection against virus infection. However, they skewed CD8 responses to a Tc17 phenotype. Thus, the apparent lack of help was actually immune deviation. This skewing depended on both IL‐21 and IL‐23. To overcome this effect, we inhibited Th17 induction by blocking TGF‐β. Anti‐TGF‐β allowed the IL‐1β adjuvant to enhance CD8⁺ T‐cell responses without skewing the phenotype to Tc17, thereby providing an approach to harness the benefit of common IL‐1‐inducing adjuvants like alum without immune deviation.     

Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells

Summary: Th3 CD4⁺ regulatory cells were identified during the course of investigating mechanisms associated with oral tolerance. Different mechanisms of tolerance are induced following oral antigen administration, including active suppression, clonal anergy and deletion. Low doses favor active suppression whereas high doses favor anergy/deletion. Th3 regulatory cells form a unique T‐cell subset which primarily secretes transforming growth factor (TGF)‐β, provides help for IgA and has suppressive properties for both Th1 and Th2 cells. Th3 type cells are distinct from the Th2 cells, as CD4⁺ TGF‐β‐secreting cells with suppressive properties have been generated from interleukin (IL)‐4‐deficient animals. In vitro differentiation of Th3 cells from Th precursors from T‐cell antigen receptor (TCR) transgenic mice is enhanced by culture with TGF‐β, IL‐4, IL‐10, and anti‐IL‐12. Th3 CD4⁺ myelin basic protein regulatory clones are structurally identical to Th1 encephalitogenic clones in TCR usage, MHC restriction and epitope recognition, but produce TGF‐β with various amounts of IL‐4 and IL‐10. Because Th3 regulatory cells are triggered in an antigen‐specific fashion but suppress in an antigen‐non‐specific fashion, they mediate “bystander suppression” when they encounter the fed autoantigen at the target organ. In vivo induction of Th3 cells and low dose oral tolerance is enhanced by oral administration of IL‐4. Anti‐CD86 but not anti‐CD80 blocks the induction of Th3 cells associated with low dose oral tolerance. Th3 regulatory cells have been described in other systems (e.g. recovery from experimental allergic encephalomyelitis) but may be preferentially generated following oral antigen administration due to the gut immunologic milieu that is rich in TGF‐β and has a unique class of dendritic cells. CD4⁺CD25⁺ regulatory T‐cell function also appears related to TGF‐β.     

Commensal A4 bacteria inhibit intestinal Th2‐cell responses through induction of dendritic cell TGF‐β production

It has been shown that while commensal bacteria promote Th1, Th17 and Treg cells in lamina propria (LP) in steady‐state conditions, they suppress mucosal Th2 cells. However, it is still unclear whether there are specific commensal organisms down‐regulating Th2 responses, and the mechanism involved. Here we demonstrate that commensal A4 bacteria, a member of the Lachnospiraceae family, which produce an immunodominant microbiota CBir1 antigen, inhibits LP Th2‐cell development. When transferred into the intestines of RAG^?/? mice, CBir1‐specific T cells developed predominately towards Th1 cells and Th17 cells, but to a lesser extent into Th2 cells. The addition of A4 bacterial lysates to CD4⁺ T‐cell cultures inhibited production of IL‐4. A4 bacteria stimulated dendritic cell production of TGF‐β, and blockade of TGF‐β abrogated A4 bacteria inhibition of Th2‐cell development in vitro and in vivo. Collectively, our data show that A4 bacteria inhibit Th2‐cell differentiation by inducing dendritic cell production of TGF‐β.     

Role of T cell TGF‐β signaling in intestinal cytokine responses and helminthic immune modulation

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL‐10 or TGF‐β, that are important in suppressing colitis. Helminths induce mucosal T cell IL‐10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL‐10‐dependent manner in WT mice. Helminths also stimulate mucosal TGF‐β release. As TGF‐β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF‐β signaling in helminthic modulation of intestinal immunity. T cell TGF‐β signaling is interrupted in TGF‐β receptor II dominant negative (TGF‐βRII DN) mice by T‐cell‐specific over‐expression of a TGF‐βRII DN. We studied LPMC responses in WT and TGF‐βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF‐β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL‐10 secretion requires intact T cell TGF‐β‐signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF‐βRII DN mice. Thus, T cell TGF‐β signaling is essential for helminthic stimulation of mucosal IL‐10 production, helminthic modulation of intestinal IFN‐γ generation and H. polygyrus‐mediated suppression of chronic colitis.     

In vitro‐induced Th17 cells fail to induce inflammation in vivo and show an impaired migration into inflamed sites

Recently, IL‐17 produced by Th17 cells was described as pro‐inflammatory cytokine with an eminent role in autoimmune diseases, e.g. rheumatoid arthritis. A lack of IL‐17 leads to amelioration of collagen‐induced arthritis. IL‐17 induction in naïve CD4⁺ T cells depends on IL‐6 and TGF‐β and is enhanced by IL‐23. The in vivo inflammatory potential of in vitro‐primed Th17 cells however, remains unclear. Here, we show that, although IL‐17 neutralisation results in amelioration of murine OVA‐induced arthritis, in vitro‐primed Th17 cells cannot exacerbate arthritic symptoms after adoptive transfer. Furthermore, Th17 cells cannot induce an inflammatory delayed type hypersensitivity reaction because they fail to migrate into inflamed sites, possibly due to the lack of CXCR3 expression. Also, re‐isolated Th17 cells acquired IFN‐γ expression, indicating instability of the Th17 phenotype. Taken together, the data show that IL‐6, TGF‐β and IL‐23 might not provide sufficient signals to induce “fully qualified” Th17 cells.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

Curcumin induces the tolerogenic dendritic cell that promotes differentiation of intestine‐protective regulatory T cells

The gut is home to a large number of Treg, with both CD4⁺ CD25⁺ Treg and bacterial antigen‐specific Tr1 cells present in normal mouse intestinal lamina propria. It has been shown recently that intestinal mucosal DC are able to induce Foxp3⁺ Treg through production of TGF‐β plus retinoic acid (RA). However, the factors instructing DC toward this mucosal phenotype are currently unknown. Curcumin has been shown to possess a number of biologic activities including the inhibition of NF‐κB signaling. We asked whether curcumin could modulate DC to be tolerogenic whose function could mimic mucosal DC. We report here that curcumin modulated BM‐derived DC to express ALDH1a and IL‐10. These curcumin‐treated DC induced differentiation of naïve CD4⁺ T cells into Treg resembling Treg in the intestine, including both CD4⁺CD25⁺ Foxp3⁺ Treg and IL‐10‐producing Tr1 cells. Such Treg induction required IL‐10, TGF‐β and retinoic acid produced by curcumin‐modulated DC. Cell contact as well as IL‐10 and TGF‐β production were involved in the function of such induced Treg. More importantly, these Treg inhibited antigen‐specific T‐cell activation in vitro and inhibited colitis due to antigen‐specific pathogenic T cells in vivo.     

Induction, function and regulation of IL-17-producing T cells

Recent reports have provided convincing evidence that IL‐17‐producing T cells play a key role in the pathogenesis of organ‐specific autoimmune diseases, a function previously attributed exclusively to IFN‐γ‐secreting Th1 cells. Furthermore, it appears that IL‐17‐producing T cells can also function with Th1 cells to mediate protective immunity to pathogens. Although much of the focus has been on IL‐17‐secreting CD4⁺ T cells, termed Th17 cells, CD8⁺ T cells, γδ T cells and NKT cells are also capable of secreting IL‐17. The differentiation of Th17 cells from naïve T cells appears to involve signals from TGF‐β, IL‐6, IL‐21, IL‐1β and IL‐23. Furthermore, IL‐1α or IL‐1β in synergy with IL‐23 can promote IL‐17 secretion from memory T cells. The induction or function of Th17 cells is regulated by cytokines secreted by the other major subtypes of T cells, including IFN‐γ, IL‐4, IL‐10 and at high concentrations, TGF‐β. The main function of IL‐17‐secreting T cells is to mediate inflammation, by stimulating production of inflammatory cytokines, such as TNF‐α, IL‐1β and IL‐6, and inflammatory chemokines that promote the recruitment of neutrophils and macrophages.     

Essential and synergistic roles of IL1 and IL6 in human Th17 differentiation directed by TLR ligand-activated dendritic cells

Requirements for human Th17 differentiation in the context of activated dendritic cells (DCs) are still emerging. Here, we demonstrate that several Toll-like receptor (TLR) ligands, particularly LPS and a synthetic lipoprotein, activate human DCs to direct increased human Th17 differentiation. Based on neutralization studies, IL1, IL6, and TGFβ contributed to human Th17 differentiation induced by LPS-activated DCs. Furthermore, TLR ligand-activated DCs produced high levels of IL6 and low levels of IL1β. In an antigen presenting cell (APC)-free system, the minimum requirements identified for human Th17 differentiation from adult naive CD4⁺ T cells, depleted of CD25⁺ cells, were TGFβ and high levels of IL1β. However, in the presence of the physiologically low levels of IL1 such as those produced by DCs, both TGFβ and IL6 were also essential. These results help to explain the conflicting reports in the literature on the roles of IL1 and IL6 on human Th17 differentiation.     

Synergistic effect of TGF‐β superfamily members on the induction of Foxp3+ Treg

TGF‐β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF‐β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF‐β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP‐2 and ‐4, are unable to induce non‐regulatory T cells to become Foxp3⁺ Treg. Neutralization studies with Noggin have revealed that BMP‐2/4 and the BMP receptor signaling pathway is not required for TGF‐β to induce naïve CD4⁺CD25^? cells to express Foxp3; however, BMP‐2/4 and TGF‐β have a synergistic effect on the induction of Foxp3⁺ Treg. BMP‐2/4 affects non‐Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ Treg induced by TGF‐β. Data further advocate that TGF‐β is a key signaling factor for Foxp3⁺ Treg development. In addition, the synergistic effect of BMP‐2/4 and TGF‐β indicates that the simultaneous manipulation of TGF‐β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.