The pruritogenic mediator endothelin‐1 shifts the dendritic cell–T‐cell response toward Th17/Th1 polarization

Endothelin‐1 (ET‐1) is associated with skin diseases such as atopic dermatitis (AD) and psoriasis. ET‐1 is enhanced in the skin of patients AD and psoriasis. In addition, plasma levels of ET‐1 are elevated in AD and psoriasis. Although both AD and psoriasis are T‐cell–mediated skin diseases, the association between ET‐1 and the T‐cell immune response has not been clarified. To evaluate the role of ET‐1 in inflammatory skin disease, we sought to investigate the effects of ET‐1 on the functions of dendritic cells (DCs) and subsequent immune responses. For this purpose, we immunohistochemically confirmed the upregulation of ET‐1 in the epidermis of patients with AD or psoriasis. ET‐1 directly induced phenotypic maturation of bone marrow‐derived DCs (BMDCs). In addition, ET‐1 augmented the production of several cytokines and allogeneic stimulatory capacity of BMDCs. Interestingly, ET‐1–activated BMDCs primed T cells to produce Th1 and Th17 cytokines, but not Th2 cytokines. These findings indicate that ET‐1 polarizes the DC–T‐cell response toward Th17/1 differentiation and may augment the persistent course of inflammatory skin diseases.     

Tolerogenic dendritic cells produced by lentiviral‐mediated CD40‐ and interleukin‐23p19‐specific shRNA can ameliorate experimental autoimmune encephalomyelitis by suppressing T helper type 17 cells

Down‐regulation of soluble or membrane‐bound co‐stimulatory molecules by RNAi in dendritic cells can prevent the activation of immune responses. Therefore, this study was designed to evaluate the therapeutic efficacy of bone marrow‐derived DCs (BMDCs) transduced with lentiviral vectors to permanently expressed shRNA specific for CD40 (CD40LV‐DCs) and/or p19 subunit of interleukin (IL)‐23 (p19LV‐DCs) mRNAs in experimental autoimmune encephalomyelitis (EAE). In‐vitro studies showed that double‐transduced BMDCs (CD40⁺p19LV‐DCs) resemble tolerogenic DCs due to profound down‐regulation of CD40, lower expression of proinflammatory cytokines (IL‐6 and IL‐12), increased IL‐10 production and stronger stimulation of myelin oligodendrocyte glycoprotein (MOG)_35–55‐specific T cells for production of IL‐10 compared with CD40LV‐DCs, p19LV‐DCs and BMDCs transduced with control lentiviral vector (CoLV‐DCs). Moreover, injection of transduced CD40⁺p19LV‐ BMDCs in EAE mice resulted in more reduction in clinical score, significant reduction in IL‐17 or increased production of IL‐10 by mononuclear cells derived from the lymph nodes or spinal cord compared with CoLV‐DCs‐treated EAE mice. In conclusion, simultaneous knock‐down of CD40 and IL‐23 production by BMDCs may represent a promising therapeutic tool for the treatment of IL‐17‐dependent autoimmune diseases, including multiple sclerosis.     

Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection

Autophagy can mediate antiviral immunity. However, it remains unknown whether autophagy regulates the immune response of dendritic cells (DCs) to influenza A (H1N1) pdm09 infection. In this study, we found that infection with the H1N1 virus induced DC autophagy in an endocytosis‐dependent manner. Compared with autophagy‐deficient Beclin‐1^+/? mice, we found that bone‐marrow‐derived DCs from wild‐type mice (WT BMDCs) presented a more mature phenotype on H1N1 infection. Wild‐type BMDCs secreted higher levels of interleukin‐6 (IL‐6), tumour necrosis factor‐ α (TNF‐α), interferon‐β (IFN‐β), IL‐12p70 and IFN‐γ than did Beclin‐1^+/? BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs exhibited increased activation of extracellular signal‐regulated kinase, Jun N‐terminal kinase, p38, and nuclear factor‐κB as well as IFN regulatory factor 7 nuclear translocation. Blockade of autophagosomal and lysosomal fusion by bafilomycin A1 decreased the co‐localization of H1N1 viruses, autophagosomes and lysosomes as well as the secretion of IL‐6, TNF‐α and IFN‐β in H1N1‐infected BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs were more efficient in inducing allogeneic CD4⁺ T‐cell proliferation and driving T helper type 1, 2 and 17 cell differentiation while inhibiting CD4⁺ Foxp3⁺ regulatory T‐cell differentiation. Moreover, WT BMDCs were more efficient at cross‐presenting the ovalbumin antigen to CD8⁺ T cells. We consistently found that Beclin‐1^+/? BMDCs were inferior in their inhibition of H1N1 virus replication and their induction of H1N1‐specific CD4⁺ and CD8⁺ T‐cell responses, which produced lower levels of IL‐6, TNF‐α and IFN‐β in vivo. Our data indicate that autophagy is important in the regulation of the DC immune response to H1N1 infection, thereby extending our understanding of host immune responses to the virus.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

Protein tyrosine phosphatase PTPN22 regulates IL‐1β dependent Th17 responses by modulating dectin‐1 signaling in mice

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno‐receptors. Fungal β‐glucan receptor dectin‐1 signals via Syk, and dectin‐1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin‐1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22^?/? mutant mice, were pulsed with OVA_323‐339 and the dectin‐1 agonist curdlan and co‐cultured in vitro with OT‐II T‐cells or adoptively transferred into OT‐II mice, and T‐cell responses were determined by immunoassay. Dectin‐1 activated Ptpn22^?/? BMDCs enhanced T‐cell secretion of IL‐17 in vitro and in vivo in an IL‐1β dependent manner. Immunoblotting revealed that compared to WT, dectin‐1 activated Ptpn22^?/? BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin‐1 activation of BMDCs expressing Ptpn22^R619W (the mouse orthologue of human PTPN22^R620W) also resulted in increased IL‐1β secretion and T‐cell dependent IL‐17 responses, indicating that in the context of dectin‐1 Ptpn22^R619W operates as a loss‐of‐function variant. These findings highlight PTPN22 as a novel regulator of dectin‐1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.     

Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed‐infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T‐cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi‐maturation of human monocytes‐derived DCs (moDCs), and elevated levels of IL‐10, IL‐27 and IL‐6 but not IL‐12p70 and TNF‐α. Consistently, HMOS‐conditioned human moDCs promoted Treg generation from naïve CD4⁺ T cells. Interestingly, HMOS limited LPS‐induced maturation of human moDCs, while maintained IL‐10 and IL‐27 secretion and reduced LPS‐induced production of IL‐12p70, IL‐6 and TNF‐α. Furthermore, HMOS+LPS‐stimulated DCs induced a higher frequency of Tregs and increased IL‐10 production, while a reduction in Tbet+Th1 frequency and IFN‐γ production was detected as compared to LPS‐DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC‐SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.     

IL‐25 exhibits disparate roles during Th2‐cell differentiation versus effector function

A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL‐25 has been shown to enhance Th2‐type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL‐25^?/? C57BL/6 mice with G4 IL‐4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL‐4‐independent Th2‐cell differentiation. These assays were used to determine whether IL‐25 was critical for the formation of Th2 cells. We found there was no physiological role for IL‐25 in either the differentiation of Th2 cells or their development to effector or memory Th2‐cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL‐25 and its proposed role in promoting Th2‐cell responses.     

Co‐operative suppression of inflammatory responses in human dendritic cells by plant proanthocyanidins and products from the parasitic nematode Trichuris suis

Interactions between dendritic cells (DCs) and environmental, dietary and pathogen antigens play a key role in immune homeostasis and regulation of inflammation. Dietary polyphenols such as proanthocyanidins (PAC) may reduce inflammation, and we therefore hypothesized that PAC may suppress lipopolysaccharide (LPS) ‐induced responses in human DCs and subsequent T helper type 1 (Th1) ‐type responses in naive T cells. Moreover, we proposed that, because DCs are likely to be exposed to multiple stimuli, the activity of PAC may synergise with other bioactive molecules that have anti‐inflammatory activity, e.g. soluble products from the helminth parasite Trichuris suis (TsSP). We show that PAC are endocytosed by monocyte‐derived DCs and selectively induce CD86 expression. Subsequently, PAC suppress the LPS‐induced secretion of interleukin‐6 (IL‐6) and IL‐12p70, while enhancing secretion of IL‐10. Incubation of DCs with PAC did not affect lymphocyte proliferation; however, subsequent interferon‐γ production was markedly suppressed, while IL‐4 production was unaffected. The activity of PAC was confined to oligomers (degree of polymerization ≥ 4). Co‐pulsing DCs with TsSP and PAC synergistically reduced secretion of tumour necrosis factor‐α, IL‐6 and IL‐12p70 while increasing IL‐10 secretion. Moreover, both TsSP and PAC alone induced Th2‐associated OX40L expression in DCs, and together synergized to up‐regulate OX40L. These data suggest that PAC induce an anti‐inflammatory phenotype in human DCs that selectively down‐regulates Th1 response in naive T cells, and that they also act cooperatively with TsSP. Our results indicate a novel interaction between dietary compounds and parasite products to influence immune function, and may suggest that combinations of PAC and TsSP can have therapeutic potential for inflammatory disorders.     

T‐cell‐derived,but not B‐cell‐derived,IL‐10 suppresses antigen‐specific T‐cell responses in Litomosoides sigmodontis‐infected mice

IL‐10, a cytokine with pleiotropic functions is produced by many different cells. Although IL‐10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth‐induced immune evasion. Here, we show that B cells and T cells produce IL‐10 during murine Litomosoides sigmodontis infection. IL‐10‐deficient mice produced increased amounts of L. sigmodontis‐specific IFN‐γ and IL‐13 suggesting a suppressive role for IL‐10 in the initiation of the T‐cell response to infection. Using cell type‐specific IL‐10‐deficient mice, we dissected different functions of T‐cell‐ and B‐cell‐derived IL‐10. Litomosoides sigmodontis‐specific IFN‐γ, IL‐5, and IL‐13 production increased in the absence of T‐cell‐derived IL‐10 at early and late time points of infection. In contrast, B‐cell‐specific IL‐10 deficiency did not lead to significant changes in L. sigmodontis‐specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag‐specific cellular responses during L. sigmodontis infection is suppressed by T‐cell‐derived IL‐10 and not by B‐cell‐derived IL‐10.     

Exosomes from heat‐stressed tumour cells inhibit tumour growth by converting regulatory T cells to Th17 cells via IL‐6

Exosomes derived from heat‐stressed tumour cells (HS‐TEXs), which contain abundant heat shock protein (HSP) 70, strongly induce antitumour immune responses. HSP70‐induced interleukin (IL)‐6 promotes IL‐17 expression and causes rejection of established prostate tumours. However, it remains unclear whether HS‐TEXs exhibit antitumour effects by converting regulatory T cells (T_regs) into T helper type 17 (Th17) cells. In this study, we found that compared with TEXs, HS‐TEXs were more potent in stimulating secretion of IL‐6 from dendritic cells. In vitro, IL‐6 blocked tumour cell‐derived transforming growth factor beta 1‐induced T_reg differentiation and promoted Th17 cell differentiation. HS‐TEXs exerted strong antitumour effects, converting T_regs into Th17 cells with high efficiency, a process that was entirely dependent upon IL‐6. Neutralization of IL‐17 completely abolished the antitumour effect of TEXs, but only partially inhibited that of HS‐TEXs. In addition, we found higher levels of IL‐6 and IL‐17 in serum from tumour patients treated with hyperthermia, and an increase in Th17 cells and a decrease in T_regs was detected in peripheral blood mononuclear cells isolated from these patients after hyperthermia. Therefore, our results demonstrate that HS‐TEXs possess a powerful capacity to convert immunosuppressive T_regs into Th17 cells via IL‐6, which contributes to their potent antitumour effect.     

Activation of the aryl hydrocarbon receptor affects activation and function of human monocyte‐derived dendritic cells

Aryl hydrocarbon receptor (AhR) is well known for mediating the toxic effects of dioxin‐containing pollutants, but has also been shown to be involved in the natural regulation of the immune response. In this study, we investigated the effect of AhR activation by its endogenous ligands 6‐formylindolo[3,2‐b]carbazole (FICZ) and 2‐(1′H‐indole‐3′‐carbonyl)‐thiazole‐4‐carboxylic acid methyl ester (ITE) on the differentiation, maturation and function of monocyte‐derived DCs in Behçet's disease (BD) patients. In this study, we showed that AhR activation by FICZ and ITE down‐regulated the expression of co‐stimulatory molecules including human leucocyte antigen D‐related (HLA‐DR), CD80 and CD86, while it had no effect on the expression of CD83 and CD40 on DCs derived from BD patients and normal controls. Lipopolysaccharide (LPS)‐treated dendritic cells (DCs) from active BD patients showed a higher level of interleukin (IL)‐1β, IL‐6, IL‐23 and tumour necrosis factor (TNF)‐α production. FICZ or ITE significantly inhibited the production of IL‐1β, IL‐6, IL‐23 and TNF‐α, but induced IL‐10 production by DCs derived from active BD patients and normal controls. FICZ or ITE‐treated DCs significantly inhibited the T helper type 17 (Th17) and Th1 cell response. Activation of AhR either by FICZ or ITE inhibits DC differentiation, maturation and function. Further studies are needed to investigate whether manipulation of the AhR pathway may be used to treat BD or other autoimmune diseases.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

Mannan Derivatives Instruct Dendritic Cells to Induce Th1/Th2 Cells Polarization via Differential Mitogen‐Activated Protein Kinase Activation

Mannan derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Immunization with antigen conjugated to oxidized mannan (OM) or reduced mannan (RM) have induced differential immune responses in mice. Yet, the adjuvant effect and differences in molecular profiles of OM and RM on APCs is unresolved. Here, we investigated the response of mouse bone marrow‐derived DCs to OM and RM. OM and RM stimulated DCs to produce differential Th1/Th2‐inducing cytokines in vitro. OM and RM‐activated DCs stimulated allogeneic T‐cell Th1 and Th2 polarization reaction. OM instruct DCs to stimulate Th1 responses via IL‐12p70 production, which depends on the phosphorylation of p38, RM barely induce IL‐12p70, but IL‐10 and IL‐4, and magnitude of ERK phosphorylation, which results in a Th2 bias. These findings indicate that OM and RM were potent adjuvant capable of directly initiating DC activation Th1 and Th2 polarization respectively.     

TSLP production by dendritic cells is modulated by IL‐1β and components of the endoplasmic reticulum stress response

Thymic stromal lymphopoietin (TSLP) produced by epithelial cells acts on dendritic cells (DCs) to drive differentiation of T_H2‐cells, and is therefore important in allergic disease pathogenesis. However, DCs themselves make significant amounts of TSLP in response to microbial products, but little is known about the key downstream signals that induce and modulate this TSLP secretion from human DCs. We show that human monocyte derived DC (mDC) secretion of TSLP in response to Candida albicans and β‐glucans requires dectin‐1, Syk, NF‐κB, and p38 MAPK signaling. In addition, TSLP production by mDCs is greatly enhanced by IL‐1β, but not TNF‐α, in contrast to epithelial cells. Furthermore, TSLP secretion is significantly increased by signals emanating from the endoplasmic reticulum (ER) stress response, specifically the unfolded protein response sensors, inositol‐requiring transmembrane kinase/endonuclease 1 and protein kinase R‐like ER kinase, which are activated by dectin‐1 stimulation. Thus, TSLP production by mDCs requires the integration of signals from dectin‐1, the IL‐1 receptor, and ER stress signaling pathways. Autocrine TSLP production is likely to play a role in mDC‐controlled immune responses at sites removed from epithelial cell production of the cytokine, such as lymphoid tissue.     

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.