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.     

MicroRNA223 promotes pathogenic T‐cell development and autoimmune inflammation in central nervous system in mice

Multiple sclerosis (MS) is an incurable central nervous system autoimmune disease. Understanding MS pathogenesis is essential for the development of new MS therapies. In the present study, we identified a novel microRNA (miR) that regulates experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression of miR223 was up‐regulated specifically in spinal cords and lymphoid organs but not in other examined tissues. A global miR223 knockout (miR223^?/?) in mice led to a significant delay in EAE onset, reduction in spinal cord lesion, and lessening of neurological symptoms. These protective effects could be reproduced in bone marrow chimeras reconstituted with miR223^?/? haematopoietic stem cells. We also found that miR223 deficiency reduced T helper type 1 (Th1) and Th17 infiltration into spinal cords. To address underlying mechanisms, we investigated the role of miR223 in regulating the function, development and interaction of the major immune cells. Expression of the genes associated with dendritic cell (DC) activation (CD86 and MHC II) and Th1 and Th17 differentiation [interleukin‐12 (IL‐12) and IL‐23, respectively] was significantly decreased in the spleens of miR223^?/? mice bearing EAE. The miR223^?/? DCs expressed significantly lower levels of basal and lipopolysaccharide‐induced IL‐12 and IL‐23 compared with the wild‐type DCs. These data are consistent with the observed lower efficiency of miR223^?/? DCs to support Th1 and Th17 differentiation from naive T cells over‐expressing an EAE antigen‐specific T‐cell receptor. Our data suggest that miR223 promotes EAE, probably through enhancing DC activation and subsequently the differentiation of naive T cells toward Th1 and Th17 effector cells.     

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen‐specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcγ receptor (FcγR)‐mediated antigen uptake and enhance antigen presentation resulting in augmented T‐cell proliferation. We examined the impact of antigen presentation and T‐cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene‐deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcγR‐deficient mice. Lung DC of WT, but not FcγR‐deficient mice, induced increased antigen‐specific CD4⁺ T‐cell proliferation when pulsed with anti‐OVA IgG immune complexes. Intranasal application of anti‐OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen‐specific T‐cell proliferation in vivo. Finally, antigen‐specific IgG in the serum of sensitized mice led to a significant increase of antigen‐specific CD4⁺ T‐cell proliferation induced by WT, but not FcγR‐deficient, lung DC. We conclude that FcγR‐mediated enhanced antigen presentation and T‐cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.     

Regulatory role of DC‐derived osteopontin in systemic allergen sensitization

Osteopontin (OPN) is a secreted phosphoglycoprotein with a wide range of functions, and is involved in various pathophysiological conditions. However, the role of OPN in IgE and Th2‐associated allergic responses remains incompletely defined. The aim of this study was to elucidate the role of OPN in systemic allergen sensitization in mice. When compared with OPN^+/+ mice, significantly increased levels of OVA‐induced IgE were found in OPN^?/? mice. OPN^?/? DC demonstrated an increased capacity to enhance Th2 cytokine production in CD4⁺ T cells from sensitized OPN^+/+ mice. Furthermore, significantly reduced levels of IL‐12p70 expression were seen in LPS‐stimulated OPN^?/? DC as compared with the WT DC, and the reduction was reversible by the addition of recombinant OPN (rOPN). rOPN was able to suppress OVA‐induced IL‐13 production in the cultures of CD4 and OPN^?/? DC, but this inhibitory activity was neutralized by the addition of anti‐IL‐12 Ab. In addition, administration of rOPN in vivo suppressed OVA‐specific IgE production; however, this suppressive effect was abrogated in IL‐12‐deficient mice. These results indicate that DC‐derived OPN plays a regulatory role in the development of systemic allergen sensitization, which is mediated, at least in part, through the production of endogenous IL‐12.     

Administration of polysaccharides from Antrodia camphorata modulates dendritic cell function and alleviates allergen‐induced T helper type 2 responses in a mouse model of asthma

Asthma is a chronic disease characterized by airway inflammation caused by the dysregulated production of cytokines secreted by allergen‐specific type 2 T helper (Th2) cells. Antrodia camphorata is a commonly used fungus in Asian folk medicine, and A. camphorata polysaccharides are reported to possess anti‐cancer activities. In this study, the immunomodulatory effects of purified fractionated polysaccharides (GF2) from A. camphorata on dendritic cells (DCs) and their potential preventive effects against ovalbumin (OVA) ‐induced asthma were investigated. In the presence of GF2, lipopolysaccharide (LPS) ‐activated DCs exhibited up‐regulated expression of major histocompatibility complex (MHC) class II and co‐stimulatory molecules, as well as enhanced interleukin‐10 (IL‐10) and IL‐12 production. GF2 treatment on LPS‐activated DCs suppressed naïve CD4⁺ T‐cell proliferation and Th2 cell polarization with IL‐10 production in an allogeneic mixed lymphocyte reaction. In animal experiments, a high dose of GF2 efficiently reduced expression levels of OVA‐specific immunoglobulin G1 (IgG1) and IgE. However, lower doses of GF2 significantly enhanced OVA‐specific IgG2a production. Our data also showed that administration of GF2 dose‐dependently inhibited the development of airway hyperresponsiveness, airway eosinophilia and Th2 responses. OVA‐specific CD4⁺ T cells from higher doses of GF2‐treated mice had significantly lower proliferative capacities compared with control mice. Moreover, treatment with GF2 significantly increased the high levels of IL‐10 and low levels of interferon‐γ produced by T cells. Taken together, these data indicate that administration of A. camphorata polysaccharides (GF2) may have therapeutic potential when used as an adjuvant for the immunomodulatory treatment of allergic asthma.     

Suppressor of cytokine signalling 1 (SOCS1) is a physiological regulator of the asthma response

Background The molecular determinants of the severity and persistence of allergic asthma remain poorly understood. Suppressor of cytokine signalling 1 (SOCS1) is a negative regulator of IL‐4‐dependent pathways in vitro and might therefore control T‐helper type 2 (Th2) immunity associated traits, such as IgE levels, mucin production, IL‐5 and IL‐13 induction, and eosinophilic mucosal inflammation, which are implicated in allergic asthma. Objective To investigate the role of SOCS1 in regulating Th2‐associated disease traits in a murine sub‐chronic aeroallergen‐driven asthma model. Methods Following sensitization and challenge with ovalbumin (OVA), bronchoalveolar lavage and serum were collected from mice lacking the Socs1 gene on an IFN‐γ null background (Socs1^?/?Ifnγ^?/?). The composition of infiltrating cells in the lung, serum IgE and IgG1 levels and cytokine levels were analysed. Results Serum IgE levels and infiltrating eosinophils were considerably increased in the lungs of OVA‐treated Socs1^?/?Ifnγ^?/? mice compared with Ifnγ^?/? and C57BL/6 controls. Expression of the Th2 cytokines, IL‐4, IL‐5 and IL‐13 was increased in CD4⁺ cells and lung tissue from OVA‐treated Socs1^?/?Ifnγ^?/? mice. IgE, IL‐5 levels and infiltrating eosinophils were also elevated in saline‐treated Socs1^?/?Ifnγ^?/? mice, suggesting that in the absence of SOCS1, mice are already biased towards a Th2 response. It is at present unclear whether the elevated cytokine levels are sufficient to result in the exacerbated Th2 response to OVA challenge or whether enhanced intra‐cellular signalling also contributes. Surprisingly, of the various IL‐4/IL‐13 responsive genes tested, only Arginase I appeared to be modestly up‐regulated in the lungs of OVA‐treated Socs1^?/?Ifnγ^?/? mice, suggesting that regulation by SOCS1 occurs primarily in haematopoietic cells and not in the airway epithelium. Conclusions Together these results indicate that SOCS1 is an important regulator of the Th2 response.     

T cells are necessary for ILC2 activation in house dust mite‐induced allergic airway inflammation in mice

Allergic asthma is a chronic inflammation of the airways mediated by an adaptive type 2 immune response. Upon allergen exposure, group 2 innate lymphoid cells (ILC2s) can be rapidly activated and represent an early innate source of IL‐5 and IL‐13. Here, we used a house dust mite (HDM)‐driven asthma mouse model to study the induction of ILC2s in allergic airway inflammation. In BALF, lungs, and lymph nodes, ILC2 activation is critically dependent on prior sensitization with HDM. Importantly, T cells are required for ILC2 induction, whereby T‐cell activation precedes ILC2 induction. During HDM‐driven allergic airway inflammation the accumulation of ILC2s in BALF is IL‐33 independent, although infiltrating ILC2s produce less cytokines in Il33^?/? mice. Transfer of in vitro polarized OVA‐specific OT‐II Th2 cells alone or in combination with Th17 cells followed by OVA and HDM challenge is not sufficient to induce ILC2, despite significant eosinophilic inflammation and T‐cell activation. In this asthma model, ILC2s are therefore not an early source of Th2 cytokines, but rather contribute to type 2 inflammation in which Th2 cells play a key role. Taken together, ILC2 induction in HDM‐mediated allergic airway inflammation in mice critically depends on activation of T cells.     

Cross‐presenting dendritic cells are required for control of Leishmania major infection

Leishmania major infection induces self‐healing cutaneous lesions in C57BL/6 mice. Both IL‐12 and IFN‐γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3 ^?/? ) mice (C57BL/6 background) that lack the major IL‐12 producing and cross‐presenting CD8α⁺ and CD103⁺ DC subsets. Batf3^?/? mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3^?/? mice secreted less IFN‐γ, but more Th2‐ and Th17‐type cytokines, mirrored by increased serum IgE and Leishmania‐specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α⁺ DCs isolated from lymph nodes of L. major‐infected mice induced significantly more IFN‐γ secretion by L. major‐stimulated immune T cells than CD103⁺ DCs. We next developed CD11c‐diptheria toxin receptor: Batf3^?/? mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf‐3‐dependent DCs from day 17 or wild‐type mice depleted of cross‐presenting DCs from 17–19 days after infection maintained significantly larger lesions similar to mice whose Batf‐3‐dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf‐3‐dependent DCs in protection against L. major.     

Competition between colitogenic Th1 and Th17 cells contributes to the amelioration of colitis

Th17 cells and Th1 cells coordinate to play a critical role in the formation of inflammatory bowel diseases. To examine how Th17 and Th1 cells are regulated at inflammatory sites, we used Th1‐dominant CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? and Th1/Th17‐mixed IL‐10^?/? mice. Interestingly, not only did colitic RAG‐2^?/? mice that were parabiosed with WT mice show significant amelioration of colitis, but amelioration of disease was also observed in those parabiosed with colitic IL‐10^?/? mice. To assess the interference between Th1 and Th17 colitogenic T cells, we co‐transferred colitogenic CD4⁺ T cells from the lamina propria (LP) of CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? mice and IL‐10^?/? mice into RAG‐2^?/? mice. Surprisingly, the co‐transferred RAG‐2^?/? mice showed a vast cellular infiltration of LP CD4⁺ T cells similar to that seen in RAG‐2^?/? mice re‐transferred with the cells from colitic RAG‐2^?/? mice alone, but the co‐transferred RAG‐2^?/? mice did not have the wasting symptoms, which are also absent in RAG‐2^?/? mice transferred with cells from colitic IL‐10^?/? mice alone. Furthermore, the percentages of Th1 and Th17 cells originating from IL‐10^?/? mice and those of Th1 cells originating from colitic RAG‐2^?/? mice were all significantly decreased in the co‐transferred mice as compared with the singly‐transferred paired RAG‐2^?/? mice, suggesting that Th1 and Th17 cells are in competition, and that their orchestration results in a merged clinical phenotype of the two types of murine colitis.     

All‐trans‐retinoic acid ameliorates experimental allergic encephalomyelitis by affecting dendritic cell and monocyte development

Experimental allergic encephalomyelitis (EAE) can be induced in animal models by injecting the MOG_35–55 peptide subcutaneously. Dendritic cells (DCs) that are located at the immunization site phagocytose the MOG_35–55 peptide. These DCs mature and migrate into the nearest draining lymph nodes (dLNs), then present antigen, resulting in the activation of naive T cells. T helper type 1 (Th1) and Th17 cells are the primary cells involved in EAE progression. All‐trans‐retinoic acid (AT‐RA) has been shown to have beneficial effects on EAE progression; however, whether AT‐RA influences DC maturation or mediates other functions is unclear. In the present study, we showed that AT‐RA led to the down‐regulation of MHC class II, CD80 (B7‐1) and CD86 (B7‐2) expressed on the surface of DCs that were isolated from dLNs or spleen 3 days post‐immunization in an EAE model. Changes to DC function influenced Th1/Th17 subset polarization. Furthermore, the number of CD44⁺ monocytes (which might trigger EAE progression) was also significantly decreased in dLNs, spleen, subarachnoid space and the spinal cord parenchyma after AT‐RA treatment. These findings are the first to demonstrate that AT‐RA impairs the antigen‐presenting capacity of DCs, leading to down‐regulation of pathogenic Th1 and Th17 inflammatory cell responses and reducing EAE severity.     

A novel Flt3‐deficient HIS mouse model with selective enhancement of human DC development

Humanized mice harboring human immune systems (HIS) represent a platform to study immune responses against pathogens and to screen vaccine candidates and novel immunotherapeutics. Innate and adaptive immune responses are suboptimal in HIS mice, possibly due to poor reconstitution of human antigen‐presenting cells, including dendritic cells (DCs). DC homeostasis is regulated by cytokine availability, and Flt3‐ligand (Flt3L) is one factor that conditions this process. Mouse myelopoiesis is essentially normal in most current HIS models. As such, developing mouse myeloid cells may limit human DC reconstitution by reducing available Flt3L and by cellular competition for specific “niches.” To address these issues, we created a novel HIS model that compromises host myeloid cell development via deficiency in the receptor tyrosine kinase Flk2/Flt3. In Balb/c Rag2^?/?Il2rg^?/?Flt3^?/? (BRGF) recipients, human conventional DCs and plasmacytoid DCs develop from hCD34⁺ precursors and can be specifically boosted with exogenous Flt3L. Human DCs that develop in this context normally respond to TLR stimulation, and improved human DC homeostasis is associated with increased numbers of human NK and T cells. This new HIS‐DC model should provide a means to dissect human DC differentiation and represents a novel platform to screen immune adjuvants and DC targeting therapies.     

15‐lipoxygenase metabolites play an important role in the development of a T‐helper type 1 allergic inflammation induced by double‐stranded RNA

Background We recently demonstrated that the T‐helper type 1 (Th1) immune response plays an important role in the development of non‐eosinophilic inflammation induced by airway exposure of an allergen plus double‐stranded RNA (dsRNA). However, the role of lipoxygenase (LO) metabolites in the development of Th1 inflammation is poorly understood. Objective To evaluate the role of LO metabolites in the development of Th1 inflammation induced by sensitization with an allergen plus dsRNA. Methods A Th2‐allergic inflammation mouse model was created by an intraperitoneal injection of lipopolysaccharide‐depleted ovalbumin (OVA, 75 μg) and alum (2 mg) twice, and the Th1 model was created by intranasal application of OVA (75 μg) and synthetic dsRNA [10 μg of poly(I : C)] four times, followed by an intranasal challenge with 50 μg of OVA four times. The role of LO metabolites was evaluated using two approaches: a transgenic approach using 5‐LO^?/? and 15‐LO^?/? mice, and a pharmacological approach using inhibitors of cysteinyl leucotriene receptor‐1 (cysLTR1), LTB4 receptor (BLT1), and 15‐LO. Results We found that the Th1‐allergic inflammation induced by OVA+dsRNA sensitization was similar between 5‐LO^?/? and wild‐type (WT) control mice, although Th2 inflammation induced by sensitization with OVA+alum was reduced in the former group. In addition, dsRNA‐induced Th1 allergic inflammation, which is associated with down‐regulation of 15‐hydroxyeicosateraenoic acids production, was not affected by treatment with cysLTR1 or BLT1 inhibitors, whereas it was significantly lower in 12/15‐LO^?/? mice compared with WT control mice. Moreover, dsRNA‐induced allergic inflammation and the recruitment of T cells following an allergen challenge were significantly inhibited by treatment with a specific 15‐LO inhibitor (PD146176). Conclusion 15‐LO metabolites appear to be important mediators in the development of Th1‐allergic inflammation induced by sensitization with an allergen plus dsRNA. Our findings suggest that the 15‐LO pathway is a novel therapeutic target for the treatment of virus‐associated asthma characterized by Th1 inflammation.     

T‐cell‐specific deletion of gp130 renders the highly susceptible IL‐10‐deficient mouse resistant to intestinal nematode infection

Gp130 is the common receptor of the IL‐6 family of cytokines and is involved in many biological processes, including acute phase response, inflammation and immune reactions. To investigate the role of gp130 under inflammatory conditions, T‐cell‐specific conditional gp130 mice were first bred to the IL‐10‐deficient background and were then infected with the gastrointestinal nematode Trichuris muris. While IL‐10^?/? mice were highly susceptible to T. muris, developed a mixed Th1/Th17 response and displayed severe inflammation of the caecum, infection of mice with an additional T‐cell‐specific deletion of gp130 signalling completely reversed the phenotype. These mice showed an accelerated worm expulsion that was associated with the rapid generation of a strong Th2 immune response and a significant increase in Foxp3‐expressing Treg. Therefore, gp130 signalling in T cells regulates a switch between proinflammatory and pathogenic Th1/Th17 cells and regulatory Th2/Treg in vivo. Taken together, the data demonstrate that gp130 signalling in T cells is a positive regulator of inflammatory processes, favouring the Th1/Th17 axis.     

APRIL (TNFSF13) regulates collagen‐induced arthritis,IL‐17 production and Th2 response

A proliferation‐inducing ligand (APRIL or TNFSF13) shares receptors with B‐cell activation factor of the TNF family (BAFF) on B and T cells. Although much is known about the function of APRIL in B cells, its role in T cells remains unclear. Blocking both BAFF and APRIL suggested that BAFF and/or APRIL contributed to collagen‐induced arthritis (CIA); however, the role of APRIL alone in CIA remained unresolved. We show here that, in vitro, our newly generated APRIL^?/? mice exhibited increased T‐cell proliferation, enhanced Th2 cytokine production under non‐polarizing conditions, and augmented IL‐13 and IL‐17 production under Th2 polarizing conditions. Upon immunization with OVA and aluminum potassium sulfate, APRIL^?/? mice responded with an increased antigen‐specific IgG1 response. We also show that in APRIL^?/? mice, the incidence of CIA was significantly reduced compared with WT mice in parallel with diminished levels of antigen‐specific IgG2a autoantibody and IL‐17 production. Our data indicate that APRIL plays an important role in the regulation of cytokine production and that APRIL‐triggered signals contribute to arthritis. Blockade of APRIL thus may be a valuable adjunct in the treatment of rheumatoid arthritis.     

In vivo targeting of human DC‐SIGN drastically enhances CD8+ T‐cell‐mediated protective immunity

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T‐cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC‐SIGN (DC‐specific‐ICAM3‐grabbing‐nonintegrin/CD209) is a C‐type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC‐SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC‐SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti‐DC‐SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti‐DC‐SIGN antibodies conjugated to OVA induced strong and persistent antigen‐specific CD4⁺ and CD8⁺ T‐cell responses, which efficiently protected from infection with OVA‐expressing Listeria monocytogenes. Thus, we propose DC targeting via DC‐SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.     

c-Rel promotes type 1 and type 17 immune responses during Leishmania major infection

Recent studies demonstrated the crucial role of c‐Rel in directing Treg lineage commitment and its involvement in T helper 1 (Th1) cell‐mediated autoimmune inflammation. We thus wondered whether these opposite functions of c‐Rel influence the course of antiparasitic immune responses against Leishmania major, an accepted model for the impact of T‐cell subsets on disease outcome. Here we show that c‐Rel‐deficient (rel^?/?) mice infected with L. major displayed dramatically exacerbated leishmaniasis and enhanced parasite burdens. In contrast to WT mice, IFN‐γ and IL‐17 production in response to L. major antigens was severely impaired in rel^?/? mice. Reconstitution of Rag1^?/? T‐cell deficient mice with rel^?/? CD4⁺ T cells followed by L. major infection demonstrated that c‐Rel‐deficient T cells mount normal Th1 responses and are able to contain the infection. Similarly, Th1 differentiation of naïve CD4⁺ cells in vitro was normal. Notably, a selective defect in IL‐12 and IL‐23 production was observed in rel^?/? DCs compared with their WT counterparts. In conclusion, our data suggest that the expression of c‐Rel in myeloid cells is essential for clearance of L. major and that this c‐Rel‐mediated effect is dominant over the lack of Tregs.