HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

Increased levels of circulating soluble CD226 in multiple sclerosis.

BACKGROUND: The glycoprotein CD226 plays a key role in regulating immune cell function. Soluble CD226 (sCD226) is increased in sera of patients with several chronic inflammatory diseases but its levels in neuroinflammatory diseases such as multiple sclerosis (MS) are unknown. OBJECTIVE: To investigate the presence and functional implications of sCD226 in persons with multiple sclerosis (pwMS) and other neurological diseases. METHODS: The mechanisms of sCD226 production were first investigated by analyzing CD226 surface expression levels and supernatants of CD3/CD226-coactivated T cells. The role of sCD226 on dendritic cell maturation was evaluated. The concentration of sCD226 in the sera from healthy donors (HD), pwMS, neuromyelitis optica (NMO), and Alzheimer's disease (AD) was measured. RESULTS: CD3/CD226-costimulation induced CD226 shedding. Addition of sCD226 to dendritic cells during their maturation led to an increased production of the pro-inflammatory cytokine interleukin (IL)-23. We observed a significant increase in sCD226 in sera from pwMS and NMO compared to HD and AD. In MS, levels were increased in both relapsing-remitting multiple sclerosis (RRMS) and secondary-progressive multiple sclerosis (SPMS) compared to clinically isolated syndrome (CIS). CONCLUSION: Our data suggest that T-cell activation leads to release of sCD226 that could promote inflammation and raises the possibility of using sCD226 as a biomarker for neuroinflammation.

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.

The anthelmintic drug praziquantel promotes human Tr1 differentiation.

Praziquantel (PZQ) is an anthelminthic human and veterinary drug used to treat trematode and cestode worms. Changes in immune responses have been demonstrated in humans following curative PZQ treatment of schistosome infections. These changes have been attributed to the removal of immunosupressive worms and immune responses to parasite antigens exposed from dying worms. To date, there has been no study investigating the potential direct effect of PZQ on the host immune cells. Herein, we analyzed the effect of PZQ on human CD4+ T cells classically costimulated by CD3/CD28 or costimulated by the complement regulator CD46 to induce Type 1 regulatory T cells (Tr1). Our results show that PZQ enhanced T-cell proliferation, increased secretion of IL-17 and IL-10 but had no effect on secretion of GM-CSF or IFNγ. Moreover, PZQ increased the coexpression of CD49b and LAG-3, a hallmark of Tr1 cells, suggesting increased Tr1 differentiation. Indeed, supernatants from PZQ-treated cells were able to decrease bystander T-cell activation, and this was partly reduced when blocking IL-10. Hence, our study demonstrates that PZQ directly modulates human T-cell activation and promotes Tr1 differentiation, suggesting that PZQ may have immunomodulatory functions in parasite-unrelated human inflammatory diseases.

Monocyte:T-cell interaction regulates human T-cell activation through a CD28/CD46 crosstalk.

T-cell activation requires engagement of the T-cell receptor and of at least one costimulatory molecule. The key role of CD28 in inducing T-cell activation was reported several decades ago and the molecular mechanisms involved have now been well described. The complement regulator CD46 also acts as a costimulatory molecule for T cells but, in contrast to CD28, has the ability to drive T-cell differentiation from producing some IFNγ to secreting some potent anti-inflammatory IL-10, acquiring a so-called Type I regulatory phenotype (Tr1). Proteolytic cleavage of CD46 occurs upon costimulation and is important for T-cell activation and IL-10 production. The observation that CD46 cleavage was reduced when PBMCs were costimulated compared with purified CD4(+) T cells led us to hypothesize that interactions between different cell types within the PBMCs were able to modulate the CD46 pathway. We show that CD46 downregulation is also reduced when CD4(+) T cells are cocultured with autologous monocytes. Indeed, monocyte: T-cell cocultures impaired CD46-mediated T-cell differentiation and coactivation, by reducing downregulation of surface CD46, lowering induction of the early activation marker CD69, as well as reducing the levels of IL-10 secretion. Blocking of CD86 could partly restore CD69 expression and cytokine secretion, demonstrating that the CD28: CD86 pathway regulates CD46 activation. Direct concomitant ligation of CD28 and CD46 on CD4(+) T cells also modulated CD46 expression and regulated cytokine production. These data identify a crosstalk between two main costimulatory pathways and provide novel insights into the regulation of human T-cell activation.

Recombinant Ad35 adenoviral proteins as potent modulators of human T cell activation.

The protein CD46 protects cells from complement attack by regulating cleavage of C3b and C3d. CD46 also regulates the adaptive immune response by controlling T cell activation and differentiation. Co-engagement of the T cell receptor and CD46 notably drives T cell differentiation by switching production of IFNγ to secretion of anti-inflammatory IL-10. This regulatory pathway is altered in several chronic inflammatory diseases highlighting its key role for immune homeostasis. The manipulation of the CD46 pathway may therefore provide a powerful means to regulate immune responses. Herein, we investigated the effect of recombinant proteins derived from the fiber knob of the adenovirus serotype 35 (Ad35) that uses CD46 as its entry receptor, on human T cell activation. We compared the effects of Ad35K++, engineered to exhibit enhanced affinity to CD46, and of Ad35K-, mutated in the binding site for CD46. Ad35K++ profoundly affects T cell activation by decreasing the levels of CD46 at the surface of primary T cells, and impairing T cell co-activation, shown by decreased CD25 expression, reduced proliferation and lower secretion of IL-10 and IFNγ. In contrast, Ad35K- acts a potent coactivator of T cells, enhancing T cell proliferation and cytokine production. These data show that recombinant Ad35 proteins are potent modulators of human T cell activation, and support their further development as potential drugs targeting T cell responses. This article is protected by copyright. All rights reserved.

Prostaglandin E2 affects T cell responses through modulation of CD46 expression.

The ubiquitous protein CD46, a regulator of complement activity, promotes T cell activation and differentiation toward a regulatory Tr1-like phenotype. The CD46-mediated differentiation pathway is defective in several chronic inflammatory diseases, underlying the importance of CD46 in controlling T cell function and the need to understand its regulatory mechanisms. Using an RNA interference-based screening approach in primary T cells, we have identified that two members of the G protein-coupled receptor kinases were involved in regulating CD46 expression at the surface of activated cells. We have investigated the role of PGE(2), which binds to the E-prostanoid family of G protein-coupled receptors through four subtypes of receptors called EP 1-4, in the regulation of CD46 expression and function. Conflicting roles of PGE(2) in T cell functions have been reported, and the reasons for these apparent discrepancies are not well understood. We show that addition of PGE(2) strongly downregulates CD46 expression in activated T cells. Moreover, PGE(2) differentially affects T cell activation, cytokine production, and phenotype depending on the activation signals received by the T cells. This was correlated with a distinct pattern of the PGE(2) receptors expressed, with EP4 being preferentially induced by CD46 activation. Indeed, addition of an EP4 antagonist could reverse the effects observed on cytokine production after CD46 costimulation. These data demonstrate a novel role of the PGE(2)-EP4 axis in CD46 functions, which might at least partly explain the diverse roles of PGE(2) in T cell functions.

RNA interference screen in primary human T cells reveals FLT3 as a modulator of IL-10 levels.

Functional studies of human primary immune cells have been hampered by the lack of tools to silence gene functions. In this study, we report the application of a lentiviral RNA interference library in primary human T cells. Using a subgenomic short hair RNA library targeting approximately 1000 signaling genes, we identified novel genes that control the levels of IL-10 produced. IL-10 is a potent anti-inflammatory cytokine secreted by several cell types, including T regulatory type 1 cells, a subset of T regulatory cells that exert their suppressive activity through IL-10 secretion. FLT3, a known hematopoeitic growth factor, was found to be a negative regulator of IL-10 levels in activated T cells. This was based on several observations. First, FLT3 and its ligand (FL) were both induced by T cell activation. Second, silencing of FLT3 led to increased IL-10 levels, whereas addition of FL suppressed IL-10 secretion and increased FLT3 surface levels. Third, engagement of CD46, a known inducer of T regulatory type 1 cells, upregulated surface FLT3, and secreted FL, which then inhibited IL-10 production by T cells. Hence, FL and FLT3 form a novel regulatory feedback loop that limits IL-10 production in T cells. Our results identified FLT3 as a new regulator of T cell function and offer a strategy to genetically dissect specific pathways in T cells.

Immune complex-induced apoptosis and concurrent immune complex clearance are anti-inflammatory neutrophil functions.

Persistent neutrophilic inflammation drives host damage in autoimmune diseases that are characterized by abundant immune complexes. Insoluble immune complexes (iICs) potently activate pro-inflammatory neutrophil effector functions. We and others have shown that iICs also promote resolution of inflammation via stimulation of neutrophil apoptosis. We demonstrate here that iICs trigger FcγRIIa-dependent neutrophil macropinocytosis, leading to the rapid uptake, and subsequent degradation of iICs. We provide evidence that concurrent iIC-induced neutrophil apoptosis is distinct from phagocytosis-induced cell death. First, uptake of iICs occurs by FcγRII-stimulated macropinocytosis, rather than phagocytosis. Second, production of reactive oxygen species, but not iIC-internalization is a pre-requisite for iIC-induced neutrophil apoptosis. Our findings identify a previously unknown mechanism by which neutrophils can remove pro-inflammatory iICs from the circulation. Together iIC clearance and iIC-induced neutrophil apoptosis may act to prevent the potential escalation of neutrophilic inflammation in response to iICs.

Vitamin D/CD46 Crosstalk in Human T Cells in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), in which T-cell migration into the CNS is key for pathogenesis. Patients with MS exhibit impaired regulatory T cell populations, and both Foxp3+ Tregs and type I regulatory T cells (Tr1) are dysfunctional. MS is a multifactorial disease and vitamin D deficiency is associated with disease. Herein, we examined the impact of 1,25(OH)2D3 on CD4+ T cells coactivated by either CD28 to induce polyclonal activation or by the complement regulator CD46 to promote Tr1 differentiation. Addition of 1,25(OH)2D3 led to a differential expression of adhesion molecules on CD28- and CD46-costimulated T cells isolated from both healthy donors or from patients with MS. 1,25(OH)2D3 favored Tr1 motility though a Vitamin D-CD46 crosstalk highlighted by increased VDR expression as well as increased CYP24A1 and miR-9 in CD46-costimulated T cells. Furthermore, analysis of CD46 expression on T cells from a cohort of patients with MS supplemented by vitamin D showed a negative correlation with the levels of circulating vitamin D. Moreover, t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis allowed the visualization and identification of clusters increased by vitamin D supplementation, but not by placebo, that exhibited similar adhesion phenotype to what was observed in vitro. Overall, our data show a crosstalk between vitamin D and CD46 that allows a preferential effect of Vitamin D on Tr1 cells, providing novel key insights into the role of an important modifiable environmental factor in MS.

Alterations in CD46-mediated Tr1 regulatory T cells in patients with multiple sclerosis.

Loss of Treg function appears to be a critical factor in the pathogenesis of human autoimmune diseases. Attention has focused on defects of CD4(+)CD25(high) Tregs, and techniques have been developed to determine their function. In contrast, the role of Tr1 regulatory T cells, which secrete the antiinflammatory cytokine IL-10, in autoimmune disease has not been well assessed. CD46 is a newly defined costimulatory molecule for T cell activation, and CD46-costimulated human T cells induce a Tr1 Treg phenotype with considerable amounts of IL-10 secretion. Here, we examined the role of Tr1 cells in patients with multiple sclerosis (MS) by stimulating CD4(+) T cells with anti-CD3 and -CD46 mAbs and measuring IL-10 secretion. There were striking defects in the induction of Tr1 cells with CD46 costimulation as measured by IL-10 but not IFN-gamma secretion in patients with MS compared with healthy subjects. This loss of Tr1 cell-associated IL-10 secretion was specific to CD46 and not CD28 costimulation and was associated with an altered regulation of the CD46-Cy2 isoform that differentially regulates T cell function in a CD46-transgenic murine model. These data demonstrate a second major Treg defect in human autoimmune disease associated with the CD46 pathway.

T-cell regulation by CD46 and its relevance in multiple sclerosis.

CD46 is a complement regulatory molecule expressed on every cell type, except for erythrocytes. While initially described as a regulator of complement activity, it later became a 'magnet for pathogens', binding to several viruses and bacteria. More recently, an alternative role for such complement molecules has emerged: they do regulate T-cell immunity, affecting T-cell proliferation and differentiation. In particular, CD46 stimulation induces Tr1 cells, regulatory T cells characterized by massive production of interleukin-10 (IL-10), a potent anti-inflammatory cytokine. Hence, CD46 is likely to control inflammation. Indeed, data from CD46 transgenic mice highlight a role for CD46 in inflammation, with antagonist roles depending on the cytoplasmic tail being expressed. Furthermore, recent data have shown that CD46 is defective in multiple sclerosis, IL-10 production being severely impaired in these patients. This lack of IL-10 production probably participates in the inflammation observed in patients with multiple sclerosis. This review will summarize the data on CD46 and T cells, and how CD46 is likely involved in multiple sclerosis.

Increased IL-23 secretion and altered chemokine production by dendritic cells upon CD46 activation in patients with multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). In MS, myeloid dendritic cells (mDCs) secrete elevated amounts of IL-23, a potent proinflammatory cytokine, compared to healthy donors. Here, we examined the role of CD46, a complement binding factor, in mDCs by analyzing cytokine and chemokine production in healthy donors and patients with MS. There were striking differences between these groups with increased IL-23p19, CCL3 and CCL5 production, but decreased CCL2 levels in patients. This demonstrates major differences of DC activation upon CD46 activation, with a potential role in the pathogenesis of MS.

Complement as a regulator of adaptive immunity.

The complement system is an ancient and evolutionarily conserved effector system comprising in mammals over 50 circulating and membrane bound proteins. Complement has long been described as belonging to the innate immune system; however, a number of recent studies have demonstrated its key role in the modulation of the adaptive immune response. This review does not set out to be an exhaustive list of the numerous interactions of the many complement components with adaptive immunity; rather, we will focus more precisely on the role of some complement molecules in the regulation of antigen presenting cells, as well as on their direct effect on the activation of the core adaptive immune cells, B and T lymphocytes. Recent reports on the local production and activation of complement proteins also suggest a major role in the control of effector responses. The crucial role of complement in adaptive immunity is further highlighted by several examples of dysregulation of these pathways in human diseases.

Abnormal Tr1 differentiation in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). In the recent years, accumulating evidence has supported an immunosuppressive role for regulatory T cells (Tregs). Most studies in the context of autoimmunity have focused on the defects of the CD4+CD25 high Tregs. However, we recently demonstrated an altered function of Tr1 Treg cells in MS, characterized by a lack of IL-10 secretion. Therefore, several major regulatory T cell defects are involved in human autoimmune disease. Hence, the induction of Tregs or the stimulation of Treg activity may be beneficial for the treatment of such diseases.

TCR-stimulated changes in cell surface CD46 expression generate type 1 regulatory T cells.

A lack of regulatory T cell function is a critical factor in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). Ligation of the complement regulatory protein CD46 facilitates the differentiation of T helper 1 (TH1) effector cells into interleukin-10 (IL-10)-secreting type 1 regulatory T cells (Tr1 cells), and this pathway is defective in MS patients. Cleavage of the ectodomain of CD46, which contains three N-glycosylation sites and multiple O-glycosylation sites, enables CD46 to activate T cells. We found that stimulation of the T cell receptor (TCR)-CD3 complex was associated with a reduction in the apparent molecular mass of CD46 in a manner that depended on O-glycosylation. CD3-stimulated changes in CD46 O-glycosylation status reduced CD46 processing and subsequent T cell signaling. During T cell activation, CD46 was recruited to the immune synapse in a manner that required its serine-, threonine-, and proline-rich (STP) region, which is rich in O-glycosylation sites. Recruitment of CD46 to the immune synapse switched T cells from producing the inflammatory cytokine interferon-γ (IFN-γ) to producing IL-10. Furthermore, CD4+ T cells isolated from MS patients did not exhibit a CD3-stimulated reduction in the mass of CD46 and thus showed increased amounts of cell surface CD46. Together, these data suggest a possible mechanism underlying the regulatory function of CD46 on T cells. Our findings may explain why this pathway is defective in patients with MS and provide insights into MS pathogenesis that could help to design future immunotherapies.

CD46 processing: a means of expression.

CD46 is a ubiquitously expressed type I transmembrane protein, first identified as a regulator of complement activation, and later as an entry receptor for a variety of pathogens. The last decade has also revealed the role of CD46 in regulating the adaptive immune response, acting as an additional costimulatory molecule for human T cells and inducing their differentiation into Tr1 cells, a subset of regulatory T cells. Interestingly, CD46 regulatory pathways are defective in T cells from patients with multiple sclerosis, asthma and rheumatoid arthritis, illustrating its importance in regulating T cell homeostasis. Indeed, CD46 expression at the cell surface is tightly regulated in many different cell types, highlighting its importance in several biological processes. Notably, CD46 is the target of enzymatic processing, being cleaved by metalloproteinases and by the presenilin/gamma secretase complex. This processing is required for its functions, at least in T cells. This review will summarize the latest updates on the regulation of CD46 expression and on its effects on T cell activation.

Calcitriol modulates the CD46 pathway in T cells.

The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. In this pilot study, we examined whether active vitamin D (1,25(OH)(2)D(3) or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated CD4+ T cells from patients with MS. In healthy T cells, calcitriol profoundly affects the phenotype of CD46-costimulated CD4+ T cells, by increasing the expression of CD28, CD25, CTLA-4 and Foxp3 while it concomitantly decreased CD46 expression. Similar trends were observed in MS CD4+ T cells except for CD25 for which a striking opposite effect was observed: while CD25 was normally induced on MS T cells by CD46 costimulation, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNγ ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Hence, we show that calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS.