β‐Glucan enhances antitumor immune responses by regulating differentiation and function of monocytic myeloid‐derived suppressor cells

Myeloid‐derived suppressor cells (MDSCs) accumulate in tumor‐bearing hosts and play a major role in tumor‐induced immunosuppression, which hampers effective immuno‐therapeutic approaches. β‐Glucans have been reported to function as potent immuno‐modulators to stimulate innate and adaptive immune responses, which contributes to their antitumor property. Here, we investigated the effect of particulate β‐glucans on MDSCs and found that β‐glucan treatment could promote the differentiation of M‐MDSCs (monocytic MDSCs) into a more mature CD11c⁺ F4/80⁺ Ly6C^low population via dectin‐1 pathway in vitro, which is NF‐κB dependent, and the suppressive function of M‐MDSCs was significantly decreased. Treatment of orally administered yeast‐derived particulate β‐glucan drastically downregulated MDSCs but increased the infiltrated DCs and macrophages in tumor‐bearing mice, thus eliciting CTL and Th1 responses, inhibiting the suppressive activity of regulatory T cells, thereby leading to the delayed tumor progression. We show here for the first time that β‐glucans induce the differentiation of MDSCs and inhibit the regulatory function of MDSCs, therefore revealing a novel mechanism for β‐glucans in immunotherapy and suggesting their potential clinical benefit.     

Impaired antigen‐specific lymphocyte priming in mice after Toll‐like receptor 4 activation via induction of monocytic myeloid‐derived suppressor cells

In sepsis, the pathology involves a shift from a proinflammatory state toward an immunosuppressive phase. We previously showed that an agonistic anti‐TLR4 antibody induced long‐term endotoxin tolerance and suppressed antigen‐specific secondary IgG production when primed prior to immunization with antigen. These findings led us to speculate that TLR4‐induced innate tolerance due to primary infection causes an immunosuppressive pathology in sepsis. Therefore, the mechanism underlying impaired antigen‐specific humoral immunity by the TLR4 antibody was investigated. We showed, in a mouse model, that primary antigen‐specific IgG responses were impaired in TLR4 antibody‐induced tolerized mice, which was the result of reduced numbers of antigen‐specific GC B cells and plasma cells. Ovalbumin‐specific CD4 and CD8 T‐cell responses were impaired in TLR4 antibody‐injected OT‐I and ‐II transgenic mice ex vivo. Adoptive transfer studies demonstrated suppression of OVA‐specific CD4 and CD8 T‐cell responses by the TLR4 antibody in vivo. The TLR4 antibody induced Gr1⁺CD11b⁺ myeloid‐derived suppressor cell (MDSC) expansion with suppression of T‐cell activation. Monocytic MDSCs were more suppressive and exhibited higher expression of PD‐L1 and inducible nitric oxidase compared with granulocytic MDSCs. In conclusion, immune tolerance conferred by TLR4 activation induces the expansion of monocytic MDSCs, which impairs antigen‐specific T‐cell priming and IgG production.     

Gr‐1 antibody induces STAT signaling,macrophage marker expression and abrogation of myeloid‐derived suppressor cell activity in BM cells

The Gr‐1 (RB6‐8C5) Ab binds with high affinity to mouse Ly‐6G molecules and to a lower extent to Ly‐6C and has been widely used for cell depletion in infected or tumor‐bearing mice. Here we found that Gr‐1 treatment of BM cells in vitro and in vivo showed no depleting effects. The epitope recognized by the Gr‐1 Ab overlapped with Ly‐6G (1A8 Ab) but not Ly‐6C (ER‐MP20 Ab). In vitro the Gr‐1 Ab transmitted signals via STAT‐1, STAT‐3 and STAT‐5 into BM cells, similar to GM‐CSF. In healthy mice injected with the Gr‐1 Ab, the Ab remained attached to the surface of myeloid cells for at least four days. Gr‐1 Ab induced myeloid cell expansion, upregulation of macrophage markers, but not the DC marker CD11c. Suppressor activity of two distinct Gr‐1^high and Gr‐1^low expressing BM‐myeloid‐derived suppressor cell subsets was transiently ablated by Gr‐1 Ab injection. Depleting effects of Gr‐1 Ab could only be observed on inflammatory Ly‐6C^intLy‐6G^high neutrophils from the peritoneal cavity, which occurred via apoptosis and was associated with the absence of Mcl‐1 expression. Together, Gr‐1 Ab induces signals leading to myelopoiesis and affects myeloid‐derived suppressor cell activity, suggesting functional roles for Ly‐6C/G molecules in macrophage differentiation and neutrophil apoptosis.     

Tumour‐activated liver stromal cells regulate myeloid‐derived suppressor cells accumulation in the liver

Regulating mechanisms underlying hepatic myeloid‐derived suppressor cell (MDSC) accumulation remain to be described. Here, we provide evidence for the involvement of tumour‐activated liver stromal cells in the process of hepatic MDSCs migration and accumulation. Our data showed an elevated frequency of MDSCs in the liver of tumour‐bearing mice. Moreover, tumour‐activated liver stromal cells promote MDSC migration into the liver site. Further investigation indicated higher levels of cytokine and chemokine expression in liver stromal cells after exposure to the tumour‐conditioned supernatant. Notably, the expression levels of proinflammatory factors, mainly including macrophage colony stimulating factor (M‐CSF), transforming growth factor‐β (TGF‐β), monocyte chemotactic protein‐1 (MCP‐1) and stromal‐derived factor‐1 (SDF‐1), increased after treatment with tumour‐conditioned supernatant, and blockade of MCP‐1 or SDF‐1 decreased the proportion of tumour infiltrated MDSCs in mice co‐transplanted with liver stromal cells and tumour cells, but not in mice with only tumour cells injection. These findings demonstrate that tumour‐activated liver stromal cells produce higher levels of chemokines and cytokines, which may contribute to MDSC accumulation into the liver site in patients with liver cancer.     

Myeloid‐derived suppressor cell activation by combined LPS and IFN‐γ treatment impairs DC development

Myeloid‐derived suppressor cells (MDSC) and DC are major controllers of immune responses against tumors or infections. However, it remains unclear how DC development and MDSC suppressor activity both generated from myeloid precursor cells are regulated. Here, we show that the combined treatment of BM‐derived MDSC with LPS plus IFN‐γ inhibited the DC development but enhanced MDSC functions, such as NO release and T‐cell suppression. This was not observed by the single treatments in vitro. In the spleens of healthy mice, we identified two Gr‐1^lowCD11b^highLy‐6C^highSSC^lowMo‐MDSC and Gr‐1^highCD11b^lowPMN‐MDSC populations with suppressive potential, whereas Gr‐1^highCD11b^high neutrophils and Gr‐1^lowCD11b^highSSC^low eosinophils were not suppressive. Injections of LPS plus IFN‐γ expanded these populations within the spleen but not LN leading to the block of the proliferation of CD8⁺ T cells. At the same time, their capacity to develop into DC was impaired. Together, our data suggest that spleens of healthy mice contain two subsets of MDSC with suppressive potential. A two‐signal‐program through combined LPS and IFN‐γ treatment expands and fully activates MDSC in vitro and in vivo.     

Human fibrocytic myeloid‐derived suppressor cells express IDO and promote tolerance via Treg‐cell expansion

By restraining T‐cell activation and promoting Treg‐cell expansion, myeloid‐derived suppressor cells (MDSCs) and tolerogenic DCs can control self‐reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor‐free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4‐day culture with FDA‐approved cytokines (recombinant human‐GM‐CSF and recombinant human‐G‐CSF). This MDSC subset, characterized by the expression of MDSC‐, DC‐, and fibrocyte‐associated markers, promotes Treg‐cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.     

Hierarchy of immunosuppressive strength among myeloid‐derived suppressor cell subsets is determined by GM‐CSF

CD11b⁺/Gr‐1⁺ myeloid‐derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8⁺ T‐cells. Two major MDSC subsets were recently shown to play an equal role in MDSC‐induced immune dysfunctions: monocytic‐ and granulocytic‐like. We isolated three fractions of MDSC, i.e. CD11b⁺/Gr‐1^high, CD11b⁺/Gr‐1^int, and CD11b⁺/Gr‐1^low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b⁺/Gr‐1^int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8⁺ T‐cell activation, while CD11b⁺/Gr‐1^high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b⁺/Gr‐1^int but not CD11b⁺/Gr‐1^high cells were also immunosuppressive in vivo following adoptive transfer. CD11b⁺/Gr‐1^low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM‐CSF was necessary to induce preferential expansion of both CD11b⁺/Gr‐1^int and CD11b⁺/Gr‐1^low subsets in the spleen of tumor‐bearing mice and mediate tumor‐induced tolerance whereas G‐CSF, which preferentially expanded CD11b⁺/Gr‐1^high cells, did not create such immunosuppressive environment. GM‐CSF also acted on granulocyte–macrophage progenitors in the bone marrow inducing local expansion of CD11b⁺/Gr‐1^low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor‐released GM‐CSF.     

The immunosuppressive tumour network: myeloid‐derived suppressor cells,regulatory T cells and natural killer T cells

Myeloid‐derived suppressor cells (MDSC) and regulatory T (Treg) cells are major components of the immune suppressive tumour microenvironment (TME). Both cell types expand systematically in preclinical tumour models and promote T‐cell dysfunction that in turn favours tumour progression. Clinical reports show a positive correlation between elevated levels of both suppressors and tumour burden. Recent studies further revealed that MDSCs can modulate the de novo development and induction of Treg cells. The overlapping target cell population of Treg cells and MDSCs is indicative for the importance and flexibility of immune suppression under pathological conditions. It also suggests the existence of common pathways that can be used for clinical interventions aiming to manipulate the TME. Elimination or reprogramming of the immune suppressive TME is one of the major current challenges in immunotherapy of cancer. Interestingly, recent findings suggest that natural killer T (NKT) cells can acquire the ability to convert immunosuppressive MDSCs into immunity‐promoting antigen‐presenting cells. Here we will review the cross‐talk between MDSCs and other immune cells, focusing on Treg cells and NKT cells. We will consider its impact on basic and applied cancer research and discuss how targeting MDSCs may pave the way for future immunocombination therapies.     

Tumor‐induced CD11b+ Gr‐1+ myeloid‐derived suppressor cells exacerbate immune‐mediated hepatitis in mice in a CD40‐dependent manner

Immunosuppressive CD11b⁺Gr‐1⁺ myeloid‐derived suppressor cells (MDSCs) accumulate in the livers of tumor‐bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune‐mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α‐galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor‐free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b⁺Gr‐1⁺ cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN‐γ‐dependent upregulation of CD40 on hepatic CD11b⁺Gr‐1⁺ cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor‐induced CD11b⁺Gr‐1⁺ MDSCs as well as enhanced reactive oxygen species (ROS)‐mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40^?/? tumor‐induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor‐induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40‐dependent manner.     

Functional characterization of myeloid‐derived suppressor cell subpopulations during the development of experimental arthritis

Recent evidence indicates the existence of subpopulations of myeloid‐derived suppressor cells (MDSCs) with distinct phenotypes and functions. Here, we characterized the role of MDSC subpopulations in the pathogenesis of autoimmune arthritis in a collagen‐induced arthritis (CIA) mouse model. The splenic CD11b⁺Gr‐1⁺ MDSC population expanded in CIA mice, and these cells could be subdivided into polymorphonuclear (PMN) and mononuclear (MO) MDSC subpopulations based on Ly6C and Ly6G expression. During CIA, the proportion of splenic MO‐MDSCs was increased in association with the severity of joint inflammation, while PMN‐MDSCs were decreased. MO‐MDSCs expressed higher levels of surface CD40 and CD86 protein, but lower levels of Il10, Tgfb1, Ccr5, and Cxcr2 mRNA. PMN‐MDSCs exhibited a more potent capacity to suppress polyclonal T‐cell proliferation in vitro, compared with MO‐MDSCs. Moreover, the adoptive transfer of PMN‐MDSCs, but not MO‐MDSCs, decreased joint inflammation, accompanied by reduced levels of serum cytokine secretion and the frequencies of Th1 and Th17 cells in draining lymph nodes. These results suggest that there could be a shift from potently suppressive PMN‐MDSCs to poorly suppressive MO‐MDSCs during the development of experimental arthritis, which might reflect the failure of expanded MDSCs to suppress autoimmune arthritis.     

Arginase‐1 is neither constitutively expressed in nor required for myeloid‐derived suppressor cell‐mediated inhibition of T‐cell proliferation

Although previous reports suggest that tumor‐induced myeloid‐derived suppressor cells (MDSC) inhibit T cells by L‐arginine depletion through arginase‐1 activity, we herein show that arginase‐1 is neither inherently expressed in MDSC nor required for MDSC‐mediated inhibition. Employing Percoll density gradients, large expansions of MDSC in the bone marrow of tumor‐bearing mice were isolated and demonstrated potent inhibition in T‐cell proliferation activated by TCR‐ligation, Concanavalin A, PMA plus ionomycin, or IL‐2. Despite demonstrating characteristic immunosuppressive capacity, these MDSC exhibit no arginase‐1 expression and/or exert their inhibitory effects independent of arginase‐1 activity. However, arginase‐1 expression in MDSC can be induced by exposure to TCR‐activated T cells or their culture medium, but not T cells activated by other means or growing tumor cells. Further investigation reveals multiple cytokines secreted by TCR‐activated T cells as orchestrating two signaling‐relay axes, IL‐6‐to‐IL‐4 and GM‐CSF/IL‐4‐to‐IL‐10, leading to arginase‐1 expression in MDSC. Specifically, IL‐6 signaling increases IL‐4R, enabling IL‐4 to induce arginase‐1 expression; similarly, GM‐CSF in concert with IL‐4 induces IL‐10R, allowing IL‐10‐mediated induction. Surprisingly, our study indicates that induction of arginase‐1 expression is not conducive to the critical MDSC‐mediated inhibition toward T cells, which is rather dependent on direct cell contacts undiminished by PD‐L1 blockade or SIRPα deficiency.     

Sepsis‐induced expansion of granulocytic myeloid‐derived suppressor cells promotes tumour growth through Toll‐like receptor 4

Severe sepsis remains a frequent and dreaded complication in cancer patients. Beyond the often fatal short‐term outcome, the long‐term sequelae of severe sepsis may also impact directly on the prognosis of the underlying malignancy in survivors. The immune system is involved in all stages of tumour development, in the detection of transforming and dying cells and in the prevention of tumour growth and dissemination. In fact, the profound and sustained immune defects induced by sepsis may constitute a privileged environment likely to favour tumour growth. We investigated the impact of sepsis on malignant tumour growth in a double‐hit animal model of polymicrobial peritonitis, followed by subcutaneous inoculation of MCA205 fibrosarcoma cells. As compared to their sham‐operated counterparts, post‐septic mice exhibited accelerated tumour growth. This was associated with intratumoural accumulation of CD11b⁺Ly6G^high polymorphonuclear cells (PMNs) that could be characterized as granulocytic myeloid‐derived suppressor cells (G‐MDSCs). Depletion of granulocytic cells in post‐septic mice inhibited the sepsis‐enhanced tumour growth. Toll‐like receptor (TLR)‐4 (Tlr4) and Myd88 deficiencies prevented sepsis‐induced expansion of G‐MDSCs and tumour growth. Our results demonstrate that the myelosuppressive environment induced by severe bacterial infections promotes malignant tumour growth, and highlight a critical role of CD11b⁺Ly6G^high G‐MDSCs under the control of TLR‐dependent signalling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Atorvastatin promotes the expansion of myeloid‐derived suppressor cells and attenuates murine colitis

Statins, widely prescribed as cholesterol‐lowering drugs, have recently been extensively studied for their pleiotropic effects on immune systems, especially their beneficial effects on autoimmune and inflammatory disorders. However, the mechanism of statin‐induced immunosuppression is far from understood. Here, we found that atorvastatin promoted the expansion of myeloid‐derived suppressor cells (MDSCs) both in vitro and in vivo. Atorvastatin‐derived MDSCs suppressed T‐cell responses by nitric oxide production. Addition of mevalonate, a downstream metabolite of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, almost completely abrogated the effect of atorvastatin on MDSCs, indicating that the mevalonate pathway was involved. Along with the amelioration of dextran sodium sulphate (DSS) ‐induced murine acute and chronic colitis, we observed a higher MDSC level both in spleen and intestine tissue compared with that from DSS control mice. More importantly, transfer of atorvastatin‐derived MDSCs attenuated DSS acute colitis and T‐cell transfer of chronic colitis. Hence, our data suggest that the expansion of MDSCs induced by statins may exert a beneficial effect on autoimmune diseases. In summary, our study provides a novel potential mechanism for statins‐based treatment in inflammatory bowel disease and perhaps other autoimmune diseases.     

Polyunsaturated fatty acids promote the expansion of myeloid‐derived suppressor cells by activating the JAK/STAT3 pathway

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects that could prove beneficial in clinical therapies for certain autoimmune and inflammatory disorders. However, the mechanism of PUFA‐mediated immunosuppression is far from understood. Here, we provide evidence that PUFAs enhance the accumulation of myeloid‐derived suppressor cells (MDSCs), a negative immune regulator. PUFA‐induced MDSCs have a more potent suppressive effect on T‐cell responses than do control MDSCs. These observations were found both in cultured mouse bone marrow cells in vitro and in vivo in mice fed diets enriched in PUFAs. The enhanced suppressive activity of MDSCs by PUFAs administration was coupled with a dramatic induction of nicotinamide adenine dinucleo‐ tide phosphate oxidase subunit p47^phox and was dependent on reactive oxygen species (ROS) production. Mechanistic studies revealed that PUFAs mediate its effects through JAK‐STAT3 signaling. Inhibition of STAT3 phosphorylation by JAK inhibitor JSI‐124 almost completely abrogated the effects of PUFAs on MDSCs. Moreover, the effects of PUFAs on MDSCs and the underlying mechanisms were confirmed in tumor‐bearing mice. In summary, this study sheds new light on the immune modulatory role of PUFAs, and demonstrates that MDSCs expansion may mediate the effects of PUFAs on the immune system.     

Neutrophils promote motility of cancer cells via a hyaluronan-mediated TLR4/PI3K activation loop

Inflammation is a component of tumour progression mechanisms. Neutrophils are a common inflammatory infiltrate in many tumours, but their regulation and functions in neoplasia are not understood. We recently demonstrated that pro-inflammatory IL-17-producing cells recruited blood neutrophils into the peritumoural stroma of hepatocellular carcinoma by epithelium-derived CXC chemokines. Here we show that a substantial population of neutrophils accumulates in the peritumoural stroma of hepatocellular, cervical, colorectal, and gastric carcinomas, and that this correlates with metastases in hepatocellular and gastric carcinomas. Exposure of neutrophils to culture supernatants from several types of solid tumour cells (TSN) resulted in sustained survival and pro-tumourigenic effects of cells. Kinetic experiments reveal that, shortly after exposure to TSN, neutrophils began to provoke activation and then produced significant inflammatory cytokines and expressed more anti-apoptotic Mcl-1 but less pro-apoptotic Bax. These long-lived neutrophils effectively enhanced the cancer cell motility via a contact-dependent mechanism; this effect, together with early activation and subsequent longevity of TSN-exposed neutrophils, could be reversed by blocking the activation of PI3K/Akt signalling in neutrophils. Moreover, we found that hyaluronan (HA) fragments constitute a common factor produced by various tumours that mimics the effect of TSN to induce long-lived neutrophils and subsequent malignant cell migration. The effects of TSN were inhibited by function blocking interactions between HA and its receptor TLR4 on neutrophils, suggesting that this is a key signalling pathway involved. These results indicate that HA derived from malignant cells educates neutrophils to adopt an activated phenotype, and in that way stimulates the metastasis of malignant cells, which represents a positive regulatory loop between tumours and their stroma during neoplastic progression.     

Tumor‐infiltrating IL‐17‐producing γδ T cells support the progression of tumor by promoting angiogenesis

Based on the evidence that IL‐17 is a key cytokine involved in various inflammatory diseases, we explored the critical role of IL‐17‐producing γδ T cells for tumor development in tumor‐bearing mouse model. IL‐17^?/? mice exhibited a significant reduction of tumor growth, concomitantly with the decrease of vascular density at lesion area, indicating a pro‐tumor property of IL‐17. Among tumor‐infiltrating lymphocytes (TIL), γδ T cells were the major cellular source of IL‐17. Analysis of TCR repertoires in TIL‐γδ T cells showed that circulating γδ T cells, but not skin resident Vγ5⁺γδ T cells, produced IL‐17. Neutralizing antibodies against IL‐23, IL‐6, and TGF‐β, which were produced within the tumor microenvironment, inhibited the induction of IL‐17‐producing γδ T cells. IL‐17 production by tumor‐infiltrating γδ T cells was blocked by anti‐γδTCR or anti‐NKG2D antibodies, indicating that these ligands, expressed within the tumor microenvironment, are involved in γδ T‐cell activation. The IL‐17‐producing TIL‐γδ T cells exhibited reduced levels of perforin mRNA expression, but increased levels of COX‐2 mRNA expression. Together, our findings support the novel concept that IL‐17‐producing γδ T cells, generated in response to tumor microenvironment, act as tumor‐promoting cells by inducing angiogenesis.     

Serum amyloid A induces interleukin‐6 in dermal fibroblasts via Toll‐like receptor 2, interleukin‐1 receptor‐associated kinase 4 and nuclear factor‐κB

Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute‐phase protein that is elevated up to 1000‐fold in times of infection or inflammation. This acute‐phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine‐like manner, possibly through toll‐like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin‐6 (IL‐6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL‐6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA‐induced IL‐6 secretion and that this was also mediated through the TLR adaptor protein IL‐1 receptor‐associated kinase 4. The effect is nuclear factor‐κB‐mediated because blockade of nuclear factor‐κB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over‐expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL‐6 signalling in systemic sclerosis via enhanced TLR2 signalling.     

IFN‐β therapy modulates B‐cell and monocyte crosstalk via TLR7 in multiple sclerosis patients

The implication of B lymphocytes in the immunopathology of multiple sclerosis (MS) is increasingly recognized. Here we investigated the response of B cells to IFN‐β, a first‐line therapy for relapsing‐remitting MS patients, upon stimulation with TLR. IFN‐β restored the frequency of TLR7‐induced IgM and IgG‐secreting cells in MS patients to the levels found in healthy donors, showing a specific deficiency in the TLR7 pathway. However, no difference was observed in the TLR9 response. Furthermore, in MS‐derived PBMCs, TLR7‐mediated production of IL‐6 and the ex vivo expression of B‐cell‐activating factor of the TNF family, two crucial cytokines for B‐cell differentiation and survival, were induced by IFN‐β. Depletion of monocytes, which are key producers of both IL‐6 and B‐cell‐activating factor of the TNF family, showed that TLR7‐mediated B‐cell differentiation into Ig‐secreting cells is strongly dependent on the cross‐talk between B cells and monocytes. Accordingly, impaired expression of TLR7 mRNA was observed in PBMCs and monocytes isolated from MS‐affected individuals as compared with those from healthy donors, which was rescued by IFN‐β therapy. Collectively, our data unveil a novel TLR7‐regulated mechanism in in vivo IFN‐β‐stimulated whole leukocytes that could be exploited to define new TLR7‐based strategies for the treatment of MS.