TLR‐mediated STAT3 and ERK activation controls IL‐10 secretion by human B cells

IL‐10‐producing B cells have a regulatory effect in various mouse models for immune‐mediated disorders via secretion of IL‐10, a potent immunoregulatory cytokine. However, currently, the signaling pathways that regulate IL‐10 production in B cells are not well understood. Here, we show that TLR signaling, but not BCR activation or CD40 ligation, induces potent production of IL‐10 in human B cells. We demonstrate that the activation of STAT3 and ERK is required for TLR‐induced IL‐10 production by B cells, since inhibition of STAT3 or ERK activation abrogates TLR‐induced IL‐10 production. We also uncover a novel function of the TLR‐MyD88‐STAT3 pathway in B cells, namely controlling IL‐10 production, in addition to the known role for this pathway in antibody production. Furthermore, IFN‐α, a member of the type I IFN family, differentially modulates TLR7/8‐ and TLR9‐activated STAT3 and ERK in B cells, which provides an explanation for our findings that IFN‐α enhances TLR7/8‐induced, but not TLR9‐induced IL‐10 production. These results yield insights into the mechanisms by which TLR signaling regulates IL‐10 production in B cells and how type I IFN modulates TLR‐mediated IL‐10 production by B cells, therefore providing potential targets to modulate the function of IL‐10‐producing B cells.     

TLR ligands of ryegrass pollen microbial contaminants enhance Th1 and Th2 responses and decrease induction of Foxp3hi regulatory T cells

Microbial contamination of grass pollens could affect sensitization, subsequent allergic response, and efficacy of allergen‐specific immunotherapy. We investigated whether bacterial immunomodulatory substances can direct PBMC responses of allergic and nonatopic subjects against ryegrass pollen (RGP) toward Th1, Th2, or regulatory T (Treg) cells. Aqueous extracts of RGP with high or low LPS were fractionated into large and small molecular weight (MW) components by diafiltration. CFSE‐labeled PBMCs from allergic and nonatopic subjects were stimulated with RGP extracts (RGPEs) and analyzed for cytokine secretion and T‐cell responses. High LPS RGPE increased IFN‐γ⁺ Th1 and IL‐4⁺ Th2 effector cell induction and consistently decreased CD4⁺Foxp3^hi Treg‐cell induction. IL‐10‐producing T‐cell frequency was unaltered, but IL‐10 secretion was increased by high LPS RGPE. RGPE‐stimulation of TLR‐transfected cell lines revealed that high LPS pollen also contained a TLR2‐ligand, and both batches a TLR9‐ligand. Beta‐1,3‐glucans were detected in large and small MW fractions and were also T‐cell stimulatory. In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not convert an established Th2‐ into a Th1‐response. Instead, proinflammatory responses are exacerbated and Foxp3^hi Treg‐cell induction is decreased. These findings show that adjuvants for specific immunotherapy should enhance Treg cells rather than target immune deviation from Th2 to Th1.     

TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells into the primary repertoire

Toll-like receptors (TLR) have been shown to play an essential role in the generation of autoantibodies in mouse models of autoimmunity, but the timing and context of these effects are poorly understood. One hypothesis is that TLR ligands assist in the positive selection of self-reactive B cells into the primary repertoire and, in this way, distinguish between immunogenic and tolerogenic forms of self-antigen. To explore this idea we generated hen egg lysozyme-specific immunoglobulin (Ig(HEL)) and isotype class-switching anti-HEL mice deficient in MyD88, TLR4 or TLR9 signalling and studied B cell development and autoantibody secretion in the presence or absence of an intracellular form of self-antigen HEL that positively selects B1 cells. Our findings show that TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells in the primary B cell repertoire, nor is MyD88 required for the generation of isotype-switched antibodies in the absence of antigen. These results suggest that the significant effects of TLR on autoimmunity occur in the established repertoire and not during B cell development.     

The Toll‐like receptor 9 signalling pathway regulates MR1‐mediated bacterial antigen presentation in B cells

Mucosal‐associated invariant T (MAIT) cells are conserved T cells that express a semi‐invariant T‐cell receptor (Vα7.2 in humans and Vα19 in mice). The development of MAIT cells requires the antigen‐presenting MHC‐related protein 1 (MR1), as well as commensal bacteria. The mechanisms that regulate the functional expression of MR1 molecules and their loading with bacterial antigen in antigen‐presenting cells are largely unknown. We have found that treating B cells with the Toll‐like receptor 9 (TLR9) agonist CpG increases MR1 surface expression. Interestingly, activation of TLR9 by CpG‐A (but not CpG‐B) enhances MR1 surface expression. This is limited to B cells and not other types of cells such as monocytes, T or natural killer cells. Knocking‐down TLR9 expression by short hairpin RNA reduces MR1 surface expression and MR1‐mediated bacterial antigen presentation. CpG‐A triggers early endosomal TLR9 activation, whereas CpG‐B is responsible for late endosomal/lysosomal activation of TLR9. Consistently, blocking endoplasmic reticulum to Golgi protein transport, rather than lysosomal acidification, suppressed MR1 antigen presentation. Overall, our results indicate that early endosomal TLR9 activation is important for MR1‐mediated bacterial antigen presentation.     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.     

IFN‐γ stimulates CpG‐induced IL‐10 production in B cells via p38 and JNK signalling pathways

The production of IL‐10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN‐γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL‐10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN‐γ on Toll‐like receptor (TLR)‐induced IL‐10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN‐γ inhibited IL‐10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL‐10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN‐γ on TLR9‐induced IL‐10 was restricted to B cells. In line with the increased IL‐10, B cells stimulated with CpG and IFN‐γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen‐activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 – an inhibitor of p38 and JNK activity – is downregulated after combined stimulation with IFN‐γ and CpG. Our data may represent a novel immunoregulatory role of IFN‐γ in B cells after triggering of TLR9, by stimulating IL‐10 production.     

Bruton's tyrosine kinase regulates TLR9 but not TLR7 signaling in human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) represent a key cell type for both innate and adaptive immunity. PDCs express both TLR7 and TLR9 and the recognition of nucleic acids by these two receptors triggers the production of a large amount of type‐I IFN and the induction of PDC maturation into APCs. This unique feature of PDCs is at the basis of clinical development of both TLR7 and TLR9 agonists for infectious diseases, allergy, cancer, and asthma. However, TLR7 and TLR9 recognition of self‐nucleic acids is linked to many autoimmune diseases including lupus, and a better understanding of the signaling pathways of these two receptors in PDCs is thus important. We have identified Bruton's tyrosine kinase (Btk) as an important player for TLR9 but not TLR7 signaling in human PDCs. Blocking Btk using a specific inhibitor leads to the reduction of all TLR9‐induced responses in PDCs, including cytokine production and expression of costimulatory molecules, while this has no impact on the TLR7 response. This identifies Btk as a key molecule in TLR9 signaling in PDCs and is the first demonstration that the TLR7 and TLR9 pathways can be dissociated in human PDCs.     

Freeze-and-thaw-disrupted tumour cells impair the responsiveness of DC to TLR stimulation

Cancer immunotherapy aims at inducing immune responses against tumour-associated antigens that mediate the eradication of tumour cells. For successful vaccination against antigens expressed by the tumour, the immune system has to be provided with sufficient amounts of these antigens in connection with strong immunostimulatory signals such as toll-like receptor (TLR) ligands. Tumour cells represent a convenient source of relevant tumour-associated antigens but can have suppressive properties. In this study, we explored how different forms of tumour cell material influence the activation of dendritic cells (DC), which play a crucial role in the induction of anti-tumour immune responses. We show that freeze-and-thaw-disrupted tumour cells inhibit DC activation in response to TLR stimulation, a phenomenon that is only partially seen with non-disrupted control cells. This suppression of DC stimulation is independent of tumour cell- and species-specific factors. We tested the hypothesis that phosphatidylserine on cells with disrupted membrane integrity mediates inhibition of TLR-induced DC activation. Our experimental evidence indicates that phosphatidylserine is not involved in the inhibition of TLR-mediated DC activation by freeze-and-thaw-disrupted cells. The inhibitory activity associated with disrupted tumour cells could explain why such preparations are less effective tumour vaccines than apoptotic tumour cells.     

Identification of two subpopulations of purified human blood B cells, CD27- CD23+ and CD27high CD80+, that strongly express cell surface Toll-like receptor 9 and secrete high levels of interleukin-6

B‐cell expression of certain Toll‐like receptors (TLRs) is important in linking innate and adaptive immune responses in normal and pathological conditions. The expression of TLR9 plays a role in the recognition of conserved pathogen motifs in a manner that is dependent on B‐cell localization, deduced from B‐cell phenotype. The nature of TLR9 function is unclear. A first step in unravelling the function of this pattern recognition receptor is to discover the precise nature of the cell types that express TLR9. This study used three‐colour flow cytometry to characterize the B lymphocytes from human peripheral blood mononuclear cells (PBMCs) that express TLR9 on the surface. We sorted TLR9‐positive B and non‐B cells from the PBMC population and detected TLR9 expression on naïve and memory B cells. Moreover, we identified two discrete subpopulations of B cells: CD19⁺ CD27^? CD23⁺ cells and CD19⁺ CD27^high CD80⁺ cells. These subpopulations expressed high levels of membrane TLR9 and exhibited a strong in vitro response to binding a relevant CpG motif by secreting high levels of interleukin‐6 (compared to controls). Our finding that this pattern recognition receptor is expressed on a variety of cell subsets adds to the current understanding of the functional complexity of B‐cell membrane TLR9.     

Innate responses of B cells

In this review, we describe the non-antibody-mediated functions of B cells within the immune system. In addition to antibody production, B cells also present antigen to T cells, programme T cell differentiation and regulate effector T cell responses and much of this is mediated by the cytokines they make. We focus on the potential of B cells to perform these functions simply as a result of activation via 'innate' receptors (e.g. Toll-like receptors) and often independently of BCR ligation. We feel an appreciation of these broad and often antigen-nonspecific functions is important at a time when there is an increasing use of B cell depletion as a therapy for autoimmune disease.     

Phosphorothioate‐modified CpG oligodeoxynucleotides mimic autoantigens and reveal a potential role for Toll‐like receptor 9 in receptor revision

Re‐expression of recombinase activating genes (RAG) in mature B cells may support autoreactivity by enabling revision of the B‐cell receptor (BCR). Recent reports suggest that administration of Toll‐like receptor 9 (TLR9) ‐stimulating CpG oligodeoxynucleotides (ODN) could trigger the manifestation of autoimmune disease and that TLR are involved in the selection processes eliminating autoreactive BCR. The mechanisms involved remain to be elucidated. This prompted us to ask, whether TLR9 could be involved in receptor revision. We found that phosphorothioate‐modified CpG ODN (CpG^PTO) induced expression of Ku70 and re‐expression of RAG‐1 in human peripheral blood B lymphocytes and Igλ expression in sorted Igκ⁺ B cells. Further results revealed unselective binding specificity of CpG^PTO‐induced immunoglobulin and suggested that CpG^PTO engage and/or mimic IgM receptor signalling, an important prerequisite for the initialization of receptor editing or revision. Altogether, our data describe a potential role for TLR9 in receptor revision and suggest that CpG^PTO could mimic chromatin‐bearing autoantigens by simultaneously engaging the BCR and TLR9 on IgM⁺ B cells.     

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis,selectively induces pro‐inflammatory cytokines and IL‐10 and is regulated by TNF‐α, IL‐10 and IFN‐γ

Leukocyte immunoglobulin‐like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane‐bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross‐linking of LILRA5 on monocytes induced production of pro‐inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common γ chain of the FcR and LILRA5 cross‐linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro‐inflammatory cytokines as well as IL‐10. LILRA5 mRNA and protein expression was tightly regulated by TNF‐α, IL‐10 and IFN‐γ. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.     

Modification of TLR-induced activation of human dendritic cells by type I IFN: synergistic interaction with TLR4 but not TLR3 agonists

Upon detection of direct and indirect signs of infection, dendritic cells (DC) undergo functional changes that modify their ability to elicit immune responses. Type I interferon (IFN-alpha/beta), which includes a large family of closely related infection-inducible cytokines, represents one indirect signal that can act as a DC stimulus. We have investigated the ability of IFN-alpha/beta subtypes to affect DC function and to influence DC responses to Toll-like receptor (TLR) agonists (i.e., direct infection-associated signals). Subtle differences were observed among 15 subtypes of IFN-alpha/beta in the ability to stimulate expression of maturation markers and chemokines by human monocyte-derived DC, with IFN-omega being the most unique in its effects. Pre-treatment with IFN-alpha/beta did not alter the ability of DC to mature in response to subsequent contact with TLR agonists, but did modulate their secretion of chemokines. Conversely, IFN-alpha/beta was shown to act synergistically with TLR4 but not TLR3 agonists for the induction of maturation and chemokine production when DC were exposed to IFN-alpha/beta and TLR ligands simultaneously. Taken together, these results indicate a complex role for IFN-alpha/beta in regulating DC function during the course an infection, which varies according to IFN-alpha/beta subtype and the timing of exposure to other stimuli.     

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.     

Toll‐like receptors – sentries in the B‐cell response

Toll‐like receptors (TLR) play a central role in the initiation of the innate immune response to pathogens. Upon recognition of molecular motifs specific for microbial molecules TLR mediate pro‐inflammatory cytokine secretion and enhance antigen presentation; in B cells they further promote expansion, class switch recombination and immunoglobulin secretion. As a result of their adjuvant properties, TLR ligands have become an integral component of antimicrobial vaccines. In spite of this, little is known of the direct effects of TLR engagement on B‐lymphocyte function. The scope of this review is to outline the differences in TLR expression and reactivity in murine and human B‐cell subsets and to provide an overview of the currently available literature. We will further discuss the possible roles of TLR in regulating B‐cell effector functions and shaping antibody‐mediated defence against microbial pathogens in vivo.     

TLR9 signaling is essential for the innate NK cell response in murine cutaneous leishmaniasis

Mice deficient for the TLR adaptor molecule MyD88 succumb to a local infection with Leishmania (L.) major. However, the TLR(s) that contribute to the control of this intracellular parasite remain to be defined. Here, we show that TLR9 was required for the induction of IL-12 in bone marrow-derived DC by intact L. major parasites or L. major DNA and for the early IFN-gamma expression and cytotoxicity of NK cells following infection with L. major in vivo. During the acute phase of infection TLR9-/- mice exhibited more severe skin lesions and higher parasite burdens than C57BL/6 wild-type controls. Although TLR9 deficiency led to a transient increase of IL-4, IL-13 and arginase 1 mRNA and a reduced expression of iNOS at the site of infection and in the draining lymph nodes, it did not prevent the development of Th1 cells and the ultimate resolution of the infection. We conclude that TLR9 signaling is essential for NK cell activation, but dispensable for a protective T cell response to L. major in vivo.     

DX5+CD4+ T cells modulate cytokine production by CD4+ T cells towards IL‐10 via the production of IL‐4

CD4⁺ Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune‐modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5⁺CD4⁺ T cells, which upon adoptive transfer show potent regulatory properties in murine collagen‐induced arthritis as well as in delayed‐hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5⁺CD4⁺ T cells on other CD4⁺ T cells in vitro. Although proliferation of naïve CD4⁺ T cells upon antigenic triggering was not altered in the presence of DX5⁺CD4⁺ T cells, there was a striking difference in cytokine production. In the presence of DX5⁺CD4⁺ T cells, an IL‐10‐producing CD4⁺ T‐cell response was induced instead of a predominant IFN‐γ‐producing Th1 response. This modulation did not require cell–cell contact. Instead, IL‐4 produced by DX5⁺CD4⁺ T cells was primarily involved in the inhibition of IFN‐γ and promotion of IL‐10 production by CD4⁺ T cells. Together, our data indicate that DX5⁺CD4⁺ T cells modulate the outcome of Th‐responses by diverting Th1‐induction into Th responses characterized by the production of IL‐10.     

FCRL3 promotes TLR9‐induced B‐cell activation and suppresses plasma cell differentiation

Fc receptor‐like (FCRL) molecules are preferentially expressed by B lymphocytes and possess tyrosine‐based immunoregulatory function. Although they generally inhibit B‐cell receptor signaling, their influence on other activation pathways remains largely unexplored. In humans, FCRL3 encodes a type I transmembrane protein harboring both cytoplasmic ITAM and ITIM elements that can repress B‐cell receptor activation. Despite this inhibitory property, mounting associations for FCRL3 with autoimmune and lympho‐proliferative disorders imply a role for it in promoting B‐cell pathogenesis. Here, we explore the influence of FCRL3 on B‐cell responses to innate TLR9 stimulation. A detailed survey of blood B‐cell populations found that FCRL3 expression increased as a function of differentiation and was higher among memory subsets with innate‐like features. FCRL3 ligation augmented CpG oligodeoxynucleotide TLR9‐mediated B‐cell proliferation, activation, and survival, but surprisingly, abrogated plasma cell differentiation and antibody production. Although FCRL3 amplified the NF‐κB and mitogen‐activated protein kinase signaling cascades, it halted CpG triggered BLIMP1 induction in an ERK‐dependent fashion. These findings indicate that FCRL3 differentially modulates innate signaling in B cells and provide new insight into the potential of this disease‐associated receptor to counter‐regulate adaptive and innate immunity.