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.     

Hepatic B cells are readily activated by Toll‐like receptor‐4 ligation and secrete less interleukin‐10 than lymphoid tissue B cells

B cells perform various immunological functions that include production of antibody, presentation of antigens, secretion of multiple cytokines and regulation of immune responses mainly via their secretion of interleukin (IL)‐10. While the liver is regarded both as an important immune organ and a tolerogenic environment, little is known about the functional biology of hepatic B cells. In this study we demonstrate that, following lipopolysaccharide (LPS) stimulation in vivo, normal mouse hepatic B cells rapidly increase their surface expression of CD39, CD40, CD80 and CD86, and produce significantly elevated levels of proinflammatory interferon (IFN)‐γ, IL‐6 and tumour necrosis factor (TNF)‐α compared with splenic B cells. Moreover, LPS‐activated hepatic B cells produce very low levels of IL‐10 compared with activated splenic B cells that produce comparatively high levels of this immunosuppressive cytokine. Splenic, but not hepatic, B cells inhibited the activation of liver conventional myeloid dendritic cells (mDCs). Furthermore, compared with the spleen, the liver exhibited significantly smaller proportions of B1a and marginal zone‐like B cells, which have been shown to produce IL‐10 upon LPS stimulation. These data suggest that, unlike in the spleen, IL‐10‐producing regulatory B cells in the liver are not a prominent cell type. Consistent with this, when compared with liver conventional mDCs from B cell‐deficient mice, those from B cell‐competent wild‐type mice displayed enhanced expression of the cell surface co‐stimulatory molecule CD86, greater production of proinflammatory cytokines (IFN‐γ, IL‐6, IL‐12p40) and reduced secretion of IL‐10. These findings suggest that hepatic B cells have the potential to initiate rather than regulate inflammatory responses.     

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.     

5,7‐dihydroxy‐3,4,6‐trimethoxyflavone inhibits the inflammatory effects induced by Bacteroides fragilis enterotoxin via dissociating the complex of heat shock protein 90 and IκBα and IκB kinase‐γ in intestinal epithelial cell culture

Enterotoxin produced by enterotoxigenic Bacteroides fragilis (BFT) has been associated with mucosal inflammation and diarrhoeal diseases. In this study, the anti‐inflammatory molecular mechanism of 5,7‐dihydroxy‐3,4,6‐trimethoxyflavone (eupatilin) was characterized in an HT‐29 intestinal epithelial cell line stimulated with BFT. Pre‐treatment of HT‐29 cells with eupatilin decreased the production significantly of both interleukin (IL)‐8 and prostaglandin E₂ induced by BFT in a dose‐dependent manner. BFT‐activated nuclear factor‐kappaB (NF‐κB) signals in HT‐29 cells and pretreatment with eupatilin suppressed NF‐κB activation that resulted in the significant inhibition of IL‐8 and cyclo‐oxygenase‐2 expression. BFT‐induced phosphorylation of both IκBα and IκB kinase (IKK) signals was prevented in eupatilin‐pretreated HT‐29 cells. Transfection of siRNA for IKK‐α and IKK‐β decreased the production of IL‐8 and prostaglandin E₂; however, the transfection of IKK‐β siRNA showed a more significant reduction of BFT‐induced IκBα phosphorylation compared with that of IKK‐α siRNA. In addition, herbimycin A, a specific inhibitor of heat shock protein 90 (Hsp90), decreased the BFT‐induced activation of IKK and NF‐κB, suggesting that Hsp90 is associated with a pathway of IKK‐NF‐κB‐IL‐8/cyclo‐oxygenase‐2 gene signalling. Furthermore, eupatilin dissociated the complex between Hsp90 and IKK‐γ in BFT‐stimulated HT‐29 cells. These results suggest that eupatilin can suppress the NF‐κB signalling pathway by targeting the Hsp90‐IKK‐γ complex in intestinal epithelial cells and may attenuate BFT‐induced inflammatory responses.     

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.     

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.     

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.     

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.     

Down‐regulation of miR‐301a suppresses pro‐inflammatory cytokines in Toll‐like receptor‐triggered macrophages

In many types of tumours, especially pancreatic adenocarcinoma, miR‐301a is over‐expressed. This over‐expression results in negative regulation of the target gene of miR‐301a, the nuclear factor‐κB (NF‐κB) repressing factor (NKRF), increasing the activation of NF‐κB and production of NF‐κB‐responsive pro‐inflammatory cytokines such as interleukin‐8, interferon‐β, nitric oxide synthase 2A and cytochrome oxidase subunit 2 (COX‐2). However, in immune cells, mechanisms that regulate miR‐301a have not been reported. Similar to tumour cells, Toll‐like receptor (TLR) ‐activated macrophages produce NF‐κB‐responsive pro‐inflammatory cytokines. Therefore, it is of considerable interest to determine whether miR‐301a regulates the secretion of cytokines by immune cells. In the present study, we demonstrate that the expression of miR‐301a was decreased in TLR‐triggered macrophages. Through targeting NKRF, miR‐301a affected the activity of NF‐κB and the expression of pro‐inflammatory genes downstream of NF‐κB such as COX‐2, prostaglandin E₂ and interleukin‐6. In addition, when lipopolysaccharide‐treated macrophages were simultaneously stimulated with trichostatin A, an inhibitor of histone deacetylases, the expression of miR‐301a increased, whereas NKRF and pro‐inflammatory cytokine expression decreased. However, further investigation revealed that there was no correlation between the induction of miR‐301a and the inhibitory effect of trichostatin A on lipopolysaccharide‐induced gene expression in macrophages. In summary, our study indicates a new mechanism by which miR‐301a regulates inflammatory cytokine expression in macrophages, which may clarify the regulatory role of microRNAs in immune‐mediated inflammatory responses.     

Protection against collagen‐induced arthritis in mice afforded by the parasitic worm product,ES‐62, is associated with restoration of the levels of interleukin‐10‐producing B cells and reduced plasma cell infiltration of the joints

We have previously reported that ES‐62, a molecule secreted by the parasitic filarial nematode Acanthocheilonema viteae, protects mice from developing collagen‐induced arthritis (CIA). Together with increasing evidence that worm infection may protect against autoimmune conditions, this raises the possibility that ES‐62 may have therapeutic potential in rheumatoid arthritis and hence, it is important to fully understand its mechanism of action. To this end, we have established to date that ES‐62 protection in CIA is associated with suppressed T helper type 1 (Th1)/Th17 responses, reduced collagen‐specific IgG2a antibodies and increased interleukin‐10 (IL‐10) production by splenocytes. IL‐10‐producing regulatory B cells have been proposed to suppress pathogenic Th1/Th17 responses in CIA: interestingly therefore, although the levels of IL‐10‐producing B cells were decreased in the spleens of mice with CIA, ES‐62 was found to restore these to the levels found in naive mice. In addition, exposure to ES‐62 decreased effector B‐cell, particularly plasma cell, infiltration of the joints, and such infiltrating B cells showed dramatically reduced levels of Toll‐like receptor 4 and the activation markers, CD80 and CD86. Collectively, this induction of hyporesponsiveness of effector B‐cell responses, in the context of the resetting of the levels of IL‐10‐producing B cells, is suggestive of a modulation of the balance between effector and regulatory B‐cell responses that may contribute to ES‐62‐mediated suppression of CIA‐associated inflammation and inhibition of production of pathogenic collagen‐specific IgG2a antibodies.     

Sequence independent interferon‐α induction by multimerized phosphodiester DNA depends on spatial regulation of Toll‐like receptor‐9 activation in plasmacytoid dendritic cells

Single‐stranded versus multimeric phosphorothioate‐modified CpG oligodeoxynucleotides (ODNs) undergo differential endosomal trafficking upon uptake into plasmacytoid dendritic cells (pDCs), correlating with Toll‐like receptor‐9‐dependent pDC maturation/activation (single‐stranded B‐type CpG ODN) or interferon‐α (IFN‐α) induction (multimeric A‐type CpG ODN), respectively. As was recently shown, IFN‐α production, other than by CpG ODNs, can also be induced in a sequence‐independent manner by phosphodiester (PD) ODNs multimerized by 3′ poly‐guanosine (poly‐G) tails. We investigate here the type of endosomal trafficking responsible for IFN‐α induction by natural PD ODN ligands. We show that 3′ extension with poly‐G tails leads to multimerization of single‐stranded PD ODNs and to enhanced cellular uptake into pDCs. While monomeric PD ODNs, which induce CpG‐dependent Toll‐like receptor‐9 stimulation and pDC maturation/activation, localized to late endosomal/lysosomal compartments, the poly‐G multimerized PD ODNs, which induce CpG‐independent IFN‐α production, located to vesicles with a distinct, ‘early’ endosomal phenotype. We conclude that poly‐G‐mediated multimerization of natural PD ODNs allows for sequence‐independent, Toll‐like receptor‐9‐dependent IFN‐α induction in pDCs by combining three distinct effects: relative protection of sensitive PD ODNs from extracellular and intracellular DNase degradation, enhanced cellular uptake and preferential early endosomal compartmentation.     

In CD28‐costimulated human naïve CD4+ T cells,I‐κB kinase controls the expression of cell cycle regulatory proteins via interleukin‐2‐independent mechanisms

Stimulation of naïve CD4⁺ T cells through engagement of the T‐cell receptor (TCR) and the CD28 co‐receptor initiates cell proliferation which critically depends on interleukin (IL)‐2 secretion and subsequent autocrine signalling via the IL‐2 receptor. However, several studies indicate that in CD28‐costimulated T cells additional IL‐2‐independent signals are also required for cell proliferation. In this study, using a neutralizing anti‐human IL‐2 antibody and two selective, structurally unrelated, cell‐permeable I‐κB kinase (IKK) inhibitors, BMS‐345541 and PS‐1145, we show that in human naïve CD4⁺ T cells stimulated through a short engagement of the TCR and the CD28 co‐receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin‐dependent kinase 2 (CDK2) and the stability of the F‐box protein S‐phase kinase‐associated protein 2 (SKP2) and its co‐factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL‐2‐independent mechanisms.     

Increased immunoglobulin A in alcoholic liver cirrhosis: exploring the response of B cells to Toll‐like receptor 9 activation

Alcoholic liver cirrhosis (ALC) is characterized by increased circulating levels of immunoglobulins (Igs). ALC patients undergo bacterial translocation evidenced by the presence of bacterial DNA in peripheral blood. Bacterial pathogen‐associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN) and unmethylated cytosine‐guanine dinucleotide (CpG) DNA are ligands of Toll‐like receptor (TLR)‐4, TLR‐2 and TLR‐9, respectively. Although TLR activation results generally in the secretion of proinflammatory cytokines, activation of B cells through TLR‐7 or TLR‐9 is involved in their maturation and Ig synthesis. The aim of the present study was to assess Ig synthesis by ALC B cells under PAMP activation in order to evaluate the possible involvement of TLR pathways in the increased Ig levels, and especially the hyper‐IgA observed in ALC. CpG, in combination with interleukin (IL)‐10 or IL‐21, enhanced IgA, IgG and IgM synthesis by healthy donor (HD) PBMCs, but had only a weak effect on ALC PBMCs. Relative CpG‐induced IgA production by purified ALC B cells was less important when compared to HD B cells, in accordance with the lower TLR‐9 expression on ALC B cells compared to HD B cells, but the absolute IgA production by CpG‐activated B cells was enhanced significantly for ALC when compared to HD, in agreement with their intrinsic ability to produce spontaneously more IgA than HD. LPS and PGN had no direct activity on B cells, whereas R848 also enhanced Ig synthesis, as reported recently. Taken together, these results suggest that TLR priming of B cells could account for the hyperimmunoglobulinaemia observed in ALC patients.