Leishmania expressed lipophosphoglycan interacts with Toll‐like receptor (TLR)‐2 to decrease TLR‐9 expression and reduce anti‐leishmanial responses

Two different Toll‐like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR‐2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR‐9 has been shown to promote a host‐protective response. However, whether there is a relationship between the interaction of LPG and TLR‐2, on one hand, with the effect of TLR‐9, on the other hand, remains unknown. In this study, we report that in‐vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR‐9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti‐LPG as well as anti‐TLR‐2 antibodies prevents this reduction of TLR‐9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR‐9 expression, which is shown to be mediated by transforming growth factor (TGF)‐β and interleukin (IL)‐10. Finally, in‐vitro treatment of macrophages with anti‐LPG and/or anti‐TLR‐2 antibodies before infection reduces the number of amastigotes in macrophages and co‐treatment of mice with anti‐TLR‐2 antibodies and cytosine–phosphate–guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)‐γ‐predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR‐2 reduce anti‐leishmanial responses via cytokine‐mediated decrease of TLR‐9 expression.     

Triggering of TLR‐3, ‐4, NOD2, and DC‐SIGN reduces viral replication and increases T‐cell activation capacity of HIV‐infected human dendritic cells

A variety of signals influence the capacity of dendritic cells (DCs) to mount potent antiviral cytotoxic T‐cell (CTL) responses. In particular, innate immune sensing by pathogen recognition receptors, such as TLR and C‐type lectines, influences DC biology and affects their susceptibility to HIV infection. Yet, whether the combined effects of PPRs triggering and HIV infection influence HIV‐specific (HS) CTL responses remain enigmatic. Here, we dissect the impact of innate immune sensing by pathogen recognition receptors on DC maturation, HIV infection, and on the quality of HS CTL activation. Remarkably, ligand‐driven triggering of TLR‐3, ‐4, NOD2, and DC‐SIGN, despite reducing viral replication, markedly increased the capacity of infected DCs to stimulate HS CTLs. This was exemplified by the diversity and the quantity of cytokines produced by HS CTLs primed by these DCs. Infecting DCs with viruses harboring members of the APOBEC family of antiviral factors enhanced the antigen‐presenting skills of infected DCs. Our results highlight the tight interplay between innate and adaptive immunity and may help develop innovative immunotherapies against viral infections.     

Association of the polymorphism of the Toll‐like receptor (TLR)‐3 and TLR‐9 genes with hepatitis C virus‐specific cell‐mediated immunity outcomes among Egyptian health‐care workers

Variations in the immune response could explain resistance to hepatitis C virus (HCV) infection. Toll‐like receptor gene (TLR)‐3 is an innate detector of dsRNA viruses, and the TLR‐9 gene recognizes bacterial and viral unmethylated cytosine–phosphate–guanosine (CpG) motifs. We previously reported that the TLR‐3.rs3775290 CC genotype was associated with HCV chronicity and that the TLR‐9 gene played no major role in this infection. This study identified the role of TLR‐3.rs3775290 (c.1377C/T), TLR‐9.rs5743836 (?1237T→C) and TLR‐9.rs352140 (G2848A) gene polymorphisms in predicting the outcome of HCV‐specific cell‐mediated immunity (CMI) among Egyptian health‐care workers (HCWs). We enrolled 265 HCWs in this study and divided them into four groups. Group 1: 140 seronegative‐aviraemic HCWs; group 2: 20 seronegative‐viraemic HCWs; group 3: 35 subjects with spontaneously resolved HCV infection; and group 4: 70 chronic HCV HCWs (patients). All subjects were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis for the TLR‐3.rs3775290, TLR‐9.rs5743836 and TLR‐9.rs352140 single nucleotide polymorphisms (SNPs). We also quantified HCV‐specific CMI in the four groups using an interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assay in response to nine HCV genotype 4a, overlapping 15mer peptide pools covering the whole viral genome. No statistically significant difference was found between CMI‐responding subjects with different HCV states and TLR‐3.rs3775290 or TLR‐9.rs352140 genotypes. However, there was a significant relationship between the outcome of the HCV‐specific CMI and the TLR‐9.rs5743836 genotype among the responding subjects (P = 0·005) and the chronic HCV patients (P = 0·044). In conclusion, TLR‐9.rs5743836 SNP, but not TLR‐3.rs3775290 or TLR‐9.rs352140 genotypes, could predict the outcome of HCV‐specific CMI responses among Egyptians infected with genotype‐4.     

MYD88‐dependent and ‐independent activation of TREM‐1 via specific TLR ligands

Triggering receptor expressed on myeloid cells (TREM)‐1 plays an important role in myeloid cell‐activated inflammatory responses. Although TLR ligands such as LPS and lipoteichoic acid have been shown to upregulate TREM‐1 expression in macrophage and neutrophils, the role of specific TLR in inducing the expression of TREM‐1 remains unclear. In this study, we investigated whether the presence of TLR is necessary for the expression of TREM‐1. We show that BM‐derived macrophages from TLR4 and TLR2 KO mice failed to induce expression of TREM‐1 message and protein in response to their specific ligands. Interestingly, the expression of TREM‐1 in response to LPS is not altered in myeloid differentiation factor 88 (MyD88) KO macrophages, suggesting that downstream of TLR a MyD88‐independent pathway induces the expression of TREM‐1. Inhibiting toll/IL‐1R domain‐containing adaptor‐inducing IFN‐β (TRIF) expression by siRNA decreased TREM‐1 expression in response to LPS, suggesting that the expression of TREM‐1 in response to LPS was mediated by the TRIF signaling pathway. On the other hand, the expression of TREM‐1 in response to lipoteichoic acid is dependent on MyD88 expression. These data indicate that the expression of TREM‐1 in response to TLR ligands occurs secondary to downstream signaling events and that the presence of TLR is necessary for the expression of TREM‐1 in response to their specific ligands. However, the downstream signaling required for the expression of TREM‐1 is dependent on the stimulus and the surface receptor through which the signaling is initiated.     

OK‐432‐stimulated chemokine secretion from human monocytes depends on MEK1/2, and involves p38 MAPK and NF‐κB phosphorylation,in vitro

Interaction between the immune system and cancer cells allows for the use of biological response modifiers, like OK‐432, in cancer therapy. We have studied the involvement of monocytes (MOs) in the immune response to OK‐432 by examining MCP‐1, MIP‐1α and MIP‐1β secretion, in vitro. OK‐432‐induced IL‐6/TNF‐α secretion has previously been shown to depend on mitogen‐activated protein kinases (MAPKs) ERK1/2 and p38, and we therefore investigated the role of these MAPKs in OK‐432‐induced chemokine secretion. Here we demonstrate that pharmacological MEK1/2 kinase inhibition generally impaired chemokine secretion from MOs, whereas p38 MAPK inhibition in particular reduced MIP‐1α production. Furthermore, simultaneous inhibition of MEK1/2 and Syk kinase was seen to have an additive impact on reduced MCP‐1, MIP‐1α and MIP‐1β secretion. Based on single cell flow cytometry analyses, OK‐432, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were seen to induce p38 MAPK and NF‐κB phosphorylation in MOs with different time kinetics. LTA and LPS have been shown to induce ERK1/2 phosphorylation, whereas the levels of phosphorylated ERK1/2 remained constant following OK‐432 treatment at the time points tested. Toll‐like receptors (TLRs) recognize pathogen‐associated molecular patterns, and we demonstrate increased TLR2 cell surface levels on the MO population, most profoundly following stimulation with LTA and OK‐432. Together these results indicate that modulation of MEK1/2 and p38 MAPK signalling could affect the response to OK‐432 treatment, having the potential to improve its therapeutic potential within cancer and lymphangioma treatment.     

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.     

IRF‐5 and NF‐κB p50 co‐regulate IFN‐β and IL‐6 expression in TLR9‐stimulated human plasmacytoid dendritic cells

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN‐β) and pro‐inflammatory (IL‐6) cytokines by CpG‐stimulated human pDCs and the human CAL‐1 pDC cell line. Results show that interferon regulatory factor‐5 (IRF‐5) and NF‐κB p50 are key co‐regulators of IFN‐β and IL‐6 expression following TLR9‐mediated activation of human pDCs. The nuclear accumulation of IRF‐1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF‐8 was identified as a novel negative regulator of gene activation in CpG‐stimulated pDCs. As variants of IRF‐5 and IRF‐8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of “K” ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.     

Co‐expression of TLR‐9 and MMP‐13 is associated with the degree of tumour differentiation in prostate cancer

In vitro experiments demonstrated that stimulation of Toll‐like receptor 9 (TLR‐9) by synthetic TLR‐9 ligands induces the invasion of TLR‐9‐expressing prostate cancer cells through matrix metalloproteinase 13 (MMP‐13). However, the clinical value of TLR‐9 and MMP‐13 co‐expression in the pathophysiology of the prostate is unknown. In the study, we evaluated the expression levels and clinical significance of the TLR‐9 and MMP‐13 in a series of prostate tissues. One hundred and eighty prostate tissues including prostate cancer (PCa) (n = 137), high‐grade prostatic intraepithelial neoplasia (HPIN) (n = 18) and benign prostatic hyperplasia (BPH) (n = 25) were immunostained for the TLR‐9 and MMP‐13 markers. Subsequently, the correlation between the TLR‐9 and MMP‐13 staining scores and clinicopathological parameters was obtained. Higher expressions of TLR‐9 and MMP‐13 were found in PCa and high‐grade prostatic intraepithelial neoplasia compared to benign prostatic hyperplasia tissues. Among PCa samples, a positive relationship was revealed between the MMP‐13 expression and Gleason score (P < 0.001). There was a significant correlation between TLR‐9 expression and regional lymph node involvement (P = 0.04). The expression patterns of TLR‐9 and MMP‐13 markers demonstrated a reciprocal significant correlation between the two markers in the same series of prostate samples (P < 0.001). Furthermore, the Gleason score of TLR‐9^high/MMP‐13^high and TLR‐9^low/MMP‐13^low phenotypes showed a significant difference (P = 0.002). Higher expressions of TLR‐9 and MMP‐13 can confer aggressive behaviour to PCa. Therefore, these markers may be used as a valuable target for tailored therapy of PCa.     

Toll‐like receptors: The role in bladder cancer development,progression and immunotherapy

Bladder cancer is one of the leading causes of death worldwide. The main immune mechanisms which lead to bladder cancer development or treatment outcomes have yet to be elucidated. Toll‐like receptors (TLRs) play key roles against cancer. TLRs are expressed both on immune cells and on tumour cells and drive immune responses in progression as well as treatment of cancer. Identification of signalling pathways via TLRs could revolutionize further improvement of therapeutic strategies against cancers in the future. According to the recent studies, TLRs agonists are effective immunostimulants and have important role in induction of immune responses with immunotherapeutic potential against several diseases including cancer. They play an important role in the bladder urothelium as a part of immune defence against uropathogens. On the other hand, decreased TLRs expression was found in bladder tumours, particularly in non‐muscle‐invasive ones. Bacillus Calmette‐Guerin (BCG) (agonist of TLR2 and TLR4) is approved by US FDA for immunotherapy of bladder cancer. Despite high efficiency, immunotherapy with BCG may cause toxicity and adverse effects. Nowadays, in vitro and in vivo studies have been conducted to find alternative options for non‐responder patients. Studies on TLR agonists for bladder cancer treatment have shown promising results. In this review, we discuss recent data about mechanisms played by TLRs in bladder cancer developments as well as therapeutic application of TLR agonists in cancer treatment.     

SARM inhibits both TRIF‐ and MyD88‐mediated AP‐1 activation

SARM (sterile α‐ and armadillo‐motif‐containing protein), the fifth identified TIR (Toll–interleukin 1 receptor (IL‐1R)) domain‐containing adaptors in humans, downregulates NF‐κB and IRF3 (interferon‐regulatory factor 3)‐mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR‐domain‐containing adaptor protein inducing IFN‐β)‐dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF‐ and MyD88‐mediated, as well as basal MAPK activity, were repressed, indicating that SARM‐mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP‐1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N‐terminal and sterile alpha motif domains in the autoregulation of SARM activity.     

Characterization of innate immune signalling receptors in virus‐induced acute asthma

Background The role of toll‐like receptors (TLRs) and innate immune activation in clinical asthma exacerbations and their relationship to virus infection are unclear. Objective This study aimed to characterize TLR expression and innate immune activity during virus infection in acute asthma. Methods Subjects with acute asthma, stable asthma and healthy controls were recruited and underwent spirometry and sputum induction with isotonic saline. Selected sputum was dispersed with dithiothreitol and total and differential leucocyte counts were performed. Selected sputum was also used for quantitative real‐time PCR for TLR2, TLR3, TLR4, IL‐10 and IP‐10mRNA expression. Sputum supernatant was used for the measurement of innate immune markers, including IL‐8, matrix metalloproteinase‐9 and neutrophil elastase activity. Viruses were detected using real‐time and gel‐based PCR. Results Sputum TLR2 mRNA expression was up‐regulated in both acute and stable asthma compared with healthy controls and decreased 4–6 weeks after acute exacerbation. Sputum TLR2 mRNA expression was elevated in viral, compared with non‐viral, acute asthma. Sputum TLR3 mRNA expression was similar in controls, stable and acute asthma. However, in acute asthma, subjects with virus‐induced acute asthma had significantly higher sputum TLR3 mRNA expression. Induced sputum gene expression for IP‐10 and IL‐10 were increased in viral, compared with non‐viral, acute asthma. In virus‐induced acute asthma, levels of IP‐10 and IL‐10 mRNA expression were correlated with the mRNA expression of TLR2 and TLR3. Conclusions and Clinical Relevance Virus‐induced acute asthma leads to specific induction of TLR2, TLR3, IP‐10 and IL‐10, suggesting that signalling via TLRs may play an important role in mediating airway inflammation, via both innate and adaptive pathways, in virus‐induced exacerbations. These mediators may provide potential treatment targets for virus‐induced asthma. They may also be useful in diagnosing the nature of acute asthma exacerbations and monitoring treatment responses, which would be useful in the clinical management of asthma exacerbations. Cite this as: L. G. Wood, J. L. Simpson, P. A. B. Wark, H. Powell and P. G. Gibson, Clinical & Experimental Allergy, 2011 (41) 640–648.     

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.     

Imprinted DC mediate the immune‐educating effect of early‐life microbial exposure

It has been long proposed that exposure to environmental factors early in life may have an educating effect on the development of immune regulatory functions. However, experimental studies on this issue are limited and the related molecular and cellular basis remains unclear. Here we report that neonatal exposure to killed bacteria (Chlamydia muridarum, originally called Chlamydia trachomatis mouse pneumonitis (MoPn)) changed the pattern of the hosts' immune responses to a model allergen (OVA) in adulthood. This was associated with altered phenotype and function of DC. We found that DC from adult mice treated neonatally with UV‐killed MoPn exhibited distinct patterns of surface marker and TLR expression and cytokine production from control mice (DC from adult mice neonatally treated with vehicle, (Sham‐DC)). More importantly, DC from adult mice treated neonatally with UV‐killed MoPn induced significantly lower type‐2 antigen‐specific T‐cell responses than Sham‐DC shown in DC:T co‐culture experiments in vitro and in adoptive transfer experiments in vivo. In addition, depletion of T cells in vivo largely abolished the phenotypic and functional alterations of DC caused by bacterial exposure, suggesting the involvement of T cell in this process. Our study demonstrates a central role of DC in linking the early‐life exposure to microbial products and the balanced development of immune regulatory functions and the involvement of T cells in imprinting of the DC function.     

Different interleukin‐17‐secreting Toll‐like receptor+ T‐cell subsets are associated with disease activity in multiple sclerosis

Signalling through Toll‐like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR‐2, ‐4 and ‐9 was significantly higher on CD4⁺ and CD8⁺ T‐cells from MS patients compared to healthy individuals. Following in‐vitro activation, the proportion of interleukin (IL)‐17⁺ and IL‐6⁺ CD4⁺ and CD8⁺ T‐cells was higher in the patients. In addition, the proportion of IFN‐γ‐secreting TLR⁺ CD8⁺ T‐cells was increased in MS patients. Among different IL‐17⁺ T‐cell phenotypes, the proportion of IL‐17⁺ TLR⁺ CD4⁺ and CD8⁺ T‐cells producing IFN‐γ or IL‐6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4⁺ and CD8⁺ T‐cells with ligands for TLR‐2 (Pam3Csk4), TLR‐4 [lipopolysaccharide (LPS)] and TLR‐9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen‐associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL‐6, IFN‐γ, IL‐17 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) levels produced by Pam3Csk4‐activated CD4⁺ cells were directly associated with disease activity. A similar correlation was observed with regard to IL‐17 levels released by Pam3Csk4‐stimulated CD8⁺ T‐cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR‐2, ‐4 and ‐9 is associated with MS disease activity, and reveals a preferential ability of TLR‐2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.     

Tim‐3 exacerbates kidney ischaemia/reperfusion injury through the TLR‐4/NF‐κB signalling pathway and an NLR‐C4 inflammasome activation

T cell immunoglobulin domain and mucin domain‐containing molecule‐3 (Tim‐3), a member of the immunoglobulin superfamily, has been shown to play a crucial role in host adaptive immunity and tolerance. However, its role in kidney ischaemia–reperfusion injury (IRI) remains unknown. In this study, we investigated the role and mechanism of Tim‐3 signalling after kidney IRI. In an established murine model of kidney IRI, we found that Tim‐3 expression is enhanced on monocytes/macrophages. Anti‐Tim‐3 antibody RMT3‐23 ameliorates biochemical and histological kidney injury, reduces apoptosis and decreases macrophage infiltration and cytokine production in ischaemic kidneys. Cell culture experiments also demonstrated that the role of Tim‐3 in IRI‐induced macrophage activation leads to the secretion of proinflammatory cytokines and chemokines. In addition, Toll‐like receptor (TLR)‐4 and Nod‐like receptor (NLR) family CARD domain‐containing protein 4 (NLR‐C4) expression were enhanced after kidney IRI and decreased significantly by RMT3‐23. Tim‐3 not only promotes TLR‐mediated nuclear factor kappa B (NF‐κB) activation and cytokine and chemokine release, but also participates in NLR‐C4 inflammasome activation. Taken together, our data confirm that Tim‐3 signalling enhances injury after kidney IRI and demonstrated that Tim‐3 is involved in regulating TLR‐4/NF‐κB signalling and NLR‐C4 inflammasome activation, which provide evidence that Tim‐3 signalling is critical for kidney IRI and may provide a new means to ameliorate kidney tissue immune responses in the clinics.