Toll‐like receptor signalling in regenerative myogenesis: friend and foe

Skeletal muscle regeneration in normal and diseased muscle is regulated by multiple factors and cells present in the injured muscle micro‐environment. In addition to muscle progenitor cells, several immunocytes participate in the regenerative response. Among them, macrophages are one of the most important components of the immune response that governs the step‐wise progression of muscle regeneration. The initial role of macrophages is to phagocytose muscle cell debris and later, through their transition to an anti‐inflammatory phenotype, they promote regeneration. However, in several genetic muscle disorders, continuous muscle injury disrupts the balance between pro‐inflammatory and anti‐inflammatory macrophages, leading to an overall inflammatory milieu and inhibition of muscle regeneration. Accumulating evidence suggests that Toll‐like receptor (TLR)‐mediated signalling plays an important role in the regulation of macrophage phenotypes during regenerative myogenesis in response to both acute and chronic muscle injury. Here, we discuss the role of TLR signalling in regulating macrophage phenotypes and skeletal muscle regeneration in healthy and diseased muscle. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Patency of Litomosoides sigmodontis infection depends on Toll‐like receptor 4 whereas Toll‐like receptor 2 signalling influences filarial‐specific CD4+ T‐cell responses

BALB/c mice develop a patent state [release of microfilariae (Mf), the transmission life‐stage, into the periphery] when exposed to the rodent filariae Litomosoides sigmodontis. Interestingly, only a portion of the infected mice become patent, which reflects the situation in human individuals infected with Wuchereria bancrofti. Since those individuals had differing filarial‐specific profiles, this study compared differences in immune responses between Mf⁺ and Mf^– infected BALB/c mice. We demonstrate that cultures of total spleen or mediastinal lymph node cells from Mf⁺ mice produce significantly more interleukin‐5 (IL‐5) to filarial antigens but equal levels of IL‐10 when compared with Mf^– mice. However, isolated CD4⁺ T cells from Mf⁺ mice produced significantly higher amounts of all measured cytokines, including IL‐10, when compared with CD4⁺ T‐cell responses from Mf^– mice. Since adaptive immune responses are influenced by triggering the innate immune system we further studied the immune profiles and parasitology in infected Toll‐like receptor‐2‐deficient (TLR2^?/?) and TLR4^?/? BALB/c mice. Ninety‐three per cent of L. sigmodontis‐exposed TLR4^?/? BALB/c mice became patent (Mf⁺) although worm numbers remained comparable to those in Mf⁺ wild‐type controls. Lack of TLR2 had no influence on patency outcome or worm burden but infected Mf⁺ mice had significantly lower numbers of Foxp3⁺ regulatory T cells and dampened peripheral immune responses. Interestingly, in vitro culturing of CD4⁺ T cells from infected wild‐type mice with granulocyte–macrophage colony‐stimulating factor‐derived TLR2^?/? dendritic cells resulted in an overall diminished cytokine profile to filarial antigens. Hence, triggering TLR4 or TLR2 during chronic filarial infection has a significant impact on patency and efficient CD4⁺ T‐cell responses, respectively.     

Disordered Toll‐like receptor 2 responses in the pathogenesis of pulmonary sarcoidosis

In this study, we hypothesized that the granulomatous disorder sarcoidosis is not caused by a single pathogen, but rather results from abnormal responses of Toll‐like receptors (TLRs) to conserved bacterial elements. Unsorted bronchoalveolar lavage (BAL) cells from patients with suspected pulmonary sarcoidosis and healthy non‐smoking control subjects were stimulated with representative ligands of TLR‐2 (in both TLR‐2/1 and TLR‐2/6 heterodimers) and TLR‐4. Responses were determined by assessing resulting production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6. BAL cells from patients in whom sarcoidosis was confirmed displayed increased cytokine responses to the TLR‐2/1 ligand 19‐kDa lipoprotein of Mycobacterium tuberculosis (LpqH) and decreased responses to the TLR‐2/6 agonist fibroblast stimulating ligand‐1 (FSL)‐1. Subsequently, we evaluated the impact of TLR‐2 gene deletion in a recently described murine model of T helper type 1 (Th1)‐associated lung disease induced by heat‐killed Propionibacterium acnes. As quantified by blinded scoring of lung pathology, P. acnes‐induced granulomatous pulmonary inflammation was markedly attenuated in TLR‐2^–/– mice compared to wild‐type C57BL/6 animals. The findings support a potential role for disordered TLR‐2 responses in the pathogenesis of pulmonary sarcoidosis.     

Children with atopic histories exhibit impaired lipopolysaccharide‐induced Toll‐like receptor‐4 signalling in peripheral monocytes

Background The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll‐like receptor (TLR)‐4 on CD4⁺ cells in nasal mucosa. Objective To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4⁺ leucocytes. Methods Peripheral blood mononuclear cells from children (aged 2–18) and adults with or without a history of atopic conditions were cultured with/without IL‐4 or LPS for up to 24 h. Expression of surface TLR‐4, CD14, CD4, CD3, as well as of intracellular phosphorylated ^p42/p44ERK and p38 mitogen‐activated protein kinase (MAPK) were assessed by flow cytometry. Results A history of atopy in children was associated with impaired LPS‐induced TLR‐4‐dependent phosphorylation of ^p42/44ERK and p38 MAPK by CD4⁺ monocytes. Decreased LPS signalling was reproduced by pre‐incubation of control cells with recombinant IL‐4. LPS stimulation also decreased TLR‐4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4⁺ T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non‐atopic children, TLR‐4 expression on monocytes of children with atopic histories decreased as a function of age. Conclusions This study provides evidence for defective LPS recognition on circulating CD4⁺ leucocytes of subjects with atopic histories compared with those from non‐atopic children. CD4⁺TLR4⁺ monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR‐4‐mediated innate immune function compared with non‐atopic children. Cite this as: D. Préfontaine, A.‐A. Banville‐Langelier, P.‐O. Fiset, J. Guay, J. An, M. Mazer, Q. Hamid and B. D. Mazer, Clinical & Experimental Allergy, 2010 (40) 1648–1657.     

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.     

Toll‐like Receptor‐2 and Toll‐like Receptor‐4 Expression on Maternal Neutrophils During Pregnancy

Nitsche JF, Jiang S‐W, Brost BC. Toll‐like receptor‐2 and toll‐like receptor‐4 expression on maternal neutrophils during pregnancy. Am J Reprod Immunol 2010; 64: 427–434 Problem Toll‐like receptors (TLR) are an important part of the innate immune system and are present in a variety of human tissues. Work investigating the role of the TLR in pregnancy has thus far focused on placental tissue; however, minimal data is currently available concerning TLR expression in other tissues. Unlike placental tissue, neutrophils are easily retrievable during pregnancy and thus allow assessment of TLR’s prior to delivery. Method of study Using real time quantitative PCR this study investigated whether TLR‐2 and TLR‐4 expression on maternal neutrophils is altered throughout gestation or at the time of labor. A group of 12 non‐pregnant women and two groups of ten pregnant patients were enrolled and followed longitudinally, one group throughout gestation and one group throughout the third trimester. Results Although increased in the placenta, TLR2 and TLR4 expression on maternal neutrophils changes minimally throughout gestation. Conclusion There appears to be very little regulation of TLR2 and TLR4 at the mRNA level during normal pregnancy and labor. However, now that the normal values of TLR expression on maternal neutrophils have been determined it will be possible to compare them to those from pregnancies complicated by such conditions as preeclampsia, preterm labor, or preterm premature rupture of membranes.     

Activation profile of Toll‐like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.     

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.     

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.     

Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll‐like receptor 4 antagonist

Shigella dysenteriae causes the most severe of all infectious diarrhoeas and colitis. We infected rhesus macaques orally and also treated them orally with a small and non‐absorbable polypropyletherimine dendrimer glucosamine that is a Toll‐like receptor‐4 (TLR4) antagonist. Antibiotics were not given for this life‐threatening infection. Six days later, the clinical score for diarrhoea, mucus and blood was 54% lower, colon interleukin‐8 and interleukin‐6 were both 77% lower, and colon neutrophil infiltration was 75% less. Strikingly, vasculitis did not occur and tissue fibrin thrombi were reduced by 67%. There was no clinical toxicity or adverse effect of dendrimer glucosamine on systemic immunity. This is the first report in non‐human primates of the therapeutic efficacy of a small and orally bioavailable TLR antagonist in severe infection. Our results show that an oral TLR4 antagonist can enable controlled resolution of the infection‐related‐inflammatory response and can also prevent neutrophil‐mediated gut wall necrosis in severe infectious diarrhoeas.     

Dosage of X‐linked Toll‐like receptor 8 determines gender differences in the development of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease with a high incidence in females and a complex phenotype. Using 564Igi mice, a model of SLE with knock‐in genes encoding an autoreactive anti‐RNA Ab, we investigated how expression of Toll‐like receptors (TLRs) in B cells and neutrophils affects pathogenesis. We established that TLR signaling through MyD88 is necessary for disease. Autoantibody was produced in mice with single deletions of Tlr7, Tlr8, or Tlr9 or combined deletions of Tlr7 and Tlr9. Autoantibody was not produced in the combined absence of Tlr7 and Tlr8, indicating that TLR8 contributes to the break in tolerance. Furthermore, TLR8 was sufficient for the loss of B‐cell tolerance, the production of class‐switched autoantibody, heightened granulopoiesis, and increased production of type I IFN by neutrophils as well as glomerulonephritis and death. We show that dosage of X‐linked Tlr8 plays a major role in the high incidence of disease in females. In addition, we show that the negative regulation of disease by TLR9 is exerted primarily on granulopoiesis and type I IFN production by neutrophils. Collectively, we suggest that individual TLRs play unique roles in the pathogenesis of systemic lupus erythematosus, suggesting new targets for treatment.     

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.     

Possible evidence of systemic lupus erythematosus and periodontal disease association mediated by Toll‐like receptors 2 and 4

Toll‐like receptors (TLRs) participate in the innate immune response and trigger the immune responses of the body. Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology, characterized by an excessive autoimmune response in the body affecting the connective tissues. The disease is possibly triggered by both environmental aetiological factors and pathological organic processes such as exposure to sunlight, chronic infectious processes and genetic factors. Conversely, periodontal disease is an infectious disease caused by microorganisms in the oral cavity, resulting in a chronic inflammatory process which continuously stimulates the immune response, thus causing damage to the periodontal tissues. The expression of both TLR‐2 and TLR‐4 receptors are increased in both SLE and periodontal disease. Periodontitis might trigger excessive activation of immune response occurring in SLE by maintaining a high expression of TLRs, leading in turn to the acceleration of the onset and progression of autoimmune reactions. In addition, periodontal treatment is able to reduce the expression of these receptors and therefore the symptoms of SLE. Here we discuss the possible interaction between SLE and periodontitis, and suggest further studies evaluating common features in both factors that could explored, due to morbidity and mortality of SLE and the high incidence of periodontal infections around the world.     

Renal tubular expression of Toll‐like receptor 4 in cyclosporine nephrotoxicity

Lim BJ, Hong SW, Jeong HJ. Renal tubular expression of Toll‐like receptor 4 in cyclosporine nephrotoxicity. APMIS 2009; 117: 583–91. Exploring the expression of Toll‐like receptor (TLR) in cyclosporine (CsA)‐induced renal injury in humans, we evaluated the expression of TLR4 in both biopsied renal tissue and cultured tubular cells. Immunohistochemical stains for TLR4, heat shock protein (HSP) 47, and HSP70 were performed in both pre‐ and post‐treatment biopsies obtained from 18 patients of minimal change nephrotic syndrome or IgA nephropathy treated with CsA, and the percentage of positive tubules was compared in each case. For in vitro experiments, HK‐2 cells were treated with CsA (2, 5, and 10 μg/ml) for 24, 48, and 72 h. TLR4 mRNA and protein were measured using real‐time RT‐PCR and Western blot. In addition, hypoxic effect was added by GasPak System. The tubular expressions of TLR4 (2.2 ± 1.2% vs 4.4 ± 2.0%, p < 0.001) and HSP70 (2.6 ± 2.8% vs 6.1 ± 4.2%, p = 0.002) were increased after CsA treatment. TLR4 mRNA and protein expression were also increased in a dose‐dependent manner. Hypoxia enormously increased TLR4 expression. In summary, CsA increased tubular expression of TLR4 and its ligand HSP70. As hypoxia was shown to be a strong stimulus for TLR4 expression, it can be said that TLR4 is influenced by both direct toxicity and impediment of renal microcirculation in human CsA nephrotoxicity.     

Functional aspects of Toll‐like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii

Toll‐like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host–pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.     

Expression of Toll‐like Receptors in Genital Tract Tissues from Normal and HIV‐infected Men

Citation Pudney J, Anderson DJ. Expression of Toll‐like receptors in genital tract tissues from normal and HIV‐infected men. Am J Reprod Immunol 2011; 65: 28–43 Problem Cells of the innate immune system use Toll‐like receptors (TLRs) to recognize and respond to invading pathogens. This study was carried out to characterize TLR expression in the human male genital tract, an initial infection site for several sexually transmitted pathogens. Method of Study Immunohistochemistry was used to detect expression of TLRs 1–9 in genital tract tissues from HIV^? and HIV⁺ men. Results In HIV^? men, TLR1⁺ leukocytes were detected throughout the genital tract. Leukocytes in the penile urethra also expressed TLRs2, 3, 5, 7 and 9. Epithelial cells in most tissues did not express TLRs; exceptions were the prostate, where TLRs3 and 8 were observed on the apical surface of luminal epithelial cells, and the penile urethra, where epithelial cells expressed TLR9. In genital tissues from HIV⁺ men with AIDS, few TLR⁺ cells were detected. Conclusion Cells in the male genital tract can express a variety of TLRs. The penile urethra contained the highest number of TLR⁺ cells, indicating that this tissue plays a major role in the innate immune defense of the male genital tract. Overall, TLR expression was reduced in genital tissues from HIV⁺ men.     

The expression of Toll‐like receptors 2, 4 and 9 in kidneys of patients with anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis

Increasing evidence suggested that Toll-like receptors (TLRs) were critically involved in immune responses of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The current study aimed to investigate the expression of TLR-2, TLR-4 and TLR-9 in kidneys of patients with ANCA-associated vasculitis. Renal biopsy specimens were collected from 24 patients with AAV. The expression of TLR-2, TLR-4 and TLR-9 in kidneys was detected by immunohistochemistry. Double immunofluorescence staining was performed to detect the expression of TLRs on various kinds of cells. In renal specimens, immunohistochemical examination revealed that expression of TLR-2 and TLR-4 could be detected in the glomeruli of AAV patients, while TLR-2 and TLR-4 were scarcely detected in the glomeruli of normal controls. Double immunofluorescence staining of TLR-2, TLR-4 and CD31 indicated that TLR-4 and TLR-2 were expressed on endothelial cells in the glomeruli. In the tubulointerstitial compartment, expression of TLR-2, TLR-4 and TLR-9 could be detected in both AAV patients and normal controls. The mean optical density of TLR-2 and TLR-4 in the tubulointerstitial compartment in AAV patients were significantly higher than that in normal controls. Among AAV patients, correlation analysis showed that the mean optical density of TLR-4 in the glomeruli correlated inversely with the initial serum creatinine, the proportion of total crescents and the proportion of cellular crescents in renal specimens (r = −0·419, P = 0·041; r = −0·506, P = 0·012; r = −0·505, P = 0·012, respectively). The expression of TLR-2 and TLR-4 was dysregulated in kidneys of AAV patients. The expression of TLR-4 in glomeruli was associated with the severity of renal injury.