Association between polymorphisms in the genes encoding toll-like receptors and dectin-1 and susceptibility to invasive aspergillosis: a systematic review

Abstract Invasive aspergillosis is a common fungal infection in immunocompromised individuals. Some studies have shown that toll-like receptor and dectin-1 genetic polymorphisms may alter signaling pathways, thus increasing an individual's susceptibility to invasive aspergillosis. We investigated the pertinent literature to determine whether polymorphisms in the genes encoding toll-like receptors and dectin-1 increase the susceptibility to invasive aspergillosis. This study systematically reviewed the literature using the databases PubMed/PMC, Scopus, and Web of Science using the keywords invasive aspergillosis, polymorphism, Toll-like, and Dectin-1. From the initial search, 415 studies were found and according to our inclusion and exclusion criteria, eight studies were selected. Several studies described single-nucleotide polymorphisms (SNPs) that are associated with a greater susceptibility to invasive aspergillosis. These SNPs were found in the genes that encode toll-like receptors 1, 3, 4, and 5 and the gene that encodes dectin-1; upon activation, both cellular receptors initiate a signaling cascade that can result in the production of cytokines and chemokines. Thus, our literature review uncovered a significant association between polymorphisms in the genes that encode toll-like receptors and dectin-1 and invasive aspergillosis. More studies should be performed to better understand the relationship between toll-like receptor and dectin-1 genetic polymorphisms and invasive aspergillosis susceptibility.

Toll-Like Receptor Gene Expression during Trichinella spiralis Infection

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (T(reg)) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and T(reg) cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-beta expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and T(reg) cell mediated immune responses, although additional data are needed to convincingly prove this observation.

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury

PURPOSE: To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). RESULTS: After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome. .

Promoters, toll like receptors and microRNAs: A strange association.

Toll-like receptors (TLRs) are proteins that play key role in the innate immune system. In the present study, ~1000 base pairs upstream are taken from the transcription start site of the various TLR genes (10 known) in human. About 40 microRNAs have been identified that share 12-19 nucleotide sequence similarity with the promoter regions of 10 TLRs. It is proposed that the microRNA performs potential role in identification of promoter sequence and initiation of transcription.

Efecto de metformina sobre la expresión del factor de necrosis tumoral-α, los receptores Toll-like 2/4 y la PCR ultra sensible en sujetos obesos con diabetes tipo 2 / Effect of metformin on the expression of tumor necrosis factor-α, Toll like receptors 2/4 and C reactive protein in obese type-2 diabetic patients

Background: The pharmacological action of metformin goes beyond mere glycemic control, decreasing markers of inflammation and contributing to the reduction of oxidative stress. Aim: To evaluate biochemical, anthropometric and pro-inflammatory markers in obese type 2 diabetic patients treated or not with metformin. Patients and Methods: Obese patients with type 2 diabetes were invited to participate in the study if they were aged more than 40 years, were not receiving insulin, did not have cardiovascular diseases and were not taking anti-inflammatory drugs. A pharmacological history was taken and patients were stratified in two groups whether they were using metformin or not. A fasting blood sample was obtained to measure blood glucose, insulin, lipid levels, C reactive protein (hsCRP) and to isolate peripheral blood mononuclear cells. RNA was isolated from these cells to measure expression of tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), Toll-Like Receptor 2/4 (TLR 2/4) and beta-2-microglobulin (B2M). Results: Thirty participants were studied. Of these, 16 subjects aged 54.4 ± 5.5years were treated with metformin and 14 subjects aged 54.9 ± 6.4 years did not receive the drug. Participants receiving metformin had lower levels of hsCRP and lower mRNA relative abundance of TNF-α and TLR 2/4. There were no differences in glucose levels or lipid profile between both groups. Conclusions: Obese diabetic patients treated with metformin had lower levels of hsCRP expression of TNF-α and TLR 2/4, than their counterparts not receiving the drug.

Toll-like receptors in autoimmunity with special reference to systemic lupus erythematosus.

The Toll-like receptor (TLR) family plays a fundamental role in host innate immunity by mounting a rapid and potent inflammatory response to pathogen infection. TLRs recognize distinct microbial components and activate intracellular signaling pathways that induce expression of host inflammatory genes. Several studies have indicated that TLRs are implicated in many inflammatory and immune disorders. Extensive research in the past decade to understand TLR-mediated mechanisms of innate immunity has enabled pharmaceutical companies to begin to develop novel therapeutics for the purpose of controlling an inflammatory disease. The roles of TLRs in the development of autoimmune diseases have been studied. TLR7 and TLR9 have key roles in production of autoantibodies and/or in development of systemic autoimmune disease. It remains to be determined their role in apoptosis, in the pathogenesis of RNA containing immune complexes, differential expression of TLRs by T regulatory cells.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Ativação da Resposta Imune Inata Mediada por Receptores do Tipo Toll em Infecção por Vírus Herpes Simplex tipo 1(HSV-1) em Modelo Murino

Este trabalho foi realizado no laboratório de Imunopatologia do Centro de Pesquisa RenéRachou (CPqRR) e no Laboratório de Vírus (Labvirus) da Universidade Federal de MinasGerais (UFMG). Os herpesvirus possuem genoma DNA e são envelopados.Aproximadamente 70% dos adultos já tiveram contato com Herpes Simplex tipo 1 (HSV-1).O HSV-1 causa úlceras ou até mesmo lesões mais graves como encefalite, e após a infecção,o vírus comumente fica latente e pode ser ou não reativado. Células do sistema immuneinato, através dos “Receptores do Tipo Toll” (TLRs), reconhecem “padrões molecularesassociados a patógenos” (PAMPs) orquestrando a resposta imune contra o vírus

Quandoativados, TLRs iniciam uma cascata de sinalização que culmina na ativação de genesrelacionados à defesa imune inata (óxido nítrico-NO, citocinas e quimiocinas). Outros estudosdo grupo mostraram que animais nocautes (KO) para TLR9 ou TLR2/9 apresentam altamortalidade na infecção por HSV-1, ao contrário dos C57BL/6 selvagens (WT) e TLR2KO,que são capazes de controlar o vírus. Objetivamos pesquisar a interrelação na expressão dosTLRs 1, 2, 3, 6, 7 e 9 em animais TLR2 KO, TLR9 KO e TLR2/9 KO infectados por HSV-1e principais citocinas, células e mecanismos imune celulares contra infecção por HSV-1. Nopresente trabalho camundongos WT, TLR2KO, TLR9KO e TLR2/9KO foram infectados viaintranasal com 106 u.f.p. de HSV-1, e seus controles receberam PBS. A eutanásia dos animaisfoi realizada no 5º dia após infecção (dpi), que previamente já foi demonstrado ser o pico dainfecção neste modelo, e então gânglios trigêmeos (TG) e cérebros foram coletados. Aexpressão de TLRs 1, 2, 3, 6, 7 e 9 foi avaliada por PCR em Tempo Real. Camundongos WTinfectados apresentaram diferença significativa na expressão destes TLRs comparados comseus respectivos controles no TG, mas não no cérebro. Nos KOs, houve aumento da expressãodos TLRs tanto nos TGs (mas com níveis mais moderados comparado aos WT) quanto noscérebros. Também foi avaliada a expressão de gp91phox, p22phox e iNOS. A expressão de iNOSem C57BL/6 WT infectados foi maior que nos outros grupos, mas o mesmo não ocorreu paragp91phox, p22phox. Adicionalmente, foi mensurada a produção de NO por macrófagosintraperitoneais, através da reação de Griess, que demonstrou maior produção por macrófagosWT, comparados aos grupos TLR KOs

Ensaios de histopatologia e imunofluorescênciademonstraram maior celularidade nos TGs infectados, além de confirmar a presença de CD8,macrófagos, e aumento da produção de iNOS nos C57BL/6 WT infectados, o que não ocorreucom os camundongos TLRs KO. Em ensaio de sobrevivência com C57BL/6 WT, CCL3 KO,CD8 KO, RAG KO e iNOS KO a importância das células T e do iNOS foi confirmada, umavez que CD8 KO, RAG KO e iNOS KO tiveram 0% de sobrevida. Camundongos C57BL/6WT, RAG KO, CCL3 KO e iNOS KO tiveram seus TGs analisados por PCR em tempo realpara verificar a expressão do gene viral VP-16 e MCP-1, iNOS, IP-10, Rantes e TNF- no 5ºdpi. Estes dados indicaram a relevância de iNOS e das célúlas T na resposta inata contra oHSV-1, uma vez que os RAG KO foram irresponsivos à expressão das citocinas, enquanto osiNOS KO apresentaram uma resposta exacerbada das mesmas. Ensaio de citometria indicouque IFN produzido pelas células T CD8, nos TGs, e produzido pelas células T CD4 eNatural Killer (NK), nos linfonodos regionais, são um possível mecanismo de controle contraa infecção de HSV-1 dos animais WT infectados. Hipotetizamos que a expressão coordenadade TLRs reflete na orquestração eficiente do sistema imune inato, através da produçãoequilibrada de quimiocinas e citocinas e da atividade imune celular adequada, com produçãode NO por macrófagos e ação de células T CD8, T CD4 e NK controlando a infecção porHSV-1

Association Between Toll-Like Receptors/CD14 Gene Polymorphisms and Inflammatory Bowel Disease in Korean Population

The innate immune response in patients who develop inflammatory bowel disease (IBD) may be abnormal. However, the exact role of Toll-like receptors (TLRs) / CD14 gene in the pathogenesis of IBD has not been fully elucidated. We aimed to investigate the association between polymorphisms of TLR1, 2, 4, 6, and CD14 gene and susceptibility to IBD in Korean population. A total 144 patients of IBD (99 patients with ulcerative colitis, 45 patients with Crohn's disease) and 178 healthy controls were enrolled. Using a PCR-RFLP, we evaluated mutations of TLR1 (Arg80Thr), TLR2 (Arg753Gln and Arg677Trp), TLR4 (Asp299Gly and Thr399Ile), TLR6 (Ser249Pro) genes and the -159 C/T promoter polymorphism of CD14 gene. No TLR polymorphisms were detected in Korean subjects. T allele and TT genotype frequencies of CD14 gene were significantly higher in IBD patients than in healthy controls. In subgroup analysis, T allelic frequency was higher in pancolitis phenotype of ulcerative colitis. In Korean population, the promoter polymorphism at -159 C/T of the CD14 gene is positively associated with IBD, both ulcerative colitis and Crohn's disease.

Ativação da resposta imune inata mediada por receptores do tipo toll em infecção por vírus herpes simplex tipo 1(HSV-1) em modelo murino / Activation of the innate immune response mediated by toll receptors type in infection herpes simplex virus type 1 (HSV-1) in murine model

Este trabalho foi realizado no laboratório de Imunopatologia do Centro de Pesquisa RenéRachou (CPqRR) e no Laboratório de Vírus (Labvirus) da Universidade Federal de MinasGerais (UFMG). Os herpesvirus possuem genoma DNA e são envelopados.Aproximadamente 70% dos adultos já tiveram contato com Herpes Simplex tipo 1 (HSV-1).O HSV-1 causa úlceras ou até mesmo lesões mais graves como encefalite, e após a infecção,o vírus comumente fica latente e pode ser ou não reativado. Células do sistema immuneinato, através dos “Receptores do Tipo Toll” (TLRs), reconhecem “padrões molecularesassociados a patógenos” (PAMPs) orquestrando a resposta imune contra o vírus. Quando ativados, TLRs iniciam uma cascata de sinalização que culmina na ativação de genesrelacionados à defesa imune inata (óxido nítrico-NO, citocinas e quimiocinas). Outros estudosdo grupo mostraram que animais nocautes (KO) para TLR9 ou TLR2/9 apresentam altamortalidade na infecção por HSV-1, ao contrário dos C57BL/6 selvagens (WT) e TLR2KO,que são capazes de controlar o vírus. Objetivamos pesquisar a interrelação na expressão dosTLRs 1, 2, 3, 6, 7 e 9 em animais TLR2 KO, TLR9 KO e TLR2/9 KO infectados por HSV-1e principais citocinas, células e mecanismos imune celulares contra infecção por HSV-1. Nopresente trabalho camundongos WT, TLR2KO, TLR9KO e TLR2/9KO foram infectados viaintranasal com 106 u.f.p. de HSV-1, e seus controles receberam PBS. A eutanásia dos animaisfoi realizada no 5º dia após infecção (dpi), que previamente já foi demonstrado ser o pico dainfecção neste modelo, e então gânglios trigêmeos (TG) e cérebros foram coletados. Aexpressão de TLRs 1, 2, 3, 6, 7 e 9 foi avaliada por PCR em Tempo Real. Camundongos WTinfectados apresentaram diferença significativa na expressão destes TLRs comparados comseus respectivos controles no TG, mas não no cérebro. Nos KOs, houve aumento da expressãodos TLRs tanto nos TGs (mas com níveis mais moderados comparado aos WT) quanto noscérebros. Também foi avaliada a expressão de gp91phox, p22phox e iNOS. A expressão de iNOSem C57BL/6 WT infectados foi maior que nos outros grupos, mas o mesmo não ocorreu paragp91phox, p22phox. Adicionalmente, foi mensurada a produção de NO por macrófagosintraperitoneais, através da reação de Griess, que demonstrou maior produção por macrófagosWT, comparados aos grupos TLR KOs. Ensaios de histopatologia e imunofluorescênciademonstraram maior celularidade nos TGs infectados, além de confirmar a presença de CD8,macrófagos, e aumento da produção de iNOS nos C57BL/6 WT infectados, o que não ocorreucom os camundongos TLRs KO. Em ensaio de sobrevivência com C57BL/6 WT, CCL3 KO,CD8 KO, RAG KO e iNOS KO a importância das células T e do iNOS foi confirmada, umavez que CD8 KO, RAG KO e iNOS KO tiveram 0% de sobrevida. Camundongos C57BL/6WT, RAG KO, CCL3 KO e iNOS KO tiveram seus TGs analisados por PCR em tempo realpara verificar a expressão do gene viral VP-16 e MCP-1, iNOS, IP-10, Rantes e TNF- no 5ºdpi. Estes dados indicaram a relevância de iNOS e das célúlas T na resposta inata contra oHSV-1, uma vez que os RAG KO foram irresponsivos à expressão das citocinas, enquanto osiNOS KO apresentaram uma resposta exacerbada das mesmas. Ensaio de citometria indicouque IFN produzido pelas células T CD8, nos TGs, e produzido pelas células T CD4 eNatural Killer (NK), nos linfonodos regionais, são um possível mecanismo de controle contraa infecção de HSV-1 dos animais WT infectados. Hipotetizamos que a expressão coordenadade TLRs reflete na orquestração eficiente do sistema imune inato, através da produçãoequilibrada de quimiocinas e citocinas e da atividade imune celular adequada, com produçãode NO por macrófagos e ação de células T CD8, T CD4 e NK controlando a infecção porHSV-1.

Intrinsic and Extrinsic Regulation of Innate Immune Receptors

Pattern recognition receptors (PRRs) in innate immune cells play a pivotal role in the first line of host defense system. PRRs recognize pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) to initiate and regulate innate and adaptive immune responses. PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), which have their own features in ligand recognition and cellular location. Activated PRRs deliver signals to adaptor molecules (MyD88, TRIF, MAL/TIRAP, TRAM, IPS-1) which act as important messengers to activate downstream kinases (IKK complex, MAPKs, TBK1, RIP-1) and transcription factors (NF-kappaB, AP-1, IRF3), which produce effecter molecules including cytokines, chemokines, inflammatory enzymes, and type I interferones. Since excessive PRR activation is closely linked to the development of chronic inflammatory diseases, the role of intrinsic and extrinsic regulators in the prevention of over- or unnecessary activation of PRRs has been widely studied. Intracellular regulators include MyD88s, SOCS1, TOLLIP, A20, and CYLD. Extrinsic regulators have also been identified with their molecular targets in PRR signaling pathways. TLR dimerization has been suggested as an inhibitory target for small molecules such as curcumin, cinnamaldehyde, and sulforaphane. TBK1 kinase can be a target for certain flavonoids such as EGCG, luteolin, quercetin, chrysin, and eriodictyol to regulate TRIF-dependent TLR pathways. This review focuses on the features of PRR signaling pathways and the therapeutic targets of intrinsic and extrinsic regulators in order to provide beneficial strategies for controlling the activity of PRRs and the related inflammatory diseases and immune disorders.

Induction of metalloproteinases expression by TLR ligands in human fibroblast like synoviocytes from juvenile idiopathic arthritis patients.

Background & objective: Juvenile idiopathic arthritis (JIA) is characterized by chronic synovitis, cartilage damage and bone erosion. Both genetic and environmental factors and microbes probably play a role in pathogenesis. Microbes are recognized by Toll like receptors (TLRs) and activate innate immune response. We studied the ability of bacterial and viral products to produce matrix metalloproteinases (MMPs) and cytokines by fibroblast like synoviocytes (FLS) from patients with JIA. Methods: FLS were cultured from synovial fluid (SF) of patients with JIA and subsequently stimulated for 48 h by different TLR ligands [peptidoglycan (PG) for TLR2, poly(I-C) for TLR3, lipopolysaccharide (LPS) for TLR4, flagellin for TLR5, imiquimod for TLR7 and CpG DNA for TLR9]. Later the production of IL6, IL8, MMP-1, MMP-3, tissue inhibitors of metalloproteinase (TIMP1) was measured in the culture supernatants by ELISA. Expression of TLR2, TLR4, TLR7 and TLR9 was studied in FLS derived from JIA patients by RT-PCR. Results: IL6, IL8, MMP3 and MMP1 production was induced on stimulation of FLS with TLR2 ligand, TLR3 ligand, TLR4 ligand, TLR5 ligand but not with TLR7 ligand and TLR9 ligand. There was no effect of these ligands on the production of TIMP thus the balance was tilted in favour of MMPs after TLR ligation. TLR2, TLR4 and low expression of TLR9 was found but, no expression of TLR7 was found in FLS from JIA patients. Interpretation & conclusion: TLR pathway stimulation by microbial products or endogenous ligands could be involved in the production of MMPs in JIA and may contribute to disease pathology. Thus it may be beneficial to inhibit TLR pathway to reduce cartilage destruction.

Effects of enteropathogenic bacteria & lactobacilli on chemokine secretion & Toll like receptor gene expression in two human colonic epithelial cell lines.

Background & objectives: The intestinal epithelium is part of the innate immune system responding to contact with pathogenic or commensal bacteria. The objective of this study was to compare innate responses of intestinal epithelial cell lines to pathogenic bacteria and to lactobacilli. Methods: Two human intestinal epithelial cell lines, HT29 (enterocyte-like) and T84 (crypt-like), were exposed to pathogenic bacteria representative of non invasive (Vibrio cholerae O1 and O139), adherent (enterohaemorrhagic Escherichia coli, EHEC) or invasive (Salmonella Typhimurium and Shigella flexneri) phenotypes and to non pathogenic Lactobacillus rhamnosus GG or Lactobacillus plantarum. Interleukin-8 (IL-8) was measured in culture supernatant by ELISA, while mRNA from cells was subjected to quantitative reverse transcriptase PCR for several other chemokines (CXCL1, CCL5 and CXCL5) and for Toll-like receptors (TLR) 2, 4, 5 and 9. Results: V. cholerae, S. Typhimurium, S. flexneri and EHEC induced IL-8 secretion from epithelial cells into the medium. Salmonella, Shigella and EHEC, but not V. cholerae, significantly increased mRNA expression of CXCL1. None of the pathogens induced CCL5 or CXCL5. Salmonella and Vibrio significantly increased TLR4 expression, while Vibrio and EHEC decreased TLR5 expression. EHEC also decreased TLR9 expression. Lactobacilli attenuated the IL-8 response of the cell lines to V. cholerae, Salmonella, and EHEC but did not significantly change the IL-8 response to Shigella. Interpretation & conclusions: Distinct patterns of epithelial cell chemokine responses were induced by the bacterial pathogens studied and these were modulated by commensal lactobacilli. Alterations in TLR expression by these pathogens are likely to be important in pathogenesis.