Infection and RNA-seq analysis of a zebrafish tlr2 mutant shows a broad function of this toll-like receptor in transcriptional and metabolic control and defense to Mycobacterium marinum infection

Abstract

BackgroundThe function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae.Resultstlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/− control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2−/− larvae than in tlr2+/−, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2−/− versus tlr2+/− shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis.ConclusionThe transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.

A Novel Function of TLR2 and MyD88 in the Regulation of Leukocyte Cell Migration Behavior During Wounding in Zebrafish Larvae

“…Embryos of the tlr2 and myd88 mutants showed similar numbers of macrophages and neutrophils as their wild type siblings ( Figures 1B–E ). This result is consistent with our previous studies of the same myd88 mutant at 3 dpf and the tlr2 mutant at 2 dpf ( van der Vaart et al, 2013 ; Hu et al, 2019 ). With the aim of investigating the importance of the tlr2 and the myd88 mutations for leukocyte behavior under unchallenged condition, the CHT region was analyzed in the double transgenic lines of tlr2 and myd8 8 using CLSM by taking time-lapse images ( Figure 1A ).…”

“…However, since TLRs have not been implied in sensing meandering gradients, we assume that this function involves other receptors. Tlr2 has been shown to be essential for the regulation of cytokines and chemokines expression in both mice and zebrafish ( Moles et al, 2014 ; Hu et al, 2019 ). For instance, we have shown that a tlr2 mutant shows a significant lower expression of cxcl11aa and also of a related chemokine, cxcl11ac , during mycobacterial infection.…”

“…The tlr2 s a 19423 mutant and myd88 h u 3568 mutant lines were identified by the sequencing of an ENU-mutagenized zebrafish library ( van der Vaart et al, 2013 ; Hu et al, 2019 ). To investigate the effect of the tlr2 and the myd88 mutations on leukocyte development, double fluorescent lines tlr2 +/+ Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP) , tlr2 –/– Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP), myd88 +/+ Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP) , myd88 –/– Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP) were used.…”

“…Zebrafish larvae are a popular model for studying functions involved in wound repair ( Renshaw et al, 2006 ; Niethammer et al, 2009 ; Xie et al, 2019 ; Bernut et al, 2020 ; Katikaneni et al, 2020 ; Sommer et al, 2020b ). The availability of mutants in TLR signaling genes tlr2 and myd88 make it possible to study their roles in leukocyte migratory behavior upon tail wounding in zebrafish ( Henry et al, 2013 ; van der Vaart et al, 2013 ; Hu et al, 2019 ; Xie et al, 2019 ; Sommer et al, 2020b ). Tlr2 and Myd88 show a highly conserved structure in mammals and zebrafish ( Meijer et al, 2004 ).…”

Detection of Cutibacterium acnes in granulomas of patients with either hypersensitivity pneumonitis or vasculitis reveals that its presence is not unique for sarcoidosis

“…Interleukin-18 production can be stimulated following activation of NLRP6 [ 13 ]. Results of several studies suggest that TLR2 and interleukin-18 are at least partly involved in granuloma formation [ 14 , 15 ], and in vitro and in vivo models have indeed shown that C. acnes is able to induce granulomas [ 3 ]. It is therefore possible that C. acnes is not a specific trigger of sarcoidosis, HP and (E)GPA, but that its LTA contributes to inflammation and granuloma formation.…”