Publikationsdatenbank

Recognition of M. tuberculosis by innate immune receptors – A novel mechanism of TB pathogenesis revealed by genetic analysis of an Indian cohort and in-vitro studies (2023)

Art

Hochschulschrift

Autor

Thada, Shruthi (WE 7)

Quelle

Berlin: Mensch und Buch Verlag, 2023 — IX, 86 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/38316

Kontakt

Institut für Mikrobiologie und Tierseuchen

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51843 / 66949
mikrobiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Tuberculosis (TB) today still is one of the leading causes of death worldwide. Host-pathogen interaction is crucial for establishing protection against pathogens and involves complex processes. Mycobacterium tuberculosis (M.tb) is a successful intracellular pathogen and evades host immune system to establish a protected niche. There is a great need to develop successful anti-TB therapies, and to overcome this mechanism of M.tb potentially may lead to novel intervention strategies. In this study genetic contribution of endosomal (Toll-like receptor (TLR)-4 and -8) and cytosolic (Cyclic GMP–AMP synthase/Stimulator of interferon genes (cGAS/STING) “pattern recognition receptors (PRRs”) towards disease susceptibility has been studied. It has been shown that both, endosomal and cytosolic recognition of microbial products is critical for the initiation of innate immune response against intracellular pathogens such as M.tb. In “single nucleotide polymorphism (SNP)” analyses of an Indian TB cohort, a TLR4 mutation affecting amino acid 399 (C/T), found frequently in India was shown to be associated with TB risk. The two mutations in TLR4 (299 & 399) analysed were not in complete linkage disequilibrium in our Indian cohort (73%) unlike Caucasians (98%). The difference in genotypic distribution among different ethnicities might be due to differences in local infection pressure during the migration of mankind. Furthermore, TLR4-399T and TLR8-1A conveyed increased susceptibility towards TB in an interdependent manner, even though there is no established TLR4 ligand present in M.tb. On the other hand, we analyzed cGAS/STING SNPs in an Indian TB-cohort. This pathway is an essential defence pathway within the cytosol after M.tb internalization and it’s DNA release inducing the production of type I Interferons (IFNs). We found that the presence of rs311686 SNP upstream of cGAS provides protection from TB overall and is differently distributed in pulmonary TB patients as compared to patients with extra-pulmonary and particularly relapse cases. This SNP furthermore differs in distribution when comparing individuals with respect to Bacille Calmette-Guérin (BCG) vaccination status. Conformational changes of cGAS were found by in silico modelling with respect to rs610913 SNP. Functional analysis of wildtype and variant forms of TLR4 revealed that the double homozygous variant form (299/399) and 399 variant forms to induce higher tumor necrosis factor (TNF) levels in peripheral blood mononuclear cells (PBMCs) stimulated with Lipopolysaccharide (LPS) as compared to the wildtype and 299 mutation alone, which is in contrast to nuclear factor kappa-light-chain-enhancer of activated B cell (NF-KB) levels induced in human embryonic kidney (HEK) cells overexpressing the TLR variants. This could be because of the alternate pathway (TIR domain-containing adaptor inducing interferon-β/TRIF-related adaptor molecule (TRIF/TRAM)) which also induce type I IFNs in addition to NF-KB in HEK cells. TLR-TLR dimerization is an established pathway to recognize and fight pathogens in a better fashion. We describe here for the first time TLR4 and -8 heterodimer formation through TLR8 ligand (R848) interaction in in-sillico modeling and that the TLR4-399T variant disrupted this interaction with TLR8. This was confirmed by the co-immunoprecipitation and mass spectrometry analyses: Here we observed precipitated TLR4 with TLR8-targeted antibodies immobilized on agarose beads in TLR8 ligand stimulated HEK cells. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. This heterodimerization of TLR4 and TLR8 led to moderate activation NF-KB (in HEK cells) inducing TNF and Interleukin -12p40 (IL-12p40) (in PBMCs) along with the activation of interferon regulatory factor3 (IRF3) (in HEK cells). The variant form of TLR4-399T with TLR8 in contrast activated increased NF-KB, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. Taken together, we present evidence that the heterodimerization of TLR4 and TLR8 at the endosome is involved in M.tb recognition via TLR8 ligands, such as M.tb RNA, which induces optimal Th1 response. In summary, our findings implicate M.tb nucleic acid recognition in TB pathogenesis is an essential mechanism to understand the course of the disease. Genotyping for the genes investigated here could help in the future in TB risk stratification of individuals. This may ultimately help in prevention of disease and aid in eveloping new vaccination and treatment strategies.