Aeromonas hydrophila utilizes TLR4 topology for synchronous activation of MyD88 and TRIF to orchestrate anti-inflammatory responses in zebrafish

Abstract

Toll-like receptor 4 (TLR4) plays a critical role in host immunity against Gram-negative bacteria. It transduces signals through two distinct TIR-domain-containing adaptors, MyD88 and TRIF, which function at the plasma membrane and endosomes, respectively. Using zebrafish Aeromonas hydrophila infection model, we demonstrate that synchronization of MyD88 and TRIF dependent pathways is critical for determining the fate of infection. Zebrafish were infected with A. hydrophila, and bacterial recovery studies suggested its effective persistence inside the host. Histopathological assessment elucidates that A. hydrophila did not provoke inflammatory responses in the spleen. Immunofluorescence revealed the presence of TLR4-bound A. hydrophila on the plasma membrane at 3 h post-infection (p.i.), and inside endosomes 1 day p.i. Quantitative PCR studies suggest that TLR4 activates the downstream pathway of MyD88-IRAK4 axis at early stages followed by a shift to TRIF-TRAF6 axis at late stages of infection coupled with fold increase in NFκB. Our results implicated the involvement of p110δ isoform of PI(3)Kinase in this transition. Coupled to this, we noted that the TLR4-TRIF-NFκB axis prompted burgeoned secretion of anti-inflammatory cytokines. We observed that A. hydrophila inhibits endosome maturation and escapes to cytoplasm. Significant downregulation of cytosolic-NLR receptors further suggested that A. hydrophila represses pro-inflammatory responses in cytosol aiding its persistence. Our findings suggest a novel role of 'TLR4 topology' in A. hydrophila-induced pathogenesis. We propose that A. hydrophila manipulates translocation of TLR4 and migrates to endosome, where it triggers TRIF-dependent anti-inflammatory responses, interferes with endosomal maturation and escapes to cytosol. Inside the cytosol, A. hydrophila avoids detection by suppressing NLRs, facilitating its survival and ensuing pathogenesis.

An Update on the Genus Aeromonas: Taxonomy, Epidemiology, and Pathogenicity

“…Data from two studies showed that the expression of TLR4 might be induced following an A. hydrophila infection in a minnow Gobiocypris rarus and the blunt snout bream Megalobrama amblycephala [21,286]. Similarly, an interesting study was conducted by Srivastava et al [24] using zebrafish as a model. Table 2.…”

“…A. hydrophila, A. salmonicida, A. veronii [21,24,282,283,[286][287][288][289][290][291][292] Cytokines (TNF-α, TNF-β, IFN-γ, IL-6, IL-8, IL-10) [22,23] Epithelioma papulosum cyprini cell line (EPC); head kidney leukocytes (HKLs), human epithelial cell line (Hep-2); murine macrophage cell line (RAW 264.7); human epithelial type cell line (HeLa); human epithelial colorectal adenocarcinoma cell line (Caco-2); spleen leukocytes (SLs). The strains of these studies [282,283,286,[292][293][294]296,297,302] were identified by non-molecular methods or lack information about the identification.…”

“…Zebrafish were used in another study as a model of Aeromonas species co-infection [293], and showed a higher mortality rate under co-infection in relation to the single infection. Additionally, zebrafish were used to study the immune response against Aeromonas [24].…”

“…The presence of these virulence factors allows the bacteria to colonize, invade, and overcome the immune response mechanism of the host, resulting in an infection that generates the disease [4,5,18,20]. Several studies demonstrated the expression of different immune-related genes in the host following an Aeromonas infection, including those involved in pathogen recognition, the proteins involved in cell signaling, and apoptosis [21][22][23][24].…”

Effect of chrysophanic acid on immune response and immune genes transcriptomic profile in Catla catla against Aeromonas hydrophila

“…Combined stimulation of intracellular MAP kinase pathway and Iκβ deprivation irritates the triggering of transcription factors, like AP-1 and NF-κB; which triggers proinflammatory cytokine secretion in the B and T cells leading to B cell proliferation 84 . In zebrafish, the TLR4-induced MyD88 dependent signals triggered the regulation of anti-inflammation 85 . The results herein suggest that both MyD88-dependent and independent TLR-induced signaling pathways together www.nature.com/scientificreports/ to regulate the activation of catla lymphocytes and lymphoid organs.…”