Transcriptomic analysis of spleen-derived macrophages in response to lipopolysaccharide shows dependency on the MyD88-independent pathway in Chinese giant salamanders (Andrias davidianus).

Background: Gram-negative bacteria are the main bacterial pathogens infecting Chinese giant salamanders (Andrias davidianus; CGS) in captivity and the wild, causing substantial economic losses in the CGS industry. However, the molecular mechanisms underlying pathogenesis following infection remain unclear.

Results: Spleen-derived macrophages from healthy CGS were isolated, cultured, and identified using density gradient centrifugation and immunofluorescence. A macrophage transcriptome database was established 0, 6, and 12 h post lipopolysaccharide stimulation using RNA-sequencing. In the final database 76,743 unigenes and 4,698 differentially expressed genes (DEGs) were functionally annotated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results showed that DEGs were concentrated in toll-like receptor-nuclear factor kappa B-related immune pathways. Ten DEGs were validated 12 h after lipopolysaccharide (LPS) stimulation. Although the common LPS recognition receptor toll-like receptor 4 was not activated and the key adaptor protein MyD88 showed no significant response, we observed significant up-regulation of the following adaptors: toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β, tumour necrosis factor receptor-associated factor 6, and transforming growth factor-β activated kinase 1, which are located downstream of the non-classical MyD88 pathway.

Conclusions: In contrast to that in other species, macrophage activation in CGS could depend on the non-classical MyD88 pathway in response to bacterial infection. Our study provides insights into the molecular mechanisms regulating CGS antibacterial responses, with implications for disease prevention and understanding immune evolution in amphibians.