BMAL2 promotes eCIRP-induced macrophage endotoxin tolerance

• Published in: Frontiers in immunology Vol. 15; p. 1426682
• Main Authors: Zhou, Mian, Aziz, Monowar, Li, Jingsong, Jha, Alok, Ma, Gaifeng, Murao, Atsushi, Wang, Ping
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 13.06.2024
• Subjects: Animals; ARNTL Transcription Factors - genetics; BMAL2; circadian; Disease Models, Animal; eCIRP; Endotoxins - immunology; Humans; Immune Tolerance; Immunology; Lipopolysaccharides - immunology; macrophages; Macrophages - immunology; Macrophages - metabolism; Macrophages, Peritoneal - immunology; Macrophages, Peritoneal - metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; PD-L1; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Sepsis - immunology; Sepsis - metabolism; Triggering Receptor Expressed on Myeloid Cells-1 - genetics; Triggering Receptor Expressed on Myeloid Cells-1 - immunology; Triggering Receptor Expressed on Myeloid Cells-1 - metabolism; PD-L1; circadian; immune tolerance; macrophages; BMAL2; eCIRP
• ISSN: 1664-3224; 1664-3224
• DOI: 10.3389/fimmu.2024.1426682

The disruption of the circadian clock is associated with inflammatory and immunological disorders. BMAL2, a critical circadian protein, forms a dimer with CLOCK, activating transcription. Extracellular cold-inducible RNA-binding protein (eCIRP), released during sepsis, can induce macrophage endotoxin tolerance. We hypothesized that eCIRP induces BMAL2 expression and promotes macrophage endotoxin tolerance through triggering receptor expressed on myeloid cells-1 (TREM-1). C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20 h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24 h. The cells were then stimulated with LPS for 5 h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24 h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1 mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay. Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages. eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.