Phagosome-mediated anti-bacterial immunity is governed by the proton-activated chloride channel in peritoneal macrophages

Phagosome-mediated degradation is a well-conserved and highly effective innate immune response against pathogen infections. However, its role in shaping immune responses remains underappreciated. The molecular mechanisms underlying how phagosomes mature are also poorly understood. Here, we identify proton-activated chloride (PAC) channel as a key negative regulator of phagosome maturation. PAC deletion facilitates phagosomal acidification and protease activation, leading to augmented bactericidal activity in peritoneal macrophages upon Escherichia coli infection. Moreover, enhanced phagosome bacterial degradation in PAC-deficient macrophages promotes downstream STING-IRF3 activation and type 1 interferon (IFN) responses through the release of bacterial ligands. Such ligand release also activates the inflammasome, leading to gasdermin D (GSDMD) cleavage and secretion. The secreted N-terminus GSDMD directly participates in bacterial killing by forming GSDMD pores on bacterial surfaces. Eventually, this macrophage hyperactivation is terminated by pyroptotic cell death. In mice, PAC deletion in macrophages reduced overall peritoneal inflammation and proinflammatory neutrophil and monocyte infiltrations, as well as improved survival after peritoneal infection. Our study thus provides new insights into mechanisms of phagosome maturation and the dynamics of host defense response following phagosome-mediated bacterial degradation. Mice were subjected to intraperitoneal E. coli infection for 30 minutes and underwent peritoneal lavage to acquire peritoneal cells. Cells were washed with ice-cold PBS and collected in FACS buffer. Cells further underwent surface staining for LPM gating. Subsequently, Sony MA900 Multi-Application Cell Sorter was used for isolating LPM cells. 3 samples from PBS- and infection-treated mice from control or Cx3cr1-PAC−/− mice were collected. Total RNA was extracted using the RNeasy Micro kit (Qiagen). In brief, libraries were constructed using the NEBNext Ultra II RNA library Prep Kit for Illumina (New England Biolabs) and sequenced on an illumina NovaSeq 6000 System. Reads were mapped to GRCm38/mm10 reference mouse genome with Hisat2 v2.0.5. FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. Fragments per kilobase per million mapped reads (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Further differential expression analysis, Gene Set Enrichment analysis, Gene Ontology analysis, and KEGG pathway analysis were carried out by clusterProfiler R package.