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

Keywords

• Phagosome

• Proton-activated chloride (PAC/TMEM206) channel

• Large peritoneal macrophages (LPMs)

• STING/IFN signalling

• Inflammasome and gasdermin D

Main Findings

Efficient microbial killing by  macrophages requires phagosomal acidification. This process is driven by  the vacuolar-type ATPase (V-ATPase), which pumps protons into the  compartment; however, the accumulation of positive charge eventually  stalls the pump. Previous studies have identified chloride channel (CLC)  exchangers, which relieve this electrical brake by importing Cl-,  allowing V-ATPase to fully acidify the compartment. In this preprint,  Cheng et al. reveal a  counter-regulatory mechanism: the proton-activated chloride channel  (PAC/TMEM206), which exports luminal Cl-, reinforcing the charge barrier  and restricting phagosomal acidification.

The authors first re-analysed a  published proteomics dataset generated by label-free mass-spectrometry  of bead-induced phagosomes from murine bone-marrow-derived macrophages.  PAC channel emerged as a top candidate annotated as endo-lysosomal  localization and strongly expressed in macrophage lineages.  Immunofluorescence subsequently demonstrated PAC channel localization to  bead-, zymosan-, and E. coli-containing  phagosomes in THP-1-differentiated macrophages and RAW264.7 cells.  Intriguingly, Lipopolysaccharide (LPS) stimulation down-regulated Pacc1 transcripts and attenuated PAC channel currents, implying that  inflammatory priming naturally silences the channel to promote efficient  phagosome maturation.

To examine the function of PAC channel during infection, Cheng et al. intraperitoneally injected GFP-expressing E. coliinto two independent conditional knockouts (Pacc1fl/fl crossed with either Cx3cr1-Cre or LysM-Cre).  Flow-cytometric tracking of GFP signal within large peritoneal  macrophages revealed no difference in bacterial uptake but a strong  increase in intracellular killing by the PAC channel-deficient LPMs. To  confirm that this phenotype was macrophage-intrinsic, resident  macrophages were depleted with clodronate-loaded multilamellar liposomes  and replaced with purified donor LPMs. Only PAC channel-deficient donor  cells retained the accelerated bactericidal activity, confirming a  cell-autonomous role.

Ratiometric phagosome reporters  then linked the accelerated killing phenotype to enhanced luminal  acidification. Zymosan dual-labelled with pH-sensitive pHrodo-Green and  pH-insensitive Alexa Fluor 633 revealed that phagosomes in PAC  channel-deficient BMDMs and LPMs acidified more rapidly and to a lower  pH than wild-type cells. Parallel assays supported a concordant rise in  protease activity, consistent with increased phagosomal acidification.

RNA-seq analyses of wild-type versus Cx3cr1-Pacc1-/-  LPMs highlighted enrichment of interferon-stimulated and other  inflammatory programmes. ELISA confirmed increased IFNβ secretion from  PAC channel-deficient LPMs after E. colichallenge,  while immunoblotting linked the output to upstream activation of the  STING-IRF3 pathway. Treating macrophages with E64d, a broad cathepsin  inhibitor that stalls phagosomal proteolysis, attenuated STING and IRF3  phosphorylation and abolished IFNβ release, connecting heightened  protease activity to increased interferon signalling.

PAC channel-deficient cells also secreted more cleaved gasdermin D (GSDMD-NT). E. coli cultured in supernatants from PAC channel-deficient LPMs grew poorly  compared to those exposed to wild-type supernatants, an effect lost when  the supernatant was derived from Gsdmd-/- LPMs. Additionally, in the clodronate-depletion/adoptive-transfer model, Gsdmd-/-  LPMs failed to replicate the enhanced bacterial clearance conferred by  PAC channel-deficient LPMs. Together these data establish a model  whereby PAC channel loss leads to enhanced phagosomal acidification,  greater proteolysis, increased GSDMD release, and enhanced bacterial  killing.

Finally, the authors challenged mice with a carbapenem-resistant E. coli strain. PAC channel-deficient animals cleared bacteria more rapidly  from both the peritoneal cavity and the bloodstream, exhibited a faster  decline in pro-inflammatory mediators, and showed a strong survival  advantage. These findings position the PAC channel as a promising target  for treating antibiotic-resistant intra-abdominal infections.

Limitations & Suggestions

The study is comprehensive and  technically rigorous, yet several points could be addressed to further  strengthen the central message. Future work along these lines may refine  the mechanistic insights and broaden translational relevance:

1. Direct phagosomal Cl- flux: The  conclusion that PAC exports Cl- from phagosomal compartments is  inferred in this work from pH and protease read-outs. A Cl- reporter  (e.g. ClopHensor) that enables the direct quantification of luminal  [Cl-] in wild-type versus PAC channel-deficient macrophages could  further substantiate the model.

2. Validation in primary human macrophages: All  the functional data rely on murine cells or THP-1-differentiated  macrophages. Repeating the acidification assays on blood-derived human  macrophages with PAC channel knocked-down would strengthen clinical  applicability.

3. Identity of the inflammasome sensor(s): Enhanced  caspase-1 activation is demonstrated, but whether this is a consequence  of increased LPS leakage into the cytosol remains unresolved. Employing  NLRC4-, NLRP3-, and caspase-11-deficient BMDMs would help pinpoint the  dominant inflammasome pathway and the immunoligand(s) responsible for  its activation.

4. Mechanism of LPS-mediated PAC repression: LPS  down-regulates PAC channel, yet the transcriptional or  post-translational mechanism remains unexplored. Future studies could  employ promotor analyses to clarify how inflammatory priming silences  the channel.

5. Consequences of acute elevations in pyroptosis: Intra-abdominal  infection can resolve microbiologically yet leave behind fibrotic  complications. Short-term bacterial clearance and survival improve;  however, it will be essential to show that PAC-channel depletion does  not worsen peritoneal adhesions, diffuse fibrosis, or mesothelial damage  by histopathological analysis weeks after infection.

Significance/Novelty

This preprint helps to reframe our understanding of phagosome biology by identifying PAC/TMEM206 as a  proton-gated “brake” on acidification, operating in opposition to  classical CLC exchangers. The work links the loss of PAC channel to  increased phagosomal acidification, protease activity, immunoligand  escape, and gasdermin-D-mediated bacterial clearance in an unprecedented  way. Critically, PAC channel-deletion improves survival in miche  challenged with carbapenem-resistant E. coli, highlighting PAC  channel-inhibition as a plausible therapeutic strategy against  antibiotic-resistant intra-abdominal infections. These insights are  likely to stimulate fresh exploration of PAC channel in various immune  contexts and may ultimately inform the revision of therapies for  multidrug-resistant sepsis.

Credit

Reviewed by John Benjamin W. Duncan and edited by Kelsey Voss as part of a cross-institutional  journal club between the University of Virginia, Max-Delbrück Center  Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the  Medical University of Vienna and other life science institutes in  Vienna.

The authors declare no conflict of interests in relation to their involvement in the review.