Preprint Club
A cross-institutional Journal Club Initiative

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.