Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation.

Resident peritoneal macrophages (resMØs) are crucial for repairing peritoneal injuries and controlling infections by forming mesothelium-bound resMØ-aggregates in the peritoneal wall and omentum. Here we present a protocol to analyze these structures in mouse models of peritoneal inflammation. We describe the dissection, fixation, immunofluorescent staining, and mounting of whole peritoneal wall and omentum samples and subsequent confocal microscopy imaging of resMØ-aggregates. We also detail the steps to isolate resMØ-aggregates for additional studies, including flow cytometry and electron-microscopy-based analysis. For complete details on the use and execution of this protocol, please refer to Vega-Pérez et al. (2021).1.

Mouse Tissue-Resident Peritoneal Macrophages in Homeostasis, Repair, Infection, and Tumor Metastasis.

Large peritoneal macrophages (LPMs) are long-lived, tissue-resident macrophages, formed during embryonic life, developmentally and functionally confined to the peritoneal cavity. LPMs provide the first line of defense against life-threatening pathologies of the peritoneal cavity, such as abdominal sepsis, peritoneal metastatic tumor growth, or peritoneal injuries caused by trauma, or abdominal surgery. Apart from their primary phagocytic function, reminiscent of primitive defense mechanisms sustained by coelomocytes in the coelomic cavity of invertebrates, LPMs fulfill an essential homeostatic function by achieving an efficient clearance of apoptotic, that is crucial for the maintenance of self-tolerance. Research performed over the last few years, in mice, has unveiled the mechanisms by which LPMs fulfill a crucial role in repairing peritoneal injuries and controlling microbial and parasitic infections, reflecting that the GATA6-driven LPM transcriptional program can be modulated by extracellular signals associated with pathological conditions. In contrast, recent experimental evidence supports that peritoneal tumors can subvert LPM metabolism and function, leading to the acquisition of a tumor-promoting potential. The remarkable functional plasticity of LPMs can be nevertheless exploited to revert tumor-induced LPM protumor potential, providing the basis for the development of novel immunotherapeutic approaches against peritoneal tumor metastasis based on macrophage reprogramming.

Resident macrophage-dependent immune cell scaffolds drive anti-bacterial defense in the peritoneal cavity.

Peritoneal immune cells reside unanchored within the peritoneal fluid in homeostasis. Here, we examined the mechanisms that control bacterial infection in the peritoneum using a mouse model of abdominal sepsis following intraperitoneal Escherichia coli infection. Whole-mount immunofluorescence and confocal microscopy of the peritoneal wall and omentum revealed that large peritoneal macrophages (LPMs) rapidly cleared bacteria and adhered to the mesothelium, forming multilayered cellular aggregates composed by sequentially recruited LPMs, B1 cells, neutrophils, and monocyte-derived cells (moCs). The formation of resident macrophage aggregates (resMφ-aggregates) required LPMs and thrombin-dependent fibrin polymerization. E. coli infection triggered LPM pyroptosis and release of inflammatory mediators. Resolution of these potentially inflammatory aggregates required LPM-mediated recruitment of moCs, which were essential for fibrinolysis-mediated resMφ-aggregate disaggregation and the prevention of peritoneal overt inflammation. Thus, resMφ-aggregates provide a physical scaffold that enables the efficient control of peritoneal infection, with implications for antimicrobial immunity in other body cavities, such as the pleural cavity or brain ventricles.

Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State.

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.