Generation of precision preclinical cancer models using regulated in vivo base editing.

Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.

Airway allergy causes alveolar macrophage death, profound alveolar disorganization and surfactant dysfunction.

Respiratory disorders caused by allergy have been associated to bronchiolar inflammation leading to life-threatening airway narrowing. However, whether airway allergy causes alveolar dysfunction contributing to the pathology of allergic asthma remains unaddressed. To explore whether airway allergy causes alveolar dysfunction that might contribute to the pathology of allergic asthma, alveolar structural and functional alterations were analyzed during house dust mite (HDM)-induced airway allergy in mice, by flow cytometry, light and electron microscopy, monocyte transfer experiments, assessment of intra-alveolarly-located cells, analysis of alveolar macrophage regeneration in Cx3cr1 cre:R26-yfp chimeras, analysis of surfactant-associated proteins, and study of lung surfactant biophysical properties by captive bubble surfactometry. Our results demonstrate that HDM-induced airway allergic reactions caused severe alveolar dysfunction, leading to alveolar macrophage death, pneumocyte hypertrophy and surfactant dysfunction. SP-B/C proteins were reduced in allergic lung surfactant, that displayed a reduced efficiency to form surface-active films, increasing the risk of atelectasis. Original alveolar macrophages were replaced by monocyte-derived alveolar macrophages, that persisted at least two months after the resolution of allergy. Monocyte to alveolar macrophage transition occurred through an intermediate stage of pre-alveolar macrophage and was paralleled with translocation into the alveolar space, Siglec-F upregulation, and downregulation of CX3CR1. These data support that the severe respiratory disorders caused by asthmatic reactions not only result from bronchiolar inflammation, but additionally from alveolar dysfunction compromising an efficient gas exchange.

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.

Delayed Effects of Acute Reperfusion on Vascular Remodeling and Late-Phase Functional Recovery After Stroke.

Tissue perfusion is a necessary condition for vessel survival that can be compromised under ischemic conditions. Following stroke, delayed effects of early brain reperfusion on the vascular substrate necessary for remodeling, perfusion and maintenance of proper peri-lesional hemodynamics are unknown. Such aspects of ischemic injury progression may be critical for neurological recovery in stroke patients. This study aims to describe the impact of early, non-thrombolytic reperfusion on the vascular brain component and its potential contribution to tissue remodeling and long-term functional recovery beyond the acute phase after stroke in 3-month-old male C57bl/6 mice. Permanent (pMCAO) and transient (60 min, tMCAO) brain ischemia mouse models were used for characterizing the effect of early, non-thrombolytic reperfusion on the brain vasculature. Analysis of different vascular parameters (vessel density, proliferation, degeneration and perfusion) revealed that, while early middle cerebral artery recanalization was not sufficient to prevent sub-acute vascular degeneration within the ischemic brain regions, brain reperfusion promoted a secondary wave of vascular remodeling in the peri-lesional regions, which led to improved perfusion of the ischemic boundaries and late-phase neurological recovery. This study concluded that acute, non-thrombolytic artery recanalization following stroke favors late-phase vascular remodeling and improves peri-lesional perfusion, contributing to secondary functional recovery.