Alessandra Livraghi-Butrico

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor critical for host defense against microbial infections. To prevent aberrant activation, nuclear cGAS is restrained by nucleosome tethering or by barrier-to-autointegration factor–mediated restriction of its access to DNA. However, its physiological roles beyond enzymatic suppression in the nucleus remain poorly understood. Here, we present a genome-wide map of cGAS chromatin localization in human cells and unexpectedly find its binding beyond nucleosome tethering, where cGAS is recruited to chromatin by AP-1 transcription factors via a potentially previously uncharacterized transactivation domain. Integrated CUT & RUN , RNA-seq, and metabolomics analyses reveal that cGAS transcriptionally regulates cholesterol homeostasis. Mechanistically, cGAS promotes LGALS3 transcription, which inhibits ACAA2 activity to suppress fatty acid oxidation and acetyl-CoA production, thereby lowering intracellular cholesterol levels. Reduced cholesterol lowers plasma membrane phosphatidylinositol [3,4,5] triphosphate [PI(3,4,5)P ₃ ] levels and decreases mTOR complex 2 (mTORC2) activation. In contrast, cGAS depletion results in cholesterol accumulation and elevated PI(3,4,5)P ₃ , driving mTORC2/Akt signaling, promoting xenografted tumor growth, and conferring resistance to DNA-damaging agents. In vivo, the elevated hepatic cholesterol levels observed in cGas ^–/– mice were largely reversed by inhibition of Akt but not STING, whereas cGAS deficiency had minimal effects on lung physiology. Collectively, our findings uncover an unexpected role for nuclear cGAS in orchestrating a transcriptional program that links lipid metabolism to cell proliferation, stress response, and cholesterol homeostasis in liver.

Post-acute sequelae of SARS-CoV-2 (PASC) occurs in subsets of individuals, including those with pre-existing lung disease. To investigate PASC pathogenesis and therapeutics in a chronic bronchitis mouse model ( Scnn1b -Tg), Scnn1b -Tg and WT mice were inoculated with a mouse adapted SARS-CoV-2 virus (SARS-CoV-2MA10) and followed for 60 days. Viral titer, histology, immunohistochemistry (IHC), single-cell RNA sequencing, RNA in situ hybridization, and spatial transcriptomic profiling characterized disease pathologies. Scnn1b -Tg mice inoculated with SARS-CoV-2MA10 exhibited lower viral titers and less weight loss than WT mice. Airway epithelia of Scnn1b -Tg mice were less infected than epithelia of WT mice, reflecting increased airway mucus and enhanced epithelial antiviral activities in Scnn1b -Tg mice. However, Scnn1b -Tg mice subsequently exhibited heterogeneous airway and parenchymal disease with elevated Il33 expression characteristic of human eosinophilic pneumonia. Cohorts of infected mice were administered a monoclonal antibody targeting the IL-33 receptor (ST2) or enteral prednisone. Administration of an anti-ST2 monoclonal antibody mitigated development of eosinophilic pneumonia while enteral prednisone suppressed IL33 expression and disease. The eosinophilic pneumonia in Scnn1b -Tg mice after SARS-CoV-2MA10 infection mimics reports of eosinophilic pneumonia in humans post-SARS-CoV-2, suggesting targeting of IL-33 may be beneficial in treating post-viral eosinophilic pneumonia in humans.

The long-term pulmonary sequelae of SARS-CoV-2 respiratory infections reflect infection severity, innate and adaptive immunity, and respiratory epithelial repair. This study investigated the acute and reparative responses as a function of age and sex in primary human bronchial epithelial (HBE) cultures utilizing a 14-day SARS-CoV-2 infection protocol. SARS-CoV-2 infection peaked at 3 days post-infection (dpi) with an ∼ 2 log titer suppression at 14 dpi. SARS-CoV-2 infection induced interferon, interferon-induced gene, and cell damage responses. No age- or sex-dependent effects on SARS-CoV-2 infection were detected. Airway epithelia repaired to an abnormal mucus metaplastic/inflammatory state that reflected potentially beneficial and adverse consequences at 14 dpi. Repair processes were infection severity-dependent, not sex-dependent, and were more robust in young donor cultures. Analyses of long-COVID subjects with persistent pulmonary fibrosis or persistent bronchitic airway diseases exhibited expression of HBE 14 dpi failed repair gene signatures, including ISG gene signatures. Human airway epithelial repair post-SARS-CoV-2 is prolonged and incomplete in vitro over 14 days, and persistently abnormal repair may contribute to phenotypes of people with long-COVID pulmonary syndrome.

Asthma is a complex airway inflammatory disease characterized by immune dysregulation, with diverse cellular contributors to inflammation that result in airway hyperresponsiveness (AHR) and excessive immune responses. The role of the inflammasome in asthma is conflicted. AIM2 is an innate immune receptor that activates the inflammasome through DNA binding. The clinical relevance of AIM2 in asthma is supported by analysis of blood or endobronchial biopsies from patients with severe asthma, showing increased AIM2 but not NLRP3 expression. We investigated the role of AIM2 in allergic asthma using ovalbumin (OVA)-LPS and house dust mite (HDM)-induced models. AIM2 expression was elevated in lung homogenates and bronchoalveolar lavage fluid (BALF) cells from allergen-induced groups compared to controls. In the OVA-LPS model, deletion of Aim2 led to reduced airway smooth muscle actin expression and DNA damage, contributing to decreased AHR and lung inflammation in whole body Aim2−/− mice. Surprisingly, cell-specific deletion in CD4+ T cells but not regulatory T cells or myeloid cells significantly affected AHR, indicating AIM2 in CD4+ T cells is a main driver of pathogenesis. Additionally, in HDM-induced asthma, Aim2−/− mice also exhibited reduced AHR. Cell-specific deletion reveals that AIM2 in CD4+ T cells promotes AHR and cytokine production, while AIM2 in myeloid cells modulates IgG1 levels and IL-13-producing CD4+ T cells, and the percentage of alveolar macrophages in BALF. These findings reveal that AIM2 differentially regulates HDM-induced allergic asthma through distinct exacerbating roles in T cells and myeloid cells, highlighting its potential as a therapeutic target.

Airway mucus drives severity and mortality in diverse chronic lung diseases. The Jagged-1/Notch pathway controls the balance of ciliated versus mucous cells, but targeting the pathway systemically carries the risk of side effects. Here we developed novel, Anticalin-derived, pulmonary-delivered Jagged-1 antagonists, to inhibit airway mucus hyperproduction and obstruction in chronic lung diseases. Our preclinical data demonstrate the effectiveness of these antagonists in diminishing secretory cell and mucus levels and alleviating hallmarks of mucus obstruction.

BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O ₂ , airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na ⁺ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na ⁺ channel) subunit expression. The combination of increased Na ⁺ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA–in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Mutations of Cystic Fibrosis Transmembrane conductance Regulator ( CFTR ) lead to Cystic Fibrosis (CF), but the substantial phenotypic variations are determined by non- CFTR allelic diversity. To map novel disease phenotypes in a CF mouse model, we used Collaborative Cross (CC) mice, a highly genetically diverse mouse resource population. ΔF508-Cftr homozygosity produced a fully penetrant lethal phenotype by eight weeks in two CC lines. The lethality of CC006 ΔF508/ΔF508 was fully prenatal while CC037 ΔF508/ΔF508 showed either prenatal or postnatal lethality. Novel phenotypes of CC037 ΔF508/ΔF508 were revealed early in life including respiratory and systemic inflammatory profiles, and blood, bone marrow, pancreas, heart, and reproductive tract pathologies. Severe intestinal blockage was observed as common in other CF mouse models. These results suggest that the exploration of CF disease phenotypes in a mouse population with diverse genetic profiles is needed to map the genetic origin of currently unidentified disease traits and their potential translation to humans.

CF rats are a novel in vivo model to study CF-like chronic bacterial airway infection.https://bit.ly/3KBhKMr