Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection.

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1ß release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.

Peripheral markers of allergen-specific immune activation predict clinical allergy in eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a T-cell-mediated disease that is caused by specific foods and results in esophageal dysfunction. Existing allergy testing modalities are not helpful when attempting to identify EoE-causal foods necessitating empiric food elimination and recurrent endoscopy. The goal of this study was to identify and compare allergen-specific immune features that can be assayed in a minimally invasive manner to predict clinical food allergy in EoE. METHODS: We obtained blood samples from control subjects (n = 17), subjects with clinical EoE milk allergy (n = 17), and subjects with immunoglobulin E-mediated milk allergy (n = 9). We measured total and milk-specific plasma immunoglobulin G (IgG)4 levels and peripheral memory CD4+ T helper (TH ) cell proliferation and cytokine production after stimulation with endotoxin-depleted milk proteins. Sensitivity and specificity for predicting clinical EoE milk allergy were calculated and compared between approaches. RESULTS: Total and milk-specific IgG4 levels were not significantly different between control subjects and subjects with clinical EoE milk allergy. Stimulation with milk proteins caused TH lymphocytes from subjects with clinical EoE milk allergy to proliferate more (%P1 of 38.3 ± 4.6 vs. 12.7 ± 2.8, p < 0.0001), and produce more type 2 cytokines (%IL-4+ of 33.7 ± 2.8 vs. 6.9 ± 1.6, p < 0.0001) than cells from control subjects. Milk-dependent memory TH -cell proliferation (sensitivity and specificity of 88% and 82%, respectively) and interleukin 4 (IL-4) production (sensitivity and specificity of 100%) most strongly predicted clinical EoE milk allergy. CONCLUSIONS: Peripheral markers of allergen-specific immune activation may be useful in identifying EoE-causal foods. Assaying milk-dependent IL-4 production by circulating memory TH lymphocytes most accurately predicts clinical EoE milk allergy.

Targeting of protease activator receptor-2 (PAR-2) antagonist FSLLRY-NH2 as an asthma adjuvant therapy.

Asthma is a chronic inflammatory and multifactorial respiratory tract disease. It affects over 18 million adults and 6 million children in the USA with Puerto Ricans showing the highest prevalence (12%-19%). This airways illness can be triggered by an environmental stimulus such as grass pollen, fungi spores, cockroaches allergens, dust mites metabolic compounds, and importantly, by environmental proteases such as trypsin and tryptase. Because of the pivotal role of proteases in the onset of asthma pathophysiology, we focused this study on the serine Protease Activated Receptor-2 (PAR-2), a G-protein-coupled receptor widely expressed in cells across the respiratory tract. Herein, we measured the activation of PAR-2 on primary pulmonary bronchial/tracheal epithelial cells, human small airway epithelial cells, lung bronchial smooth muscle cells (with and without asthma). We tested human-derived eosinophils from 61 Puerto Rican participants (33 asthmatic and 28 non-asthmatic). As surrogate of PAR-2 activation or inhibition we used intracellular calcium mobilization assay. We hypothesized that following exposure of the PAR-2 agonist (AC264613), the studied human primary cell types will increase the mobilization of intracellular calcium levels. In contrast, we expected a decrease of the intracellular calcium levels upon exposure to a PAR-2 antagonist (FSLLRY-NH2). The Puerto Rican-derived eosinophils were analyzed for the proinflammatory markers MAPK/PI3K using flow cytometry (n = 8). As expected, the PAR-2 agonist significantly increased the activation of PAR-2 on the bronchial/tracheal epithelial cells, bronchial smooth muscle cells and human small airway epithelial cells (P = .01). The PAR-2 antagonist significantly decreased the intracellular calcium levels of these lung primary down to undetectable levels (P = .01). Remarkably, the asthmatic-derived eosinophils showed a striking 300% increase of intracellular calcium mobilization suggesting a severe response to the PAR-2 agonist stimuli in asthmatics. In contrast, there were no significant changes between groups after adding the PAR-2 antagonist. Our outcomes revealed that PAR-2 antagonist effectively inhibited the studied primary cells, expecting to decrease the immune response of eosinophils. Most importantly, our results reveal a promising role for the PAR-2 antagonist in targeting bronchial/tracheal epithelial cells, human small airway epithelial cells and bronchial smooth muscle cells with the potential to oblige an asthma adjuvant therapy.

The Autotransporter IcsA Promotes Shigella flexneri Biofilm Formation in the Presence of Bile Salts.

Shigella flexneri is an intracellular bacterial pathogen that invades epithelial cells in the colonic mucosa, leading to bloody diarrhea. A previous study showed that S. flexneri forms biofilms in the presence of bile salts, through an unknown mechanism. Here, we investigated the potential role of adhesin-like autotransporter proteins in S. flexneri biofilm formation. BLAST search analysis revealed that the S. flexneri 2457T genome harbors 4 genes, S1242, S1289, S2406, and icsA, encoding adhesin-like autotransporter proteins. Deletion mutants of the S1242, S1289, S2406 and icsA genes were generated and tested for biofilm formation. Phenotypic analysis of the mutant strains revealed that disruption of icsA abolished bile salt-induced biofilm formation. IcsA is an outer membrane protein secreted at the bacterial pole that is required for S. flexneri actin-based motility during intracellular infection. In extracellular biofilms, IcsA was also secreted at the bacterial pole and mediated bacterial cell-cell contacts and aggregative growth in the presence of bile salts. Dissecting individual roles of bile salts showed that deoxycholate is a robust biofilm inducer compared to cholate. The release of the extracellular domain of IcsA through IcsP-mediated cleavage was greater in the presence of cholate, suggesting that the robustness of biofilm formation was inversely correlated with IcsA processing. Accordingly, deletion of icsP abrogated IcsA processing in biofilms and enhanced biofilm formation.