Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes in PM cation fluxes, cell death, and NLRP3 inflammasome activation.

The coronavirus disease 2019 (COVID-19) pandemic presents health care challenges to asylum seekers living in congregate encampments, including those along the U.S.-Mexico border. It is necessary to understand the impact of the pandemic among this population to address health care needs, reduce transmission, and diminish COVID-19-related morbidity. Methods: Thirty interviews were conducted with asylum seekers and health care professionals in a temporary camp in Matamoros, Mexico to determine challenges, perceptions, and effects of the COVID-19 pandemic. Interviews were coded in NVivo12 by using a team-based approach. Results: The pandemic caused significant mental health burdens but no perceived adverse physical effects, with the U.S. border closure and health care access barriers as more pressing concerns. Participants reported access to information about COVID-19 but had varied levels of knowledge and adherence to disease reduction strategies due to camp conditions. Most participants believed that they had special protection from COVID-19, including strong immune systems or from God. The nongovernmental organizations providing health care and sanitation faced multiple challenges to implement new policies to manage COVID-19. The institution of required temperature checks and quarantine of COVID-19 positive patients led to distrust, decreased seeking of health care services among asylum seekers, and possible underreporting of COVID-19 cases. Conclusion: Our findings among asylum seekers in a Matamoros camp highlight the challenges to implementing disease reduction policies in low-resource congregate camps. Policies to address disease outbreaks focusing on the social determinants of health, health care access barriers, and community engagement may be more acceptable to asylum seekers, suggesting the need for effective strategies to provide prevention information that complements such measures.

Colonic epithelial barrier function is controlled by differential expression of Claudin (CLDN) proteins in the crypt‐luminal axis. We recently identified the expression of a CLDN family member, CLDN‐23, in intestinal epithelial cells (IECs). However, the role of CLDN‐23 in regulating epithelial barrier function has not been identified. Analysis of the human and murine intestinal crypt‐luminal axis revealed increased CLDN‐23 expression in differentiated IECs facing the lumen compared to the proliferative crypt‐base IECs. Similarly, CLDN‐23 expression was higher in differentiated human and murine IECs cultured as a monolayer on permeable supports compared to proliferative IECs cultured in a 3D matrigel matrix. To investigate the contribution of CLDN‐23 in regulating IEC barrier function in vivo, we generated inducible intestinal‐epithelia specific (Villin‐CreERT2) Cldn23 knockout mice (Cldn23ERΔIEC). Functional analysis displayed increased intestinal mucosal permeability to 4kDa FITC dextran, indicating compromised epithelial barrier function. To confirm this observation, we created human IECs in which CLDN‐23 expression was either silenced or enhanced in vitro. CLDN‐23 loss increased paracellular permeability to 4kDa FITC dextran and reduced transepithelial electrical resistance (TEER) consistent with a leaky barrier. Conversely, CLDN‐23 overexpression resulted in improved barrier function. Altogether, these data suggest that CLDN‐23 controls the IEC barrier function. Evaluation of CLDN protein interactions employing co‐culture of HeLa cells expressing CLDN‐23 with those expressing either CLDN‐2, CLDN‐3, or CLDN‐4 showed that CLDN‐23 interacts in trans with barrier‐forming CLDN‐3 and CLDN‐4, but not with pore‐forming CLDN‐2. Using in situ proximity ligation assay, we observed close association in cis between CLDN‐23 and barrier‐forming CLDN‐3 and CLDN‐4 in the TJ plasma membrane, but not with CLDN‐2. These findings suggest that CLDN‐23 interacts in both cis and trans with barrier‐forming CLDN‐3 and CLDN‐4 in differentiated IECs. Interestingly, the overexpression of CLDN‐23 in human IECs revealed up‐regulation of barrier‐forming CLDN‐3 protein expression in the TJ while decreasing the pore‐forming CLDN‐2 protein at this site. In conclusion, we identified that CLDN‐23 strengthens the colonic barrier properties by orchestrating the composition of CLDN protein interactions in epithelial TJs.