Biology subjects

Ciorba, M. A.

Publications and source records attributed to Ciorba, M. A..

3 recordsLinked to original sources

bioRxiv2021

Mesalamine Reduces Intestinal ACE2 Expression Without Modifying SARS-CoV-2 Infection or Disease Severity in Mice.

IntroductionCoronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium, and can induce GI symptoms similar to the human inflammatory bowel diseases (IBD). An international surveillance epidemiology study (SECURE-IBD) reported that the standardized mortality ratio trends higher in IBD patients (1.5-1.8) and that mesalamine/sulfasalazine therapy correlates with poor outcome. The goal of our study was to experimentally address the relationship between mesalamine and SARS-CoV-2 entry, replication, and/or pathogenesis. MethodsViral infection was performed with a chimeric vesicular stomatitis virus expressing SARS-CoV-2 spike protein and EGFP (VSV-SARS-CoV-2) and SARS-CoV-2 virus derived from an infectious cDNA clone of 2019n-CoV/USA_WA1/2020. Primary human ileal spheroids derived from healthy donors were grown as 3D spheroids or on 2D transwells. We assessed the effect of 10 mM mesalamine (Millipore Sigma) on viral RNA levels, as well as the expression of the SARS-CoV-2 receptor angiotensin II-converting enzyme 2 (ACE2), Transmembrane Serine Protease 2 (TMPRSS2), TMPRSS4, Cathepsin B (CTSB) and CTSL by qRT-PCR. 8-12 week old K18-ACE2 were treated orally with PBS or mesalamine at 200 mg/kg daily. Mice were inoculated intranasally with 1x103 FFU of SARS-CoV-2. Mice were weighed daily and viral titers were determined 7 days post infection (dpi) by qRT-PCR. For the intestinal viral entry model, VSV-SARS-CoV-2 was injected into a ligated intestinal loop of anesthetized K18-ACE2 mice and tissues were harvested 6 hours post-infection. ResultsWe found no change in viral RNA levels in human intestinal epithelial cells in response to mesalamine. Expression of ACE2 was reduced following mesalamine treatment in enteroids, while CTSL expression was increased. Mice receiving mesalamine lost weight at similar rates compared to mice receiving vehicle control. Mesalamine treatment did not change viral load in the lung, heart, or intestinal tissues harvested at 7 dpi. Pretreatment with mesalamine did not modulate intestinal entry of the chimeric VSV-SARS-CoV-2 in K18-ACE2 mice. ConclusionsMesalamine did not alter viral entry, replication, or pathogenesis in vitro or in mouse models. Mesalamine treatment reduced expression of the viral receptor ACE2 while concurrently increasing CTSL expression in human ileum organoids.

microbiology↗

bioRxiv2020

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich-repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2-negative cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK-pore forming -subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice. SignificanceA primary function of goblet cells (GCs) of the intestinal epithelium is to generate a protective mucus layer lining the intestinal lumen. GC dysfunction is linked to Inflammatory Bowel Disease (IBD). GC mucus secretion is thought to be dependent on contributions of an ensemble of anion and cation fluxes, although understanding remains limited. Here, it is shown in mouse colon that the Ca2+- and voltage-dependent BK-type K+ channel, specifically in association with the LRRC26 regulatory subunit, plays a critical role in normal GC function, protecting mice against chemically-induced colitis. The results demonstrate that normal K+ fluxes mediated by LRRC26-containing BK channels are required for normal GC function, potentially providing insights into the potential role of BK channels in IBD.

physiology↗

bioRxiv2020

TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

Both gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA have been frequently observed in COVID-19 patients. However, whether SARS-CoV-2 replicate in the human intestine and its clinical relevance to potential fecal-oral transmission remain unclear. Here, we demonstrate productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. In addition to TMPRSS2, another mucosa-specific serine protease, TMPRSS4, also enhanced SARS-CoV-2 spike fusogenic activity and mediated viral entry into host cells. However, newly synthesized viruses released into the intestinal lumen were rapidly inactivated by human colonic fluids and no infectious virus was recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

microbiology↗