A Review of Epithelial Ion Transporters and Their Roles in Equine Infectious Colitis.

Equine colitis is a devastating disease with a high mortality rate. Infectious pathogens associated with colitis in the adult horse include Clostridioides difficile, Clostridium perfringens, Salmonella spp., Neorickettsia risticii/findlaynesis, and equine coronavirus. Antimicrobial-associated colitis can be associated with the presence of infectious pathogens. Colitis can also be due to non-infectious causes, including non-steroidal anti-inflammatory drug administration, sand ingestion, and infiltrative bowel disease. Current treatments focus on symptomatic treatment (restoring fluid and electrolyte balance, preventing laminitis and sepsis). Intestinal epithelial ion channels are key regulators of electrolyte (especially sodium and chloride) and water movement into the lumen. Dysfunctional ion channels play a key role in the development of diarrhea. Infectious pathogens, including Salmonella spp. and C. difficile, have been shown to regulate ion channels in a variety of ways. In other species, there has been an increased interest in ion channel manipulation as an anti-diarrheal treatment. While targeting ion channels also represents a promising way to manage diarrhea associated with equine colitis, ion channels have not been well studied in the equine colon. This review provides an overview of what is known about colonic ion channels and their known or putative role in specific types of equine colitis due to various pathogens.

Prolonged oral antimicrobial administration prevents doxorubicin-induced loss of active intestinal stem cells.

Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5-driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia. These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.

Secretory Sorcery: Paneth Cell Control of Intestinal Repair and Homeostasis.

Paneth cells are professional secretory cells that classically play a role in the innate immune system by secreting antimicrobial factors into the lumen to control enteric bacteria. In this role, Paneth cells are able to sense cues from luminal bacteria and respond by changing production of these factors to protect the epithelial barrier. Paneth cells rely on autophagy to regulate their secretory capability and capacity. Disruption of this pathway through mutation of genes, such as Atg16L1, results in decreased Paneth cell function, dysregulated enteric microbiota, decreased barrier integrity, and increased risk of diseases such as Crohn's disease in humans. Upon differentiation Paneth cells migrate downward and intercalate among active intestinal stem cells at the base of small intestinal crypts. This localization puts them in a unique position to interact with active intestinal stem cells, and recent work shows that Paneth cells play a critical role in influencing the intestinal stem cell niche. This review discusses the numerous ways Paneth cells can influence intestinal stem cells and their niche. We also highlight the ways in which Paneth cells can alter cells and other organ systems.

Epithelial Regeneration After Doxorubicin Arises Primarily From Early Progeny of Active Intestinal Stem Cells.

BACKGROUND & AIMS: aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood. METHODS: We monitored DXR's effect on aISCs by enumerating Lgr5-eGFP+ and Olfm4+ crypts, cleaved caspase-3 (CASP3+) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations (Bmi1+, Hopx+, Dll1+, and Defa6+) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR. RESULTS: Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3+ cells, p<0.0001). aISCs downregulated stem cell gene expression and underwent rapid apoptosis (6 hours DXR: 63.4% of CASP3+ cells). There was minimal regenerative contribution from Bmi1+, Hopx+, Dll1+, and Defa6+-expressing populations. In homeostasis, 48.4% of early progeny cells were BrdU+, and expressed low levels of DDR transcripts. CONCLUSIONS: We show that DXR effectively depleted aISCs in the small intestine and subsequent epithelial regeneration depended on nonquiescent early progeny cells of aISCs. The chemoresistant phenotype of the early progeny cells may rely on a dampened DDR in contrast to aISCs' robust DDR, which facilitates expeditious apoptosis.

Doxorubicin increases permeability of murine small intestinal epithelium and cultured T84 monolayers.

Enteric bacteria and/or their products are necessary for doxorubicin (DXR)-induced small intestine mucosal damage. While DXR does not induce gross loss of epithelium, others have shown elevated serum endotoxin after DXR administration. However, the mechanism of movement is unknown. We hypothesized that DXR treatment resulted in increased paracellular translocation of bacteria or bacterial products through the small intestinal epithelium. We measured permeability after DXR administration using transepithelial resistance and macromolecular flux and assessed tight junctional gene expression and protein localization both in vitro using T84 cells and ex vivo using murine jejunum. DXR treatment increased flux of 4 kDa dextrans in mouse jejenum, but increased flux of 4, 10 and 20 kDa dextrans in T84 cells. Following DXR, we observed increased permeability, both in vitro and ex vivo, independent of bacteria. DXR induced increased expression of Cldn2 and Cldn4 in murine small intestine but increased only CLDN2 expression in T84 cells. DXR treatment induced disorganization of tight junctional proteins. We conclude that DXR increases paracellular transit of small macromolecules, including bacterial products, through the epithelium, by altering expression of tight junctional components and dynamic loosening of cellular tight junctions.

The area under the curve of L-lactate in neonatal foals from birth to 14 days of age.

BACKGROUND: Prior studies have shown the prognostic utility of measuring L-lactate in critically ill neonatal foals, both as single (at admission) and serial measurements. Greater prognostic sensitivity and specificity may be achieved by use of the area under the L-lactate versus time curve (LACArea ) over the first 24 hours of hospitalization, which captures both severity and duration of hyperlactatemia. Prior to application of this concept in sick equine neonates, a reference interval for LACArea should be determined. METHODS: The concentration of lactate [LAC] was measured in blood obtained via direct jugular venipuncture from clinically normal foals on Days 1 (birth-24 h of age), 3, 7, and 14 following birth at 6-hour intervals for each 24-hour period. LACArea was calculated using the trapezoidal method. Differences in LACArea by Day were determined by MANOVA with a priori Bonferroni correction, P ≤ 0.05. RESULTS: LACArea differed by Day (P = 0.001), being largest on Day 1, followed by Day 3. Days 7 and 14 were smallest and not different from each other. CONCLUSION: LACArea decreases substantially and predictably over the first week of life in normal neonatal foals. Knowing how LACArea normally changes over the first 2 weeks of life will aid in future study of LACArea as it applies to sick neonatal foals, allowing for consideration of maturational changes potentially unrelated to disease.