Anatomic plasticity and functional impacts of neural – immune and neural – epithelial signaling in the intestine

2021 Summer.Includes bibliographical references.The intestinal wall is a multicompartmental barrier tissue composed of over 25 distinct cell types with integrated and complex signaling both within and between compartments. The gut wall is also a large endocrine organ comprised of cells capable of producing dozens of peptides used for hormonal and other signaling functions. However, the mechanistic roles that neural secretions play in regulating the gut epithelial barrier in health and disease are not well known. Additionally, frequently used models available for studying intestinal function outside of the body lack the complexity to investigate neural – epithelial and neural – immune signaling interactions. Using a bifurcated approach to method development, we created two culture systems for maintaining the full thickness of the intestinal wall ex vivo. One method allows for culture of mouse or human organotypic intestinal slices that maintain the gut wall for 6 or 4 days, respectively. This system does not however, maintain a true luminal – epithelial barrier as seen in the in vivo gut. The second method, a microfluidic organotypic device (MOD) enables maintenance of explanted mouse or pig intestinal tissue for up to 3 days ex vivo, with an intestinal barrier intact. These two methods allow for investigating and cross-validating of numerous biological questions now previously possible using traditional culture models. Neuronal fiber proximity to gut epithelia has been shown, with goblet, tuft and enteroendocrine cells being closely opposed by fibers. Goblet cells secrete mucopolysaccharides, a first line of defense separating luminal microbiota from host tissue. I have recently shown that vasoactive intestinal peptide (VIP) can regulate goblet cell production in organotypic slices of mouse ileum. This peptide is also in close proximity to Paneth cells in the base of the crypt, and enteric mast cells. There were sex differences in baseline mast cell neuronal proximity, quantities, and cell size in mouse ileum. Further, mast cells showed a sex difference in responses to lipopolysaccharide challenge. Further investigation of neurosecretory factor regulation of immune and epithelial function is needed, both in goblet cells and other secretory epithelia like anti-microbial producing Paneth cells, and in immune components like mast cells. Graphical illustration of the dissertation project is included below

253 - Luke Allen Schwerdtfeger

Intestinal infections impact millions of people each year, often with ineffective treatments available. There is a lack of models capable of studying these infections that recapitulate the guts cellular environment. Addressing this, we placed biopsies from human colon into culture, and maintained the cellular environment observed in vivo, in a dish. Using this model, sex differences in colonic T-cell responses to Salmonella were observed. When culturing biopsy tissue at ~1% oxygen there was an increase in epithelial cell birth compared to atmospheric oxygen. These results signal the need for controlling oxygen and tracking sex when studying colon-microbiome interactions. Purpose: Mammalian intestines maintain a highly complex physiology that survives an extreme oxygen gradient, a mixed population of commensal and pathogenic bacteria, and a heterogeneous cell composition that includes immune, neural, and epithelial elements. Understanding disorders and developing treatment modalities for intestinal disease and infection states requires balancing multiple factors, including sex-dependence. This study begins to account for the impacts of oxygen concentrations in the context of sex, pathogen, and antibiotic exposure. Procedure: Organotypic slices from human colon biopsies provided three-dimensional environments that maintained cellular morphology and relationships, ex vivo. Biopsy slices were used to study impacts of pathogen exposure on lymphocyte counts, and oxygen and antibiotic treatments on epithelial proliferation rates. Results: Sex differences were observed in basal CD3+ T lymphocyte count in human colon, with male patients having over 2-fold more CD3+ T cells than females. When exposed to a strain of Salmonella enterica, male cell counts did not change, while there was a significant increase in CD3+ T cells in biopsy slices taken from females (P < 0.05). For intestinal mucosa, there were greater rates of epithelial cell proliferation in lowered oxygen conditions (1%) than under more standard atmospheric conditions (20%)(P < 0.05). Antibiotic treatment decreased epithelial proliferation in slices maintained in 1% oxygen, but not 20% (P < 0.05). Implications: These results show a baseline T cell sex difference in the human colon that showed a sex-dependent response to pathogen ex vivo. In addition, oxygen concentration impacted colonic epithelial cell proliferation. Together, these results point towards the need to account for oxygen concentration and sex when studying gut-microbe interactions

High incidence of a third head of biceps brachii in females

Incidence rates of a third head of biceps brachii muscle have been shown to vary based on race, sex, and side of the body. Traditionally, males were thought to have higher incidences than females, and the right limb was thought to most frequently contain an additional head of biceps brachii. Additionally, innervation of this third head has been unclear, with musculocutaneous nerve, median nerve, or a combination of both providing nerve supply. Here we present findings of the dissection of 23 cadavers (n = 46 limbs), in which 6 limbs demonstrated a third head of biceps brachii. Of the 6 limbs with a third head, 3 were left and 3 were right limbs, with one cadaver having the extra muscle head bilaterally. Incidence rates in females in the current study were observed to be higher than expected (20.8%), while males showed a 4.5% occurrence. All 6 of the additional heads of biceps brachii observed were innervated by musculocutaneous nerve. Together these data show a higher than expected number of female cadavers with a third head of biceps brachii, equal incidence between sides of the body, and uniform innervation by musculocutaneous nerve. These findings challenge our traditional understanding of the third head of biceps brachii and suggests a need for additional study of this complex anatomical variation. Keywords: Biceps brachii, Third head, Innervation, Sex, Cadaver, Variatio

192 - Ian Cook McLean

Currently, most in vitro experimental models of the intestine rely upon cell lines, and consequently, lack the diverse representation of cells present in vivo. Slices of intestine, removed from living organisms, offer a better representation of in vivo physiology. However, current techniques for maintaining intestinal tissue in vitro are not capable of recapitulating the in vivo environment. This project utilizes 3D printing and microfluidic principles to design a device that delivers differential flows of media across the two surfaces of intestinal tissue. The device will enable the investigation of complex biological questions that previous models have been unable to address

Human colon function ex vivo: Dependence on oxygen and sensitivity to antibiotic.

BackgroundHuman intestines contain a heterogeneous collection of cells that include immune, neural and epithelial elements interacting in a highly complex physiology that is challenging to maintain ex vivo. There is an extreme oxygen gradient across the intestinal wall due in part to microbiota in the lumen and close to the gut wall, which complicates the design of tissue culture systems. The current study established the use of an organotypic slice model of human intestinal tissue derived from colonoscopy biopsies to study host-microbial interactions after antibiotic treatment, and the influence of oxygen concentration on gut wall function.MethodsOrganotypic slices from human colon biopsies collected during routine colonoscopy provided three-dimensional environments that maintained cellular morphology ex vivo. Biopsy slices were used to study impacts of oxygen concentrations and antibiotic treatments on epithelial proliferation rates, and metabolites from tissue culture supernatants.ResultsImmune function was validated via demonstration of a T lymphocyte response to Salmonella enterica serovar Typhimurium. Following 24 h of Salmonella exposure there was a significant increase in CD3+ T-lymphocytes in biopsy slices. Metabolite profiling of tissue culture supernatants validated the influence of antibiotic treatment under varied oxygen culture conditions on both host and microbiome-mediated metabolism. Epithelial health was influenced by oxygen and antibiotic. Increased epithelial proliferation was measured in lowered oxygen conditions (1% = 5.9 mmHg) compared to atmospheric conditions standard at 5000 feet above sea level in Colorado (~17% = 100 mmHg). Antibiotic treatment reduced epithelial proliferation only in 5.9 mmHg oxygen cultured slices.ConclusionsA human colon organotypic slice model was established for applications ranging from gut epithelial proliferation to enteric pathogen influence on mucosal immune functions ex vivo. The results further support the need to account for oxygen concentration in primary tissue cultures, and that antibiotic use impacts gut-microbe-immune interactions