Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development.

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother's milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Vasoactive intestinal polypeptide promotes intestinal barrier homeostasis and protection against colitis in mice.

Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP's role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP's role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during colitis.

Intestinal epithelium-specific MyD88 signaling impacts host susceptibility to infectious colitis by promoting protective goblet cell and antimicrobial responses.

Intestinal epithelial cells (IECs), including secretory goblet cells, form essential physiochemical barriers that separate luminal bacteria from underlying immune cells in the intestinal mucosa. IECs are common targets for enteric bacterial pathogens, with hosts responding to these microbes through innate toll-like receptors that predominantly signal through the MyD88 adaptor protein. In fact, MyD88 signaling confers protection against several enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Since IECs are considered innately hyporesponsive, it is unclear whether MyD88 signaling within IECs contributes to this protection. We infected mice lacking MyD88 solely in their IECs (IEC-Myd88(-/-)) with S. Typhimurium. Compared to wild-type (WT) mice, infected IEC-Myd88(-/-) mice suffered accelerated tissue damage, exaggerated barrier disruption, and impaired goblet cell responses (Muc2 and RELMß). Immunostaining revealed S. Typhimurium penetrated the IECs of IEC-Myd88(-/-) mice, unlike in WT mice, where they were sequestered to the lumen. When isolated crypts were assayed for their antimicrobial actions, crypts from IEC-Myd88(-/-) mice were severely impaired in their antimicrobial activity against S. Typhimurium. We also examined whether MyD88 signaling in IECs impacted host defense against C. rodentium, with IEC-Myd88(-/-) mice again suffering exaggerated tissue damage, impaired goblet cell responses, and reduced antimicrobial activity against C. rodentium. These results demonstrate that MyD88 signaling within IECs plays an important protective role at early stages of infection, influencing host susceptibility to infection by controlling the ability of the pathogen to reach and survive at the intestinal mucosal surface.

CD4+ T cells drive goblet cell depletion during Citrobacter rodentium infection.

Both idiopathic and infectious forms of colitis disrupt normal intestinal epithelial cell (IEC) proliferation and differentiation, although the mechanisms involved remain unclear. Recently, we demonstrated that infection by the attaching and effacing murine pathogen Citrobacter rodentium leads to a significant reduction in colonic goblet cell numbers (goblet cell depletion). This pathology depends on T and/or B cells, as Rag1(-/-) mice do not suffer this depletion during infection, instead suffering high mortality rates. To address the immune mechanisms involved, we reconstituted Rag(-/-) mice with either CD4(+) or CD8(+) T cells. Both T cell subsets increased Rag1(-/-) mouse survival during infection, with mice that received CD8(+) T cells developing colonic ulcers but not goblet cell depletion. In contrast, mice that received CD4(+) T cells showed goblet cell depletion in concert with exaggerated IEC proliferation. To define the possible involvement of T cell-derived cytokines, we infected gamma interferon receptor gene knockout (IFN-γR(-/-)) mice and wild-type mice given interleukin 17A (IL-17A) neutralizing antibodies and found that IFN-γ signaling was required for both goblet cell depletion and increased IEC proliferation. Immunostaining revealed that C. rodentium cells preferentially localized to nonhyperplastic crypts containing numerous goblet cells, whereas hyperplastic, goblet cell-depleted crypts appeared protected from infection. To address whether goblet cell depletion benefits the C. rodentium-infected host, we increased goblet cell numbers using the γ-secretase inhibitor dibenzazepine (DBZ), which resulted in greatly increased pathogen burdens and mortality rates. These results demonstrate that goblet cell depletion reflects host immunomodulation of IEC homeostasis and reflects a novel host defense mechanism against mucosal-adherent pathogens.