Lactogenic immunity/probiotics: Effect on neonatal gut immunity

描述（由申请人提供）：母乳中含有促进婴儿健康和轻度腹泻的母源抗体（MatAbs），以及可溶性免疫介质（TGF2, IL4， sCD14等），其作用在很大程度上尚未被探索。轮状病毒（RV）是全世界婴儿腹泻的主要原因。新许可的口服RV疫苗在发展中国家的有效性未经证实或较低，而且高昂的费用仍然是普遍采用的障碍。虽然乳酸菌（LAB）可以减少婴儿RV腹泻，但机制尚不明确，同时提供MatAb/介质与LAB的效果尚未探索。我们的创新研究将首先评估初乳/乳和关键母体免疫介质（TGF2, IL4）对益生菌定植的影响。然后，我们确定了冷/奶和乳酸菌对新生儿肠道免疫成熟、RV腹泻的调节和肠道内稳态的单独和联合影响。双歧杆菌和乳酸菌菌株在母乳喂养和配方奶粉喂养的婴儿中有所不同。我们假设冷/牛奶促进了这两种益生菌乳酸菌的定植，需要更少或更低的乳酸菌剂量，这种协同作用增强了肠道免疫成熟和体内平衡，从而减缓了轮状病毒腹泻。初步研究表明，鼠李糖GG （LGG）在单次口服剂量后长期定殖冷/乳饲猪，这支持了我们的新想法。我们独特的新生儿非生物（Gn）猪模型是初乳剥夺（没有MatAb预冷/牛奶），允许MatAb/介质操作和无微生物（剖腹产衍生的无菌环境），允许评估定义的益生菌。重要的是，它们的胃肠道生理、粘膜免疫反应和对人RV腹泻的易感性与婴儿相似。在Aim 1中，我们确定含有MatAb/介质（或TGF2+IL4）的牛奶是否会影响a)乳酸双歧杆菌和LGG在肠道中的定植、持续和分布，以及b)新生儿肠道免疫反应的成熟。在目的2中，我们阐明了a)低滴度RV抗体（自然暴露的母猪）的冷/奶是否与乳酸菌协同调节HRV的发病机制和免疫，从而减轻HRV腹泻；b)肠致病性病毒感染是否影响肠道内稳态和乳酸菌群。我们将使用体内生物发光成像技术来研究新生儿肠道中LGG与乳酸菌的相互作用，以及感冒/牛奶或HRV感染对这些相互作用的影响。这种研究体内细菌生态学的新方法将阐明益生菌在宿主体内的相互作用。待评估的肠道和全身免疫参数包括：1)先天反应；2)针对肠道IgA的病毒特异性、实验室特异性和总同型B细胞反应；3) T细胞反应（Th1, Th2, Th3， Tr1细胞因子）。我们的创新研究将提高对新生儿免疫成熟的理解，并对新型冷/牛奶MatAb/介质与益生菌在RV保护性免疫中的有益作用提供新的见解。此外，我们的研究结果将提出限制发展中国家婴儿RV腹泻的新策略，即对母乳喂养的婴儿或孕妇短期联合给予益生菌和冷/乳介质。这种方法将促进婴儿稳定的乳酸菌定植和肠道免疫成熟，从而导致疾病的缓和。新生儿免疫反应的免疫调节和增强对食物抗原或共生物的耐受性也具有根本意义，以控制食物过敏或炎症性肠综合征以及抗生素引起的腹泻。

DESCRIPTION (provided by applicant): Breast milk contains maternal antibodies (MatAbs) that promote infant health and moderate diarrhea, and soluble immunomediators (TGF2, IL4, sCD14, etc) whose role is largely unexplored. Rotavirus (RV) is a leading cause of diarrhea in infants worldwide. Newly licensed oral RV vaccines have unproven or lower efficacies in developing countries and high costs remain obstacles to universal adoption. Although lactic acid bacteria (LAB) reduce RV diarrhea in infants, mechanisms are undefined and the effect of providing concurrent MatAb/mediators with LAB is unexplored. Our innovative studies will first assess the impact of colostrum (col)/milk and key maternal immunomediators (TGF2, IL4) on colonization by probiotics. We then determine the individual and combined effects of col/milk and LAB on immunologic maturation, moderation of RV diarrhea and homeostasis in the neonatal gut. Bifidobacteria and lactobacillus strains differ in breastfed vs formula fed infants. We hypothesize that col/milk promotes colonization by these 2 probiotic LAB, requiring fewer or lower LAB doses, and this synergism enhances gut immune maturation and homeostasis leading to moderation of rotavirus diarrhea. Preliminary studies showing that L rhamnosus GG (LGG) colonized col/milk-fed pigs long-term after a single oral dose support our novel idea. Our unique neonatal gnotobiotic (Gn) pig model is colostrum- deprived (no MatAb pre-col/milk), allowing MatAb/mediator manipulation and free of microbes (caesarean-derived, sterile housing), permitting evaluation of defined probiotics. Importantly their gastrointestinal physiology, mucosal immune responses and susceptibility to human RV diarrhea mimic that of infants. In Aim 1, we determine if col/milk containing MatAb/mediators (or TGF2+IL4) influence a) Bifidobacterium lactis and LGG colonization, persistence and distribution in the gut and b) maturation of neonatal gut immune responses. In Aim 2 we elucidate a) if col/milk with low titer RV antibody (naturally exposed sows) in concert with LAB modulates HRV pathogenesis and immunity, thereby moderating HRV diarrhea and b) if an enteropathogenic viral infection affects gut homeostasis and the LAB microflora. We will use in vivo bioluminescent imaging to study LGG interactions with/without B. lactis in the neonatal gut, and the influence of col/milk or HRV infection on these interactions. This novel approach to in vivo bacterial ecology will elucidate probiotic LAB interactions within the host. Intestinal and systemic immunologic parameters to be assessed include: 1) innate responses; 2) virus- specific, LAB-specific and total isotype B cell responses focusing on gut IgA; 3) T cell responses (Th1, Th2, Th3, Tr1 cytokines). Our innovative studies will provide improved understanding of maturation of neonatal immunity and new insights into the beneficial effects of novel col/milk MatAb/mediators in concert with probiotics in protective immunity to RV. Further, our findings will suggest new strategies to limit RV diarrhea in infants in developing countries through short-term co- administration of probiotics and col/mlk mediators to breastfed infants or pregnant mothers. This approach will promote stable LAB colonization and gut immune maturation in infants leading to disease moderation. Immunomodulation and enhancement of neonatal immune responses also have fundamental implications for tolerance to food antigens or commensals to control food allergies or inflammatory bowel syndromes as well as antibiotic-induced diarrheas.