Preterm Life in Sterile Conditions: A Study on Preterm, Germ-Free Piglets

Abstract

Preterm infants born with immature organ systems, which can impede normal development, can also be highly sensitive to different biological and/or environmental factors. Animal models could aid in investigating and understanding the effects of different conditions on the health of these immunocompromised infants. The epitheliochorial placentation of the pig prevents the prenatal transfer of protective colostral immunoglobulins. Surgical colostrum-deprived piglets are free of maternal immunoglobulins, and the cells that are normally provided via colostrum. We bred preterm germ-free piglets in sterile conditions and compared them with their term counterparts. Enterocyte development and intestinal morphology, tight junction proteins claudin-1 and occludin, pattern-recognizing receptors, adaptor molecules and coreceptors (RAGE, TLR2, TLR4, TLR9, MyD88, TRIF, MD2, and CD14), and inflammasome NLRP3 transcription were all evaluated. The production of inflammatory mediators IFN-α, IL-4, IL-6, IL-8, IL-10, IL-12/23 p40, TNF-α, IFN-γ, and high mobility group box 1 (HMGB1) in the intestine of germ-free piglets was also assessed. In the preterm germ-free piglets, the ileum showed decreased lamina propria cellularity, reduced villous height, and thinner and less distinct stratification – especially muscle layer, in comparison with their term counterparts. Claudin-1 transcription increased in the intestine of the preterm piglets. The transcription levels of pattern-recognizing receptors and adaptor molecules showed ambiguous trends between the groups. The levels of IL-6, IL-8, IL-10, and TNF-α were increased in the preterm ileum numerically (though not significantly), with statistically significant increases in the colon. Additionally, IL-12/23 p40 and IFN-γ were statistically significantly higher in the preterm colon. Both blood plasma and intestinal HMGB1 levels were nonsignificantly higher in the preterm group. We propose that the intestine of the preterm germ-free piglets showed “mild inflammation in sterile conditions.” This model, which establishes preterm, hysterectomy-derived germ-free piglets, without protective maternal immunoglobulins, can be used to study influences of microbiota, nutrition, and therapeutic interventions on the development and health of vulnerable immunocompromised preterm infants.

Colonization of preterm gnotobiotic piglets with probioticLactobacillus rhamnosusGG and its interference withSalmonellaTyphimurium

“…Conventionalization of germ‐free piglets by pig feces triggered changes leading to intestinal histology that was similar to the histology typical for conventional piglets . The lamina propria , submucosa cellularity and muscular layer thickness were increased in the LGG group in comparison with the preterm GF piglets, and histology of their ileum resembled the ileum of the term germ‐free piglets . Quite a different picture was seen in the ileum of the Salmonella ‐infected preterm piglets (ST and LGG+ST) in which inflammatory cellularity in the lymphatic vessel lumen, villus atrophy, exudate in the lumen and PMN infiltration into the lamina propria shared characteristics with Salmonella ‐infected term gnotobiotic piglets .…”

High Mobility Group Box 1 and TLR4 Signaling Pathway in Gnotobiotic Piglets Colonized/Infected with L. amylovorus, L. mucosae, E. coli Nissle 1917 and S. Typhimurium

“…Gnotobiotic piglets were obtained and bred in gnotobiotic isolators as described elsewhere [57]. Fifty-five piglets were divided to eight groups (Supplementary Figure S1): (i) GF (germ-free, n = 6); (ii) ST (one-week-old GF piglets orally infected with 1 × 10 6 CFU of S. Typhimurium for 24 h, n = 7); (iii) LA (GF orally colonized with 1 × 10 8 CFU of L. amylovorus 4 h after hysterectomy, n = 7); (iv) LA+ST (one-week-old LA-colonized piglets orally infected with 1 × 10 6 CFU of S. Typhimurium for 24 h, n = 7); (v) LM (GF orally colonized with 1 × 10 8 CFU of L. mucosae 4 h after hysterectomy, n = 7); (vi) LM+ST (one-week-old LM-colonized piglets orally infected with 1 × 10 6 CFU of S. Typhimurium for 24 h, n = 7); (vii) EcN (GF orally colonized with 1 × 10 8 CFU of E. coli Nissle 1917 4 h after hysterectomy, n = 7), and (viii) EcN+ST (one-week-old EcN-colonized piglets orally infected with 1 × 10 6 CFU of S. Typhimurium for 24 h, n = 7).…”

“…Total RNA from the terminal ileum, transverse colon, and mesenteric lymph nodes was isolated and transcribed as described elsewhere [57]. Approximately 10 mg of the tissue stored in RNAlater (Qiagen, Hilden, Germany) at −20 • C were moved to RLT buffer of the RNeasy Plus Mini kit (Qiagen) with zirconia beads (BioSpec Products, Bartlesville, OK).…”

“…Moreover, colostrum-free piglets are immunocompromised by deprivation of maternal immunoglobulins and cells [56]. Therefore, germ-free surgery-derived piglets that are reared in microbiologically controlled conditions can be used as a model of vulnerable immunocompromised infants [57]. Salmonella Typhimurium strain LT2 is a well-characterized laboratory strain [58].…”

Toll-Like Receptor 4 Signaling in the Ileum and Colon of Gnotobiotic Piglets Infected with Salmonella Typhimurium or Its Isogenic ∆rfa Mutants

“…They show lower colonization resistance in comparison with conventional animals [40,41] and can be associated with simple microbiota [42][43][44]. This suggests gnotobiotic animal models for studies of host-microbe crosstalk and microbe-microbe interferences in animals associated with defined microbiota as well as studies of less virulent bacteria without the interference of undefined microbes [45,46]. Moreover, colostrum-deprived gnotobiotic piglets lack protective and immunoregulatory maternal immunoglobulins and cells [47,48].…”