Commensal oral microbiota induces osteoimmunomodulatory effects separate from systemic microbiome in mice.

Commensal microbes critically regulate skeletal homeostasis, yet the impact of specific microbiota communities on osteoimmune response mechanisms is unknown. To discern osteoimmunomodulatory effects imparted by the commensal oral microbiota that are distinct from the systemic microbiota, osteoimmunology studies were performed in both alveolar bone and nonoral skeletal sites of specific pathogen-free (SPF) versus germ-free (GF) mice and SPF mice subjected to saline versus chlorhexidine oral rinses. SPF versus GF mice had reduced cortical/trabecular bone and an enhanced pro-osteoclastic phenotype in alveolar bone. TLR signaling and Th17 cells that have known pro-osteoclastic actions were increased in alveolar BM, but not long BM, of SPF versus GF mice. MHC II antigen presentation genes and activated DCs and CD4+ T cells were elevated in alveolar BM, but not long BM, of SPF versus GF mice. These findings were substantiated by in vitro allostimulation studies demonstrating increased activated DCs derived from alveolar BM, but not long BM, of SPF versus GF mice. Chlorhexidine antiseptic rinse depleted the oral, but not gut, bacteriome in SPF mice. Findings from saline- versus chlorhexidine-treated SPF mice corroborated outcomes from SPF versus GF mice, which reveals that the commensal oral microbiota imparts osteoimmunomodulatory effects separate from the systemic microbiome.

Rationally designed bacterial consortia to treat chronic immune-mediated colitis and restore intestinal homeostasis.

Environmental factors, mucosal permeability and defective immunoregulation drive overactive immunity to a subset of resident intestinal bacteria that mediate multiple inflammatory conditions. GUT-103 and GUT-108, live biotherapeutic products rationally designed to complement missing or underrepresented functions in the dysbiotic microbiome of IBD patients, address upstream targets, rather than targeting a single cytokine to block downstream inflammation responses. GUT-103, composed of 17 strains that synergistically provide protective and sustained engraftment in the IBD inflammatory environment, prevented and treated chronic immune-mediated colitis. Therapeutic application of GUT-108 reversed established colitis in a humanized chronic T cell-mediated mouse model. It decreased pathobionts while expanding resident protective bacteria; produced metabolites promoting mucosal healing and immunoregulatory responses; decreased inflammatory cytokines and Th-1 and Th-17 cells; and induced interleukin-10-producing colonic regulatory cells, and IL-10-independent homeostatic pathways. We propose GUT-108 for treating and preventing relapse for IBD and other inflammatory conditions characterized by unbalanced microbiota and mucosal permeability.

Listeria monocytogenes Establishes Commensalism in Germ-Free Mice Through the Reversible Downregulation of Virulence Gene Expression.

The intestine harbors a complex community of bacterial species collectively known as commensal microbiota. Specific species of resident bacteria, as known as pathobiont, have pathogenic potential and can induce apparent damage to the host and intestinal inflammation in a certain condition. However, the host immune factors that permit its commensalism under steady state conditions are not clearly understood. Here, we studied the gut fitness of Listeria monocytogenes by using germ-free (GF) mice orally infected with this food-borne pathogen. L. monocytogenes persistently exists in the gut of GF mice without inducing chronic immunopathology. L. monocytogenes at the late phase of infection is not capable of infiltrating through the intestinal barrier. L. monocytogenes established the commensalism through the reversible down regulation of virulence gene expression. CD8+ T cells were found to be sufficient for the commensalism of L. monocytogenes. CD8+ T cells responding to L. monocytogenes contributed to the down-regulation of virulence gene expression. Our data provide important insights into the host-microbe interaction and have implications for developing therapeutics against immune disorders induced by intestinal pathogens or pathobionts.

The Role of Microbiome and Genotype in Daphnia magna upon Parasite Re-Exposure.

Recently, it has been shown that the community of gut microorganisms plays a crucial role in host performance with respect to parasite tolerance. Knowledge, however, is lacking on the role of the gut microbiome in mediating host tolerance after parasite re-exposure, especially considering multiple parasite infections. We here aimed to fill this knowledge gap by studying the role of the gut microbiome on tolerance in Daphnia magna upon multiple parasite species re-exposure. Additionally, we investigated the role of the host genotype in the interaction between the gut microbiome and the host phenotypic performance. A microbiome transplant experiment was performed in which three germ-free D. magna genotypes were exposed to a gut microbial inoculum and a parasite community treatment. The gut microbiome inocula were pre-exposed to the same parasite communities or a control treatment. Daphnia performance was monitored, and amplicon sequencing was performed to characterize the gut microbial community. Our experimental results showed that the gut microbiome plays no role in Daphnia tolerance upon parasite re-exposure. We did, however, find a main effect of the gut microbiome on Daphnia body size reflecting parasite specific responses. Our results also showed that it is rather the Daphnia genotype, and not the gut microbiome, that affected parasite-induced host mortality. Additionally, we found a role of the genotype in structuring the gut microbial community, both in alpha diversity as in the microbial composition.

Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development.

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Beta-glucan's varying structure characteristics modulate survival and immune-related genes expression from Vibrio harveyi-infected Artemia franciscana in gnotobiotic conditions.

ß-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of ß-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma ß-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and ß-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of ß-glucan and the results confirmed that ß-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Understanding immune-microbiota interactions in the intestine.

The past two decades have seen an explosion in research that aims to understand how the dynamic interplay with the gut microbiota impacts host health and disease, establishing a role for the gut microbiota in a plethora of pathologies. Understanding how health-promoting microbiota are established and how beneficial host-microbiota interactions are maintained is of immense biomedical importance. Despite the enormous progress that has been made, our knowledge of the specific microbiota members that mediate these effects and the mechanisms underlying these interactions is rudimentary. The dearth of information regarding the nature of advantageous host-microbiota interactions, and the factors that cause these relationships to go awry, has hampered our ability to realize the therapeutic potential of the microbiota. Here we discuss key issues that limit current knowledge and describe a path forwards to improving our understanding of the contributions of the microbiota to host health.

Pre- and Neonatal Imprinting on Immunological Homeostasis and Epithelial Barrier Integrity by Escherichia coli Nissle 1917 Prevents Allergic Poly-Sensitization in Mice.

A steady rise in the number of poly-sensitized patients has increased the demand for effective prophylactic strategies against multi-sensitivities. Probiotic bacteria have been successfully used in clinics and experimental models to prevent allergic mono-sensitization. In the present study, we have investigated whether probiotic bacteria could prevent poly-sensitization by imprinting on the immune system early in life. We used two recombinant variants of probiotic Escherichia coli Nissle 1917 (EcN): i) EcN expressing birch and grass pollen, poly-allergen chimera construct (EcN-Chim), and ii) an "empty" EcN without allergen expression (EcN-Ctrl). Conventional mice (CV) were treated with either EcN-Chim or EcN-Ctrl in the last week of the gestation and lactation period. Gnotobiotic mice received one oral dose of either EcN-Chim or EcN-Ctrl before mating. The offspring from both models underwent systemic allergic poly-sensitization and intranasal challenge with recombinant birch and grass pollen allergens (rBet v 1, rPhl p 1, and rPhl p 5). In the CV setting, the colonization of offspring via treatment of mothers reduced allergic airway inflammation (AAI) in offspring compared to poly-sensitized controls. Similarly, in a gnotobiotic model, AAI was reduced in EcN-Chim and EcN-Ctrl mono-colonized offspring. However, allergy prevention was more pronounced in the EcN-Ctrl mono-colonized offspring as compared to EcN-Chim. Mono-colonization with EcN-Ctrl was associated with a shift toward mixed Th1/Treg immune responses, increased expression of TLR2 and TLR4 in the lung, and maintained levels of zonulin-1 in lung epithelial cells as compared to GF poly-sensitized and EcN-Chim mono-colonized mice. This study is the first one to establish the model of allergic poly-sensitization in gnotobiotic mice. Using two different settings, gnotobiotic and conventional mice, we demonstrated that an early life intervention with the EcN without expressing an allergen is a powerful strategy to prevent poly-sensitization later in life.

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.

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be actively secreted by immune cells after different immune stimuli or passively released from cells undergoing necrosis. HMGB1 amplifies inflammation, and its hypersecretion contributes to multiple organ dysfunction syndrome and death. We tested possible immunomodulatory effect of commensal Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM) or probiotic Escherichia coli Nissle 1917 (EcN) in infection of gnotobiotic piglets with Salmonella Typhimurium (ST). Transcription of HMGB1 and Toll-like receptors (TLR) 2, 4, and 9 and receptor for advanced glycation end products (RAGE), TLR4-related molecules (MD-2, CD14, and LBP), and adaptor proteins (MyD88 and TRIF) in the ileum and colon were measured by RT-qPCR. Expression of TLR4 and its related molecules were highly upregulated in the ST-infected intestine, which was suppressed by EcN, but not LA nor LM. In contrast, HMGB1 expression was unaffected by ST infection or commensal/probiotic administration. HMGB1 protein levels in the intestine measured by ELISA were increased in ST-infected piglets, but they were decreased by previous colonization with E. coli Nissle 1917 only. We conclude that the stability of HMGB1 mRNA expression in all piglet groups could show its importance for DNA transcription and physiological cell functions. The presence of HMGB1 protein in the intestinal lumen probably indicates cellular damage.

Intestinal IgA Regulates Expression of a Fructan Polysaccharide Utilization Locus in Colonizing Gut Commensal Bacteroides thetaiotaomicron.

Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism.IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.

Microbiology and immunology: An ideal partnership for a tango at the gut surface-A tribute to Philippe Sansonetti.

Over the past 20 years, the highly dynamic interactions that take place between hosts and the gut microbiota have emerged as a major determinant in health and disease. The complexity of the gut microbiota represents, however, a considerable challenge, and reductionist approaches are indispensable to define the contribution of individual bacteria to host responses and to dissect molecular mechanisms. In this tribute to Philippe Sansonetti, I would like to show how rewarding collaborations with microbiologists have guided our team of immunologists in the study of host-microbiota interactions and, thanks to the use of controlled colonisation experiments in gnotobiotic mice, toward the demonstration that segmented filamentous bacteria (SFB) are indispensable to drive the post-natal maturation of the gut immune barrier in mice. The work led with Philippe Sansonetti to set up in vitro culture conditions has been one important milestone that laid the ground for in-depth characterization of the molecular attributes of this unusual symbiont. Recent suggestions that SFB may be present in the human microbiota encourage further cross-fertilising interactions between microbiologists and immunologists to define whether results from mice can be translated to humans and, if so, how SFB may be used to promote human intestinal defences against enteropathogens. Nurturing the competences to pursue this inspiring project is one legacy of Philippe Sansonetti.

Decreased maternal serum acetate and impaired fetal thymic and regulatory T cell development in preeclampsia.

Maternal immune dysregulation seems to affect fetal or postnatal immune development. Preeclampsia is a pregnancy-associated disorder with an immune basis and is linked to atopic disorders in offspring. Here we show reduction of fetal thymic size, altered thymic architecture and reduced fetal thymic regulatory T (Treg) cell output in preeclamptic pregnancies, which persists up to 4 years of age in human offspring. In germ-free mice, fetal thymic CD4+ T cell and Treg cell development are compromised, but rescued by maternal supplementation with the intestinal bacterial metabolite short chain fatty acid (SCFA) acetate, which induces upregulation of the autoimmune regulator (AIRE), known to contribute to Treg cell generation. In our human cohorts, low maternal serum acetate is associated with subsequent preeclampsia, and correlates with serum acetate in the fetus. These findings suggest a potential role of acetate in the pathogenesis of preeclampsia and immune development in offspring.

Bridging intestinal immunity and gut microbiota by metabolites.

The gastrointestinal tract is the site of nutrient digestion and absorption and is also colonized by diverse, highly mutualistic microbes. The intestinal microbiota has diverse effects on the development and function of the gut-specific immune system, and provides some protection from infectious pathogens. However, interactions between intestinal immunity and microorganisms are very complex, and recent studies have revealed that this intimate crosstalk may depend on the production and sensing abilities of multiple bioactive small molecule metabolites originating from direct produced by the gut microbiota or by the metabolism of dietary components. Here, we review the interplay between the host immune system and the microbiota, how commensal bacteria regulate the production of metabolites, and how these microbiota-derived products influence the function of several major innate and adaptive immune cells involved in modulating host immune homeostasis.

Gut microbiota from high-risk men who have sex with men drive immune activation in gnotobiotic mice and in vitro HIV infection.

Men who have sex with men (MSM) have differences in immune activation and gut microbiome composition compared with men who have sex with women (MSW), even in the absence of HIV infection. Gut microbiome differences associated with HIV itself when controlling for MSM, as assessed by 16S rRNA sequencing, are relatively subtle. Understanding whether gut microbiome composition impacts immune activation in HIV-negative and HIV-positive MSM has important implications since immune activation has been associated with HIV acquisition risk and disease progression. To investigate the effects of MSM and HIV-associated gut microbiota on immune activation, we transplanted feces from HIV-negative MSW, HIV-negative MSM, and HIV-positive untreated MSM to gnotobiotic mice. Following transplant, 16S rRNA gene sequencing determined that the microbiomes of MSM and MSW maintained distinct compositions in mice and that specific microbial differences between MSM and MSW were replicated. Immunologically, HIV-negative MSM donors had higher frequencies of blood CD38+ HLADR+ and CD103+ T cells and their fecal recipients had higher frequencies of gut CD69+ and CD103+ T cells, compared with HIV-negative MSW donors and recipients, respectively. Significant microbiome differences were not detected between HIV-negative and HIV-positive MSM in this small donor cohort, and immune differences between their recipients were trending but not statistically significant. A larger donor cohort may therefore be needed to detect immune-modulating microbes associated with HIV. To investigate whether our findings in mice could have implications for HIV replication, we infected primary human lamina propria cells stimulated with isolated fecal microbiota, and found that microbiota from MSM stimulated higher frequencies of HIV-infected cells than microbiota from MSW. Finally, we identified several microbes that correlated with immune readouts in both fecal recipients and donors, and with in vitro HIV infection, which suggests a role for gut microbiota in immune activation and potentially HIV acquisition in MSM.

Prebiotic supplementation in frail older people affects specific gut microbiota taxa but not global diversity.

BACKGROUND: There are complex interactions between aging, frailty, diet, and the gut microbiota; modulation of the gut microbiota by diet could lead to healthier aging. The purpose of this study was to test the effect of diets differing in sugar, fat, and fiber content upon the gut microbiota of mice humanized with microbiota from healthy or frail older people. We also performed a 6-month dietary fiber supplementation in three human cohorts representing three distinct life-stages. METHODS: Mice were colonized with human microbiota and then underwent an 8-week dietary intervention with either a high-fiber/low-fat diet typical of elderly community dwellers or a low-fiber/high-fat diet typical of long-stay residential care subjects. A cross-over design was used where the diets were switched after 4 weeks to the other diet type to identify responsive taxa and innate immunity changes. In the human intervention, the subjects supplemented their normal diet with a mix of five prebiotics (wheat dextrin, resistant starch, polydextrose, soluble corn fiber, and galactooligo-saccharide) at 10 g/day combined total, for healthy subjects and 20 g/day for frail subjects, or placebo (10 g/day maltodextrin) for 26 weeks. The gut microbiota was profiled and immune responses were assayed by T cell markers in mice, and serum cytokines in humans. RESULTS: Humanized mice maintained gut microbiota types reflecting the respective healthy or frail human donor. Changes in abundance of specific taxa occurred with the diet switch. In mice with the community type microbiota, the observed differences reflected compositions previously associated with higher frailty. The dominance of Prevotella present initially in community inoculated mice was replaced by Bacteroides, Alistipes, and Oscillibacter. Frail type microbiota showed a differential effect on innate immune markers in both conventional and germ-free mice, but a moderate number of taxonomic changes occurring upon diet switch with an increase in abundance of Parabacteroides, Blautia, Clostridium cluster IV, and Phascolarctobacterium. In the human intervention, prebiotic supplementation did not drive any global changes in alpha- or beta-diversity, but the abundance of certain bacterial taxa, particularly Ruminococcaceae (Clostridium cluster IV), Parabacteroides, Phascolarctobacterium, increased, and levels of the chemokine CXCL11 were significantly lower in the frail elderly group, but increased during the wash-out period. CONCLUSIONS: Switching to a nutritionally poorer diet has a profound effect on the microbiota in mouse models, with changes in the gut microbiota from healthy donors reflecting previously observed differences between elderly frail and non-frail individuals. However, the frailty-associated gut microbiota did not reciprocally switch to a younger healthy-subject like state, and supplementation with prebiotics was associated with fewer detected effects in humans than diet adjustment in animal models.

Expansion of commensal fungus Wallemia mellicola in the gastrointestinal mycobiota enhances the severity of allergic airway disease in mice.

The gastrointestinal microbiota influences immune function throughout the body. The gut-lung axis refers to the concept that alterations of gut commensal microorganisms can have a distant effect on immune function in the lung. Overgrowth of intestinal Candida albicans has been previously observed to exacerbate allergic airways disease in mice, but whether subtler changes in intestinal fungal microbiota can affect allergic airways disease is less clear. In this study we have investigated the effects of the population expansion of commensal fungus Wallemia mellicola without overgrowth of the total fungal community. Wallemia spp. are commonly found as a minor component of the commensal gastrointestinal mycobiota in both humans and mice. Mice with an unaltered gut microbiota community resist population expansion when gavaged with W. mellicola; however, transient antibiotic depletion of gut microbiota creates a window of opportunity for expansion of W. mellicola following delivery of live spores to the gastrointestinal tract. This phenomenon is not universal as other commensal fungi (Aspergillus amstelodami, Epicoccum nigrum) do not expand when delivered to mice with antibiotic-depleted microbiota. Mice with Wallemia-expanded gut mycobiota experienced altered pulmonary immune responses to inhaled aeroallergens. Specifically, after induction of allergic airways disease with intratracheal house dust mite (HDM) antigen, mice demonstrated enhanced eosinophilic airway infiltration, airway hyperresponsiveness (AHR) to methacholine challenge, goblet cell hyperplasia, elevated bronchoalveolar lavage IL-5, and enhanced serum HDM IgG1. This phenomenon occurred with no detectable Wallemia in the lung. Targeted amplicon sequencing analysis of the gastrointestinal mycobiota revealed that expansion of W. mellicola in the gut was associated with additional alterations of bacterial and fungal commensal communities. We therefore colonized fungus-free Altered Schaedler Flora (ASF) mice with W. mellicola. ASF mice colonized with W. mellicola experienced enhanced severity of allergic airways disease compared to fungus-free control ASF mice without changes in bacterial community composition.

Early cellular responses of germ-free and specific-pathogen-free mice to Francisella tularensis infection.

Bacteria that are highly virulent, expressing high infectivity, and able to survive nebulization, pose great risk to the human population. One of these is Francisella tularensis, the etiological agent of tularemia. F. tularensis is a subject of intense scientific interest due to the fact that vaccines for its immunoprophylaxis in humans are not yet routinely available. One of the substantial obstacles in developing such vaccines is our insufficient knowledge of processes that initiate and regulate the expression of effective protective immunity against intracellular bacteria. Here, we present data documenting the different pattern of cellular behavior occurring in an environment unaffected by microbiota using the model of germ-free mice mono-associated with F. tularensis subsp. holarctica strain LVS in comparison with a classic specific-pathogen-free murine model during early stages of infection.

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

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.