Characterizing the Interactions between a Naturally Primed Immunoglobulin A and Its Conserved Bacteroides thetaiotaomicron Species-specific Epitope in Gnotobiotic Mice

The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1−/−, or Myd88−/− mice. Comparison of gnotobiotic Rag1−/− mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism.     

Host adaptive immunity alters gut microbiota

It has long been recognized that the mammalian gut microbiota has a role in the development and activation of the host immune system. Much less is known on how host immunity regulates the gut microbiota. Here we investigated the role of adaptive immunity on the mouse distal gut microbial composition by sequencing 16 S rRNA genes from microbiota of immunodeficient Rag1^−/− mice, versus wild-type mice, under the same housing environment. To detect possible interactions among immunological status, age and variability from anatomical sites, we analyzed samples from the cecum, colon, colonic mucus and feces before and after weaning. High-throughput sequencing showed that Firmicutes, Bacteroidetes and Verrucomicrobia dominated mouse gut bacterial communities. Rag1⁻ mice had a distinct microbiota that was phylogenetically different from wild-type mice. In particular, the bacterium Akkermansia muciniphila was highly enriched in Rag1^−/− mice compared with the wild type. This enrichment was suppressed when Rag1^−/− mice received bone marrows from wild-type mice. The microbial community diversity increased with age, albeit the magnitude depended on Rag1 status. In addition, Rag1^−/− mice had a higher gain in microbiota richness and evenness with increase in age compared with wild-type mice, possibly due to the lack of pressure from the adaptive immune system. Our results suggest that adaptive immunity has a pervasive role in regulating gut microbiota''s composition and diversity.     

Expression of Human Paraoxonase 1 Decreases Superoxide Levels and Alters Bacterial Colonization in the Gut of Drosophila melanogaster

Paraoxonases (PON) are a family of proteins (PON1, 2 and 3) with multiple enzymatic activities. PON1 interferes with homoserine lactone-mediated quorum sensing in bacteria and with reactive oxygen species (ROS) in humans and mice. PON1 gene mutations have been linked to multiple traits, including aging, and diseases of the cardiovascular, nervous and gastrointestinal system. The overlapping enzymatic activities in the PON family members and high linkage disequilibrium rates within their polymorphisms confound animal and human studies of PON1 function. In contrast, arthropods such as Drosophila melanogaster have no PON homologs, resulting in an ideal model to study interactions between PON genotype and host phenotypes. We hypothesized that expression of PON1 in D. melanogaster would alter ROS. We found that PON1 alters expression of multiple oxidative stress genes and decreases superoxide anion levels in normal and germ-free D. melanogaster. We also found differences in the composition of the gut microbiota, with a remarkable increase in levels of Lactobacillus plantarum and associated changes in expression of antimicrobial and cuticle-related genes. PON1 expression directly decreased superoxide anion levels and altered bacterial colonization of the gut and its gene expression profile, highlighting the complex nature of the interaction between host genotype and gut microbiota. We speculate that the interaction between some genotypes and human diseases may be mediated by the presence of certain gut bacteria that can induce specific immune responses in the gut and other host tissues.     

Gut immune maturation depends on colonization with a host-specific microbiota

Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

Host Responses to Intestinal Microbial Antigens in Gluten-Sensitive Mice

Background and Aims

Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice.

Methodology/Principal Findings

HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-γ in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota.

Conclusion

Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-γ production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.     

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression

Background

Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity.

Methodology/Principal Findings

Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals'' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer''s patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium.

Conclusion

We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.     

Infant gut microbiota is protective against cow's milk allergy in mice despite immature ileal T-cell response

Faecal microbiota of healthy infant displays a large abundance of Bifidobacterium spp. and Bacteroides spp. Although some studies have reported an association between these two genera and allergy, these findings remain a subject of debate. Using a gnotobiotic mouse model of cow's milk allergy, we investigated the impact of an infant gut microbiota – mainly composed of Bifidobacterium and Bacteroides spp. – on immune activation and allergic manifestations. The transplanted microbiota failed to restore an ileal T-cell response similar to the one observed in conventional mice. This may be due to the low bacterial translocation into Peyer's patches in gnotobiotic mice. The allergic response was then monitored in germ-free, gnotobiotic, and conventional mice after repeated oral sensitization with whey proteins and cholera toxin. Colonized mice displayed a lower drop of rectal temperature upon oral challenge with b-lactoglobulin, lower plasma mMCP-1, and lower anti-BLG IgG1 than germ-free mice. The foxp3 gene was highly expressed in the ileum of both colonized mice that were protected against allergy. This study is the first demonstration that a transplanted healthy infant microbiota mainly composed of Bifidobacterium and Bacteroides had a protective impact on sensitization and food allergy in mice despite altered T-cell response in the ileum.     

CD4+CD25+ regulatory T cells control innate immune reactivity after injury

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1(-/-)) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-alpha, IL-1beta, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1(-/-), CD4(-/-), or CD8(-/-) mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8(-/-) mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4(-/-) mice produced cytokines at significantly higher levels, equivalent to those in Rag1(-/-) mice. Moreover, reconstitution of Rag1(-/-) or CD4(-/-) mice with WT CD4(+) T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4(+) T cells into CD4(+)CD25(+) and CD4(+)CD25(-) subpopulations before their adoptive transfer into Rag1(-/-) mice showed that CD4(+)CD25(+) T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4(+)CD25(-) T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses after injury.     

Saccharomyces boulardii stimulates sIgA production and the phagocytic system of gnotobiotic mice

The effect of Saccharomyces boulardii on the immune system was evaluated, comparing germ-free Swiss/NIH mice monoassociated with the probiotic with germ-free mice. Saccharomyces boulardii colonized the gut of germ-free mice and survived the gastrointestinal conditions. An increase in sIgA production, both total and anti-S. boulardii, was observed in the intestinal contents of monoassociated mice when compared with germ-free controls. The number of Kupffer cells was significantly higher in monoassociated mice than in germ-free controls. In S. boulardii-monoassociated mice, clearance of Escherichia coli B41 was higher than in germ-free controls. TNF-alpha, IFN-gamma and IL-12 serum levels were higher at earlier time points in monoassociated mice when compared with germ-free mice. These results show that the yeast S. boulardii modulates the host immune responses. This effect may be of interest for improving the resistance to enteropathogenic bacterial infections.     

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.     

IgA anti-dextran B1355 responses.

Injection of mice bearing the Ig-1a allotype with dextran B1355 results in an IgM antibody response that is generally regarded as thymus independent. Moreover, the antibody is directed to alpha[1,3] determinants on dextran B1355 and shares cross-reacting idiotypic determinants with a lambda 1 IgA (J558) myeloma protein as well as a lambda 1 IgM (MOPC 104E) myeloma protein. In this study, we show that BALB/c (Ig-1a) mice injected with dextran B1355 produced highly significant IgA anti-dextran responses with specificity directed to the alpha[1,3] epitope. Kinetics of the IgA anti-dextran response in BALB/c mice paralleled kinetics of the IgM response. However, the magnitude of the IgA response was markedly T cell dependent and age dependent.     

重组大肠杆菌菌蜕与幽门螺杆菌蛋白质疫苗协同免疫BALB/c小鼠

目的:考察重组菌蜕和蛋白质疫苗混合后对BAB/c小鼠机体免疫反应的影响。方法:首先构建展示尿素酶B抗原表位的大肠杆菌菌蜕,同时通过融合PCR构建分子内佐剂蛋白质疫苗rLTBKAT,然后分别利用该菌蜕和蛋白质疫苗免疫BAB/c小鼠。结果:当rLTBKAT和重组菌蜕联合口服免疫BAB/c小鼠时,其抗尿素酶B抗体的效价由单独免疫时的1∶(239±23)提高到1∶(681±76),二者差异极显著(P=0.009);而其抗过氧化氢酶抗体的效价则由单独免疫时的1∶(2800±275)下降至1∶(1800±400),二者差异不显著(P=0.08)。抗体分型试验表明,单独和联合免疫时,其抗尿素酶B抗体类型均以IgG1为主,而抗过氧化氢酶抗体类型则由单独免疫时的IgG1为主转变为联合免疫时的IgG2a为主。联合免疫时,小鼠抗尿素酶B和抗过氧化氢酶IgA抗体水平均高于单独免疫时。结论:重组菌蜕和蛋白质疫苗联合免疫,可提高机体对某些抗原的免疫水平或改变其反应类型。     

The Gut Microbiota Modulates Glycaemic Control and Serum Metabolite Profiles in Non-Obese Diabetic Mice

Islet autoimmunity in children who later progress to type 1 diabetes is preceded by dysregulated serum metabolite profiles, but the origin of these metabolic changes is unknown. The gut microbiota affects host metabolism and changes in its composition contribute to several immune-mediated diseases; however, it is not known whether the gut microbiota is involved in the early metabolic disturbances in progression to type 1 diabetes. We rederived non-obese diabetic (NOD) mice as germ free to explore the potential role of the gut microbiota in the development of diabetic autoimmunity and to directly investigate whether the metabolic profiles associated with the development of type 1 diabetes can be modulated by the gut microbiota. The absence of a gut microbiota in NOD mice did not affect the overall diabetes incidence but resulted in increased insulitis and levels of interferon gamma and interleukin 12; these changes were counterbalanced by improved peripheral glucose metabolism. Furthermore, we observed a markedly increased variation in blood glucose levels in the absence of a microbiota in NOD mice that did not progress to diabetes. Additionally, germ-free NOD mice had a metabolite profile similar to that of pre-diabetic children. Our data suggest that germ-free NOD mice have reduced glycaemic control and dysregulated immunologic and metabolic responses.     

Increased Immune Response Elicited by DNA Vaccination with a Synthetic gp120 Sequence with Optimized Codon Usage

DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination.