Microbiota composition and mucosal immunity in patients with asymptomatic diverticulosis and controls.

INTRODUCTION: The etiology of diverticulosis is still poorly understood. However, in patients with diverticulitis, markers of mucosal inflammation and microbiota alterations have been found. The aim of this study was to evaluate potential differences of the gut microbiota composition and mucosal immunity between patients with asymptomatic diverticulosis and controls. METHODS: We performed a prospective study on patients who underwent routine colonoscopy for causes not related to diverticular disease or inflammatory bowel disease. Participants were grouped based on the presence or absence of diverticula. Mucosal biopsies were obtained from the sigmoid and transverse colon. Microbiota composition was analyzed with IS-pro, a 16S-23S based bacterial profiling technique. To predict if patients belonged to the asymptomatic diverticulosis or control group a partial least squares discriminant analysis (PLS-DA) regression model was used. Inflammation was assessed by neutrophil and lymphocyte counts within the taken biopsies. RESULTS: Forty-three patients were enrolled. Intestinal microbiota profiles were highly similar within individuals for all phyla. Between individuals, microbiota profiles differed substantially but regardless of the presence (n = 19) of absence (n = 24) of diverticula. Microbiota diversity in both sigmoid and transverse colon was similar in all participants. We were not able to differentiate between diverticulosis patients and controls with a PLS-DA model. Mucosal lymphocyte counts were comparable among both groups; no neutrophils were detected in any of the studied biopsies. CONCLUSIONS: Microbiota composition and inflammatory markers were comparable among asymptomatic diverticulosis patients and controls. This suggests that the gut microbiota and mucosal inflammation do not play a major role in the pathogenesis of diverticula formation.

The nasal microbiome, nasal transcriptome, and pet sensitization.

BACKGROUND: Pet allergies are common in children with asthma. Microbiota and host responses may mediate allergen sensitization. OBJECTIVE: We sought to uncover host-microbe relationships in pet allergen sensitization via joint examination of the nasal microbiome and nasal transcriptome. METHODS: We collected nasal samples from 132 children with asthma for parallel 16S rRNA and RNA sequencing. Specific IgE levels for cat and dog dander were measured. Analyses of the nasal microbiome, nasal transcriptome, and their correlations were performed with respect to pet sensitization status. RESULTS: Among the 132 children, 91 (68.9%) were cat sensitized and 96 (72.7%) were dog sensitized. Cat sensitization was associated with lower nasal microbial diversity by Shannon index (P = .021) and differential nasal bacterial composition by weighted UniFrac distance (permutational multivariate ANOVA P = .035). Corynebacterium sp and Staphylococcus epidermidis were significantly less abundant, and the metabolic process "fatty acid elongation in mitochondria" was lower in pet-sensitized versus unsensitized children. Correlation networks revealed that the nasal expression levels of 47 genes representing inflammatory processes were negatively correlated with the relative abundances of Corynebacterium sp and S epidermidis. Thus, these species were directly associated not only with the absence of pet sensitization but also with the underexpression of host gene expression of inflammatory processes that contribute to allergen sensitization. Causal mediation analyses revealed that the associations between these nasal species and pet sensitization were mediated by nasal gene expression. CONCLUSIONS: Higher abundances of nasal Corynebacterium sp and S epidermidis are associated with absence of pet sensitization and correlate with lower expression of inflammatory genes.

Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.

Natural killer T (NKT) cells are an abundant subset of liver lymphocytes activated by lipid antigens presented on CD1d molecules that are expressed by cholangiocytes. We aimed to determine if bile from patients with chronic liver diseases contains antigenic lipids that can activate NKT cells. Using murine invariant (24.7, 24.8 and DN32.D3) and non-invariant (14S.6, 14S.7 and 14S.10) NKT hybridomas we investigated the presence of lipid antigens in bile collected from the gallbladder of patients undergoing liver transplantation due to end-stage liver disease. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing. We found that the patient bile samples contain antigens that activate both invariant and non-invariant NKT hybridomas (24.7, 24.8, DN32.D3, 14S.6, 14S.7 and 14S.10), as demonstrated by activation of at least one hybridoma by eight of 10 bile samples. Activation at high dilutions suggests that some antigens are highly potent. We used the non-invariant NKT hybridoma 14S.6 to screen 21 additional patient bile samples for NKT-reactivity and demonstrated that 12 of 21 bile samples resulted in activation, three of which gave a strong activation. Four of 12 activating bile samples contained microbial DNA. Our results reveal an immunological pathway that could be of critical importance in biliary immunology.

Evaluation of the upper airway microbiome and immune response with nasal epithelial lining fluid absorption and nasal washes.

Despite being commonly used to collect upper airway epithelial lining fluid, nasal washes are poorly reproducible, not suitable for serial sampling, and limited by a dilution effect. In contrast, nasal filters lack these limitations and are an attractive alternative. To examine whether nasal filters are superior to nasal washes as a sampling method for the characterization of the upper airway microbiome and immune response, we collected paired nasal filters and washes from a group of 40 healthy children and adults. To characterize the upper airway microbiome, we used 16S ribosomal RNA and shotgun metagenomic sequencing. To characterize the immune response, we measured total protein using a BCA assay and 53 immune mediators using multiplex magnetic bead-based assays. We conducted statistical analyses to compare common microbial ecology indices and immune-mediator median fluorescence intensities (MFIs) between sample types. In general, nasal filters were more likely to pass quality control in both children and adults. There were no significant differences in microbiome community richness, α-diversity, or structure between pediatric samples types; however, these were all highly dissimilar between adult sample types. In addition, there were significant differences in the abundance of amplicon sequence variants between sample types in children and adults. In adults, total proteins were significantly higher in nasal filters than nasal washes; consequently, the immune-mediator MFIs were not well detected in nasal washes. Based on better quality control sequencing metrics and higher immunoassay sensitivity, our results suggest that nasal filters are a superior sampling method to characterize the upper airway microbiome and immune response in both children and adults.

Association of the eukaryotic vaginal virome with prophylactic antibiotic exposure and reproductive outcomes in a subfertile population undergoing in vitro fertilisation: a prospective exploratory study.

OBJECTIVE: The objective of this study was to use high-throughput sequencing to describe the vaginal eukaryotic DNA virome in patients undergoing in vitro fertilisation (IVF) to examine associations between the vaginal virome, antibiotic exposure and IVF outcomes. DESIGN: Prospective exploratory study. SETTING: Single academic fertility centre. POPULATION: Subfertile women age 18-43 years undergoing their first IVF cycle with a fresh embryo transfer. METHODS: The primary exposure was prophylactic azithromycin or no azithromycin before IVF. A mid-vaginal swab was obtained at the time of embryo transfer for virome analysis. MAIN OUTCOME MEASURES: The primary outcomes compared between exposure groups were characteristics of vaginal virome and clinical pregnancy rates. Secondary outcomes were virome associations with number of oocytes retrieved, number of blastocysts and implantation rate. RESULTS: Twenty-six women contributed a vaginal swab before embryo transfer. There were no significant differences in IVF outcomes between azithromycin groups. There was no association between viral diversity and clinical pregnancy overall. A higher diversity of herpesviruses and α-papillomaviruses was observed in samples from the azithromycin-treated group compared with the no azithromycin group (P = 0.04). In women that received azithromycin, viral diversity was higher in the group that did not achieve clinical pregnancy compared with those who did (P = 0.06). CONCLUSIONS: We demonstrate that the vaginal eukaryotic virome in women undergoing IVF is associated with antibiotic exposure. Additionally, we demonstrate an inverse trend between viral diversity and pregnancy, with a higher number of viruses detected associated with failure to achieve clinical pregnancy in the azithromycin group. TWEETABLE ABSTRACT: Higher viral diversity is associated with prophylactic antibiotic exposure in subfertile women undergoing IVF.

Peptidoglycan Recognition Protein 4 Limits Bacterial Clearance and Inflammation in Lungs by Control of the Gut Microbiota.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. Endogenous host defense molecules such as peptidoglycan recognition protein 4 (PGLYRP4) might influence the course of this disease. To the best of our knowledge, there are no reports on the relevance of PGLYRP4 in pneumonia. Therefore, wild type (WT) and PGLYRP4-deficient (PGLYRP4KO) mice were analyzed in an in vivo and in vitro experimental setting to examine the influence of PGLYRP4 on the course of pneumococcal pneumonia. Furthermore, caecal 16S rRNA microbiome analysis was performed, and microbiota were transferred to germfree WT mice to assess the influence of microbiotal communities on the bacterial burden. Mice lacking PGLYRP4 displayed an enhanced bacterial clearance in the lungs, and fewer mice developed bacteremia. In addition, an increased recruitment of immune cells to the site of infection, and an enhanced bacterial killing by stronger activation of phagocytes could be shown. This may depend partly on the detected higher expression of complement factors, interferon-associated genes, and the higher pro-inflammatory cytokine response in isolated primary PGLYRP4KO vs. WT cells. This phenotype is underlined by changes in the complexity and composition of the caecal microbiota of PGLYRP4KO compared to WT mice. Strikingly, we provided evidence, by cohousing and stable transfer of the respective WT or PGLYRP4KO mice microbiota into germfree WT mice, that the changes of the microbiota are responsible for the improved clearance of S. pneumoniae lung infection. In conclusion, the deficiency of PGLYRP4, a known antibacterial protein, leads to changes in the gut microbiota. Thus, alterations in the microbiota can change the susceptibility to S. pneumoniae lung infection independently of the host genotype.

An altered fecal microbiota profile in patients with non-alcoholic fatty liver disease (NAFLD) associated with obesity

Introduction: increasing evidence suggests a role of intestinal dysbiosis in obesity and non-alcoholic fatty liver disease (NAFLD). The advances in recent years with regard to the role of the gut microbiota raise the potential utility of new therapeutic approaches based on the modification of the microbiome. Objective: the aim of this study was to compare the bacterial communities in obese patients with or without NAFLD to those of healthy controls. Patients and methods: the fecal microbiota composition of 20 healthy adults, 36 obese patients with NAFLD and 17 obese patients without NAFLD was determined by 16S ribosomal RNA sequencing using the Illumina MiSeq system. Results: the results highlighted significant differences in the phylum Firmicutes between patients with and without NAFLD, which was a determining factor of the disease and supported its possible role as a marker of NAFLD. At the genus level, the relative abundance of Blautia, Alkaliphilus, Flavobacterium and Akkermansia was reduced in obese patients, both with or without NAFLD, compared to healthy controls. Furthermore, the number of sequences from the genus Streptococcus was significantly higher in patients with NAFLD in comparison with individuals without the disease, constituting another possible marker. Comparison of bacterial communities at the genus level by a principal coordinate analysis indicated that the bacterial communities of patients with NAFLD were dispersed and did not form a group. Conclusion: in conclusion, these results indicate the role of intestinal dysbiosis in the development of NAFLD associated with obesity. There was a differential microbiota profile between obese patients, with and without NAFLD. Thus, supporting gut microbiota modulation as a therapeutic alternative for the prevention and treatment of NAFLD

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The Formation of Glycan-Specific Natural Antibodies Repertoire in GalT-KO Mice Is Determined by Gut Microbiota.

Gut commensal bacteria are known to have a significant role in regulating the innate and adaptive immune homeostasis. Alterations in the intestinal microbial composition have been associated with several disease states, including autoimmune and inflammatory conditions. However, it is not entirely clear how commensal gut microbiota modulate and contribute to the systemic immunity, and whether circulating elements of the host immune system could regulate the microbiome. Thus, we have studied the diversity and abundance of specific taxons in the gut microbiota of inbred GalT-KO mice during 7 months of animal life by metagenetic high-throughput sequencing (16S rRNA gene, variable regions V3-V5). The repertoire of glycan-specific natural antibodies, obtained by printed glycan array technology, was then associated with the microbial diversity for each animal by metagenome-wide association studies (MWAS). Our data show that the orders clostridiales (most abundant), bacteriodales, lactobacillales, and deferribacterales may be associated with the development of the final repertoire of natural anti-glycan antibodies in GalT-KO mice. The main changes in microbiota diversity (month-2 and month-3) were related to important changes in levels and repertoire of natural anti-glycan antibodies in these mice. Additionally, significant positive and negative associations were found between the gut microbiota and the pattern of specific anti-glycan antibodies. Regarding individual features, the gut microbiota and the corresponding repertoire of natural anti-glycan antibodies showed differences among the examined animals. We also found redundancy in different taxa associated with the development of specific anti-glycan antibodies. Differences in microbial diversity did not, therefore, necessarily influence the overall functional output of the gut microbiome of GalT-KO mice. In summary, the repertoire of natural anti-carbohydrate antibodies may be partially determined by the continuous antigenic stimulation produced by the gut bacterial population of each GalT-KO mouse. Small differences in gut microbiota diversity could determine different repertoire and levels of natural anti-glycan antibodies and consequently might induce different immune responses to pathogens or other potential threats.

Differences in Systemic IgA Reactivity and Circulating Th Subsets in Healthy Volunteers With Specific Microbiota Enterotypes.

Changes in the intestinal microbiota have been associated with the development of immune-mediated diseases in humans. Additionally, the introduction of defined bacterial species into the mouse intestinal microbiota has been shown to impact on the adaptive immune response. However, how much impact the intestinal microbiota composition actually has on regulating adaptive immunity remains poorly understood. Therefore, we studied aspects of the adaptive immunity in healthy adults possessing distinct intestinal microbiota profiles. The intestinal microbiota composition was determined via Illumina sequencing of bacterial 16S rRNA genes extracted from the feces of 35 individuals. Blood B-cell and T-cell subsets from the same individuals were studied using flow cytometry. Finally, the binding of fecal and plasma Immunoglobulin A (IgA) to intestinal bacteria (associated with health and disease) Bacteroides fragilis, Prevotella copri, Bifidobacterium longum, Clostridium difficile, and Escherichia coli was analyzed using ELISA. Unsupervised clustering of microbiota composition revealed the presence of three clusters within the cohort. Cluster 1 and 2 were similar to previously-described enterotypes with a predominance of Bacteroides in Cluster 1 and Prevotella in Cluster 2. The bacterial diversity (Shannon index) and bacterial richness of Cluster 3 was significantly higher than observed in Clusters 1 and 2, with the Ruminococacceae tending to predominate. Within circulating B- and T-cell subsets, only Th subsets were significantly different between groups of distinct intestinal microbiota. Individuals of Cluster 3 have significantly fewer Th17 and Th22 circulating cells, while Th17.1 cell numbers were increased in individuals of Cluster 1. IgA reactivity to intestinal bacteria was higher in plasma than feces, and individuals of Cluster 1 had significant higher plasma IgA reactivity against B. longum than individuals of Cluster 2. In conclusion, we identified three distinct fecal microbiota clusters, of which two clusters resembled previously-described "enterotypes". Global T-cell and B-cell immunity seemed unaffected, however, circulating Th subsets and plasma IgA reactivity were significantly different between Clusters. Hence, the impact of intestinal bacteria composition on human B cells, T cells and IgA reactivity appears limited in genetically-diverse and environmentally-exposed humans, but can skew antibody reactivity and Th cell subsets.

Mucosal Barrier and Th2 Immune Responses Are Enhanced by Dietary Inulin in Pigs Infected With Trichuris suis.

Diet composition may play a crucial role in shaping host immune responses and commensal gut microbiota populations. Bioactive dietary components, such as inulin, have been extensively studied for their bioactive properties, particularly in modulating gut immune function and reducing inflammation. It has been shown that colonization with gastrointestinal parasitic worms (helminths) may alleviate chronic inflammation through promotion of T-helper cell type (Th) 2 and T-regulatory immune responses and alterations in the gut microbiome. In this study, we investigated if dietary inulin could modulate mucosal immune function in pigs during colonization with the porcine whipworm Trichuris suis. T. suis infection induced a typical Th2-biased immune response characterized by transcriptional changes in Th2- and barrier function-related genes, accompanied by intestinal remodeling through increased epithelial goblet and tuft cell proliferation. We observed that inulin also up-regulated Th2-related immune genes (IL13, IL5), and suppressed Th1-related pro-inflammatory genes (IFNG, IL1A, IL8) in the colon. Notably, inulin augmented the T. suis-induced responses with increased transcription of key Th2 and mucosal barrier genes (e.g., IL13, TFF3), and synergistically suppressed pro-inflammatory genes, such as IFNG and CXCL9. 16S rRNA sequencing of proximal colon digesta samples revealed that inulin supplementation reduced the abundance of bacterial phyla linked to inflammation, such as Proteobacteria and Firmicutes, and simultaneously increased Actinobacteria and Bacteroidetes. Interestingly, pigs treated with both inulin and T. suis displayed the highest Bacteroidetes: Firmicutes ratio and the lowest gut pH, suggesting an interaction of diet and helminth infection that stimulates the growth of beneficial bacterial species. Overall, our data demonstrate that T. suis infection and inulin co-operatively enhance anti-inflammatory immune responses, which is potentially mediated by changes in microbiota composition. Our results highlight the intricate interactions between diet, immune function and microbiota composition in a porcine helminth infection model. This porcine model should facilitate further investigations into the use of bioactive diets as immunomodulatory mediators against inflammatory conditions, and how diet and parasites may influence gut health.

Herpes simplex virus-binding IgG traps HSV in human cervicovaginal mucus across the menstrual cycle and diverse vaginal microbial composition.

IgG possesses an important yet little recognized effector function in mucus. IgG bound to viral surface can immobilize otherwise readily diffusive viruses to the mucin matrix, excluding them from contacting target cells and facilitating their elimination by natural mucus clearance mechanisms. Cervicovaginal mucus (CVM) is populated by a microbial community, and its viscoelastic and barrier properties can vary substantially not only across the menstrual cycle, but also in women with distinct microbiota. How these variations impact the "muco-trapping" effector function of IgGs remains poorly understood. Here we obtained multiple fresh, undiluted CVM specimens (n = 82 unique specimens) from six women over time, and employed high-resolution multiple particle tracking to quantify the mobility of fluorescent Herpes Simplex Viruses (HSV-1) in CVM treated with different HSV-1-binding IgG. The IgG trapping potency was then correlated to the menstrual cycle, and the vaginal microbial composition was determined by 16 s rRNA. In the specimens studied, both polyclonal and monoclonal HSV-1-binding IgG appeared to consistently and effectively trap HSV-1 in CVM obtained at different times of the menstrual cycle and containing a diverse spectrum of commensals, including G. vaginalis-dominant microbiota. Our findings underscore the potential broad utility of this "muco-trapping" effector function of IgG to reinforce the vaginal mucosal defense, and motivates further investigation of passive immunization of the vagina as a strategy to protect against vaginally transmitted infections.

Inflammatory phenotypes in patients with severe asthma are associated with distinct airway microbiology.

BACKGROUND: Asthma pathophysiology and treatment responsiveness are predicted by inflammatory phenotype. However, the relationship between airway microbiology and asthma phenotype is poorly understood. OBJECTIVE: We aimed to characterize the airway microbiota in patients with symptomatic stable asthma and relate composition to airway inflammatory phenotype and other phenotypic characteristics. METHODS: The microbial composition of induced sputum specimens collected from adult patients screened for a multicenter randomized controlled trial was determined by using 16S rRNA gene sequencing. Inflammatory phenotypes were defined by sputum neutrophil and eosinophil cell proportions. Microbiota were defined by using α- and ß-diversity measures, and interphenotype differences were identified by using similarity of percentages, network analysis, and taxon fold change. Phenotypic predictors of airway microbiology were identified by using multivariate linear regression. RESULTS: Microbiota composition was determined in 167 participants and classified as eosinophilic (n = 84), neutrophilic (n = 14), paucigranulocytic (n = 60), or mixed neutrophilic-eosinophilic (n = 9) asthma phenotypes. Airway microbiology was significantly less diverse (P = .022) and more dissimilar (P = .005) in neutrophilic compared with eosinophilic participants. Sputum neutrophil proportions, but not eosinophil proportions, correlated significantly with these diversity measures (α-diversity: Spearman r = -0.374, P < .001; ß-diversity: r = 0.238, P = .002). Interphenotype differences were characterized by a greater frequency of pathogenic taxa at high relative abundance and reduced Streptococcus, Gemella, and Porphyromonas taxa relative abundance in patients with neutrophilic asthma. Multivariate regression confirmed that sputum neutrophil proportion was the strongest predictor of microbiota composition. CONCLUSIONS: Neutrophilic asthma is associated with airway microbiology that is significantly different from that seen in patients with other inflammatory phenotypes, particularly eosinophilic asthma. Differences in microbiota composition might influence the response to antimicrobial and steroid therapies and the risk of lung infection.

El microbioma respiratorio: más alla del cultivo / The respiratory microbiome: beyond the culture

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Perinatal Bacterial Exposure Contributes to IL-13 Aeroallergen Response.

There is a high prevalence of aeroallergen sensitivity in asthmatic populations, and seroreactivity to aeroallergens early in infancy is associated with increased risk of developing asthma later in life. In addition to allergen sensitivity, asthma development has been associated with differential microbial exposure and infection in early life. We have previously shown that cord blood mononuclear cells respond to common aeroallergens (i.e., house dust mite [Der f1] and cockroach [Bla g2]) as assayed by lymphoproliferation and cytokine (IL-13 and IFN-γ) production. We hypothesized that there is a relationship between perinatal microbial exposure and response to specific aeroallergens. To test this hypothesis, we isolated DNA from cord blood serum samples with known lymphoproliferative and cytokine responses to Bla g2 and Der f1. Bacterial 16S ribosomal DNA amplicon libraries were generated and analyzed using high throughput sequencing of cord blood serum samples. In our analysis, we identified major compositional differences, including diversity and abundance of specific taxa, between groups whose IL-13 response to Der f1 and Bla g2 differed. We demonstrate a strong association between the ratio of Acinetobacter to Proteobacteria and IL-13 production and the probability of IL-13 production after allergen exposure. IL-13 concentrations in serum were also significantly correlated with the diversity of bacterial DNA. Together, these results underscore the relationship between immune responses to allergens and bacterial exposure during perinatal development.

Sampling of intestinal microbiota and targeted amplification of bacterial 16S rRNA genes for microbial ecologic analysis.

Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre-processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology.

Nucleotide-oligomerization-domain-2 affects commensal gut microbiota composition and intracerebral immunopathology in acute Toxoplasma gondii induced murine ileitis.

BACKGROUND: Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far. METHODOLOGY/PRINCIPAL FINDINGS: Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra-intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals. CONCLUSION/SIGNIFICANCE: NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner.

Three types of human CpG motifs differentially modulate and augment immunogenicity of nonviral and viral replicon DNA vaccines as built-in adjuvants.

NakedDNA vaccines given by intramuscular injection are efficient in mouse models, but they require improvement for human use. As the immunogenicity of DNA vaccines depends, to a large extent, on the presence of CpG motifs as built-in adjuvants, we addressed this issue by inserting three types of human CpG motifs (A-type, B-type, and C-type) into the backbone of nonviral DNA and viral DNA replicon vectors with distinct immunostimulatory activities on human PBMCs. The adjuvant effects of CpG modifications in DNA vaccines expressing three types of antigens (ß-Gal, AHc, or PA4) were then characterized in mice and found to significantly enhance antigen-specific humoral and cell-mediated immune responses. The three types of CpG motifs also differentially affected and modulated immune responses and protective potency against botulinum neurotoxin serotype A and Bacillus anthracis A16R challenge. Taken together, these results demonstrate that insertion of human CpG motifs can differentially modulate the immunogenicity of nonviral DNA vaccines as well as viral DNA replicon vaccines. Our study provides not only a better understanding of the in vivo activities of CpG motif adjuvants but implications for the rational design of such motifs as built-in adjuvants for DNA vectors targeting specific antigens.

Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice.

The intestinal microbiota contributes to the development of the immune system, and conversely, the immune system influences the composition of the microbiota. Toll-like receptors (TLRs) in the gut recognize bacterial ligands. Although TLR signaling represents a major arm of the innate immune system, the extent to which TLRs influence the composition of the intestinal microbiota remains unclear. We performed deep 16S ribosomal RNA sequencing to characterize the complex bacterial populations inhabiting the ileum and cecum of TLR- and MyD88-deficient mice. The microbiota of MyD88- and TLR-deficient mouse colonies differed markedly, with each colony harboring distinct and distinguishable bacterial populations in the small and large intestine. Comparison of MyD88-, TLR2-, TLR4-, TLR5-, and TLR9-deficient mice and their respective wild-type (WT) littermates demonstrated that the impact of TLR deficiency on the composition of the intestinal microbiota is minimal under homeostatic conditions and after recovery from antibiotic treatment. Thus, differences between TLR-deficient mouse colonies reflected long-term divergence of the microbiota after extended husbandry in isolation from each other. Long-term breeding of isolated mouse colonies results in changes of the intestinal microbiota that are communicated to offspring by maternal transmission, which account for marked compositional differences between WT and mutant mouse strains.

Compositional modification of nascent in vitro dental plaques by human host-defence peptides.

Salivary host-defence peptides include defensins, histatins and cathelicidin. We have investigated the effects of these peptides on the microbial composition of dental plaques. Salivary consortia, established within hydroxyapatite disc models, were exposed during development to physiological levels of human neutrophil proteins (HNP) 1 and 2; human ß defensins (hßD) 1, 2 and 3; histatins (His) 5 and 8; and cathelicidin (LL37). Effects on aggregation and microbial composition were determined using fluorescence microscopy; and differential culture with PCR-DGGE, respectively. LIVE/DEAD microscopic analysis indicated that HDPs decreased total bacterial viability, whilst ß defensins, paired HNPs, His 5, His 8 and the HDPs combined inhibited bacterial aggregation. According to differential culture, all test HDPs (except His 5) significantly decreased the abundance of Gram-negative anaerobes and lactobacilli (except HNP 2, hßD 1, paired HNPs and His 5). Combined HNPs and paired hßD 1 and 3 inhibited streptococci, whereas HNP 1, hßD 1, hßD 3, His 5 and LL37 increased streptococcal numbers. According to cluster analyses of DGGE profiles, HDP-exposed plaques were compositionally distinct from undosed controls. Thus, whilst HDPs reportedly exhibit variable potency against oral bacteria in endpoint susceptibly tests, exposure of nascent plaques can markedly influence bacterial viability, composition and microbial aggregation.