Faculty Bibliography

Commensal gut microbiota-host immune responses are experimentally delineated via gnotobiotic animal models or alternatively by antibiotic perturbation of gut microbiota. Osteoimmunology investigations in germ-free mice, revealing that gut microbiota immunomodulatory actions critically regulate physiologic skeletal development, highlight that antibiotic perturbation of gut microbiota may dysregulate normal osteoimmunological processes. We investigated the impact of antibiotic disruption of gut microbiota on osteoimmune response effects in postpubertal skeletal development. Sex-matched C57BL/6T mice were administered broad-spectrum antibiotics or vehicle-control from the age of 6 to 12 weeks. Antibiotic alterations in gut bacterial composition and skeletal morphology were sex dependent. Antibiotics did not influence osteoblastogenesis or endochondral bone formation, but notably enhanced osteoclastogenesis. Unchanged Tnf or Ccl3 expression in marrow and elevated tumor necrosis factor-α and chemokine (C-C motif) ligand 3 in serum indicated that the pro-osteoclastic effects of the antibiotics are driven by increased systemic inflammation. Antibiotic-induced broad changes in adaptive and innate immune cells in mesenteric lymph nodes and spleen demonstrated that the perturbation of gut microbiota drives a state of dysbiotic hyperimmune response at secondary lymphoid tissues draining local gut and systemic circulation. Antibiotics up-regulated the myeloid-derived suppressor cells, immature myeloid progenitor cells known for immunosuppressive properties in pathophysiologic inflammatory conditions. Myeloid-derived suppressor cell-mediated immunosuppression can be antigen specific. Therefore, antibiotic-induced broad suppression of major histocompatibility complex class II antigen presentation genes in bone marrow discerns that antibiotic perturbation of gut microbiota dysregulates critical osteoimmune cross talk.

OBJECTIVE:Gut microorganisms contribute to the metabolism of environmental toxicants, including methylmercury (MeHg). Our main objective was to investigate whether associations between biomarkers for prenatal MeHg exposure and maternal gut microbiota differed between early and late gestation. METHODS:Maternal blood and stool samples were collected during early (8.3-17 weeks, n=28) and late (27-36 weeks, n=24) gestation. Total mercury and MeHg concentrations were quantified in biomarkers, and inorganic mercury was estimated by subtraction. The diversity and structure of the gut microbiota were investigated using 16S rRNA gene profiling (n = 52). Biomarkers were dichotomized, and diversity patterns were compared between high/low mercury concentrations. Spearman's correlation was used to assess bivariate associations between MeHg biomarkers (stool, blood, and meconium), and 23 gut microbial taxa (genus or family level, >1% average relative abundance). RESULTS:Within-person and between-person diversity patterns in gut microbiota differed between early/late gestation. The overall composition of the microbiome differed between high/low MeHg concentrations (in blood and stool) during early gestation, but not late gestation. Ten (of 23) taxa were significantly correlated with MeHg biomarkers (increasing or decreasing); however, associations differed, depending on whether the sample was collected during early or late gestation. A total of 43% of associations (69/161) reversed the direction of correlation between early/late gestation. CONCLUSIONS:The time point at which a maternal fecal sample is collected may yield different associations between gut microorganisms and MeHg biomarkers, which may be due in part to remodeling of maternal microbiota during pregnancy. Our results suggest the effectiveness of dietary interventions to reduce prenatal MeHg exposure may differ between early and late gestation.

Microbial signals have been linked to autoantibody induction. Recently, we found that purified anti-CD4 autoantibodies from the plasma of chronic HIV-1-infected patients under viral-suppressed antiretroviral therapy (ART) play a pathologic role in poor CD4+ T cell recovery. The purpose of the study was to investigate the association of systemic microbiome and anti-CD4 autoantibody production in HIV. Plasma microbiome from 12 healthy controls and 22 HIV-infected subjects under viral-suppressed ART were analyzed by MiSeq sequencing. Plasma level of autoantibodies and microbial translocation (LPS, total bacterial 16S rDNA, soluble CD14, and LPS binding protein) were analyzed by ELISA, limulus amebocyte assay, and qPCR. We found that plasma level of anti-CD4 IgGs but not anti-CD8 IgGs was increased in HIV+ subjects compared to healthy controls. HIV+ subjects with plasma anti-CD4 IgG > 50 ng/mL (high) had reduced microbial diversity compared to HIV+ subjects with anti-CD4 IgG ≤ 50 ng/mL (low). Moreover, plasma anti-CD4 IgG level was associated with elevated microbial translocation and reduced microbial diversity in HIV+ subjects. The Alphaproteobacteria class was significantly enriched in HIV+ subjects with low anti-CD4 IgG compared to patients with high anti-CD4 IgG even after controlling for false discovery rate (FDR). The microbial components were different from the phylum to genus level in HIV+ subjects with high anti-CD4 IgGs compared to the other two groups, but these differences were not significant after controlling for FDR. These results suggest that systemic microbial translocation and microbiome may associate with anti-CD4 autoantibody production in ART-treated HIV disease.

Background A transmissible agent has long been suspected inthe pathogenesis of SLE, yet the potential contribution of thehuman intestinal microbiome has been little examined. Wetherefore characterized the gut microbiota of patients withSLE, with special interest in those with lupus nephritis (LN).Methods Blood and fecal samples from SLE patients wereobtained, with strict inclusion/exclusion of criteria. Fecal 16SrDNA sequencing, as well as cytokine and autoantibody assayswere performed. In addition, sera from two independent lupuscohorts were studied for validation. Biomarkers of gut leakiness were assessed.Results Compared to controls, the intestinal microbiome fromSLE patients (n=61) showed decreased species richness diversity with reductions in taxonomic complexity mostpronounced in those with high disease activity. Notably, SLEpatients had an overall 5-fold greater representation of a species in the Lachnospiracaea family of obligate anaerobic Grampositive cocci, with reciprocal contractions of two other commensal species with putative protective properties. Abundanceof the Lachnospiracaea species correlated with serum IgG to acell wall component, postulated to represent a lipoglycan,from a strain of this same species (p=0.002, n=61, Spearman)but not with 7 other strains. There was also a significantdirect correlation between SLEDAI scores and levels of thesecirculating anti-strain IgG antibodies (p=0.02, n=48). Levelsof antibodies to strain-specific bacterial antigen, treated withRNAse/DNAse/proteinase K, were significantly higher in thosewith active nephritis at time of sampling compared to SLEwithout renal activity (Cohort 1 p=0.01 n=48; Cohort 2p=0.006, n=28, Mann-Whitney). Levels of serum IgG antistrain antibodies also significantly correlated with high-titerserum IgG to native DNA (p<0.0001, n=27), and inverselycorrelated with C3 and C4 levels. High titers of these antibacterial antibodies were associated with active Class III, IVand V (overlap) LN (Cohort 3).Conclusions These findings suggest a novel paradigm for thepathogenesis of LN in which a common intestinal commensalbacteria may contribute to the immune-complex mediated disease process, with features akin to poststreptococcal GN butwithout outward signs and symptoms of clinical infection

Background: Compared with Caucasian Americans (CAs), African Americans (AAs) with colorectal cancer have poorer survival, especially younger-age patients. A robust lymphocytic reaction within colorectal cancers is strongly associated with better survival, but whether immune response impacts the disparity in colorectal cancer survival is unknown.Methods: The study population was comprised of 211 histologically confirmed colorectal cancers at the Medical University of South Carolina (Charleston, SC; 159 CAs and 52 AAs) diagnosed between Jan 01, 2000, and June 30, 2013. We constructed a lymphocyte score based on blinded pathologic assessment of the four different types of lymphocytic reactions. Cox proportional hazards regression was used to evaluate the association between the lymphocyte score and risk of death by race.Results: Colorectal cancers in AAs (vs. CAs) had a stronger lymphocytic reaction at diagnosis. A high lymphocyte score (vs. the lowest) was associated with better survival in AAs [HR 0.19; 95% confidence interval (CI), 0.04-0.99] and CAs (HR 0.47; 95% CI, 0.15-1.45). AAs with no lymphocytic reaction (vs. other categories) had poor survival HR 4.48 (1.58-12.7) whereas no difference was observed in CAs. The risk of death in AAs (vs. CA) was more pronounced in younger patients (HR 2.92; 95% CI, 1.18-7.22) compared with older (HR 1.20; 95% CI, 0.54-2.67), especially those with lymphocytic poor colorectal cancers.Conclusions: The lymphocytic reaction in tumor impacted the racial disparity in survival.Impact: Our results confirm the importance of the lymphocytic score on survival and highlight the need to fully characterize the immune environment of colorectal cancers by race. Cancer Epidemiol Biomarkers Prev; 27(7); 1-7. ©2018 AACR.

BACKGROUND:Dysbiosis of the oral microbiome can lead to local oral disease and potentially to cancers of the head, neck, and digestive tract. However, little is known regarding exogenous factors contributing to such microbial imbalance. RESULTS:We examined the impact of alcohol consumption on the oral microbiome in a cross-sectional study of 1044 US adults. Bacterial 16S rRNA genes from oral wash samples were amplified, sequenced, and assigned to bacterial taxa. We tested the association of alcohol drinking level (non-drinker, moderate drinker, or heavy drinker) and type (liquor, beer, or wine) with overall microbial composition and individual taxon abundance. The diversity of oral microbiota and overall bacterial profiles differed between heavy drinkers and non-drinkers (α-diversity richness p = 0.0059 and β-diversity unweighted UniFrac p = 0.0036), and abundance of commensal order Lactobacillales tends to be decreased with higher alcohol consumption (fold changes = 0.89 and 0.94 for heavy and moderate drinkers, p trend = 0.005 [q = 0.064]). Additionally, certain genera were enriched in subjects with higher alcohol consumption, including Actinomyces, Leptotrichia, Cardiobacterium, and Neisseria; some of these genera contain oral pathogens, while Neisseria can synthesize the human carcinogen acetaldehyde from ethanol. Wine drinkers may differ from non-drinkers in microbial diversity and profiles (α-diversity richness p = 0.048 and β-diversity unweighted UniFrac p = 0.059) after controlling for drinking amount, while liquor and beer drinkers did not. All significant differences between drinkers and non-drinkers remained after exclusion of current smokers. CONCLUSIONS:Our results, from a large human study of alcohol consumption and the oral microbiome, indicate that alcohol consumption, and heavy drinking in particular, may influence the oral microbiome composition. These findings may have implications for better understanding the potential role that oral bacteria play in alcohol-related diseases.

OBJECTIVE: A history of periodontal disease and the presence of circulating antibodies to selected oral pathogens have been associated with increased risk of pancreatic cancer; however, direct relationships of oral microbes with pancreatic cancer have not been evaluated in prospective studies. We examine the relationship of oral microbiota with subsequent risk of pancreatic cancer in a large nested case-control study. DESIGN: We selected 361 incident adenocarcinoma of pancreas and 371 matched controls from two prospective cohort studies, the American Cancer Society Cancer Prevention Study II and the National Cancer Institute Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. From pre-diagnostic oral wash samples, we characterised the composition of the oral microbiota using bacterial 16S ribosomal RNA (16S rRNA) gene sequencing. The associations between oral microbiota and risk of pancreatic cancer, controlling for the random effect of cohorts and other covariates, were examined using traditional and L1-penalised least absolute shrinkage and selection operator logistic regression. RESULTS: Carriage of oral pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were associated with higher risk of pancreatic cancer (adjusted OR for presence vs absence=1.60 and 95% CI 1.15 to 2.22; OR=2.20 and 95% CI 1.16 to 4.18, respectively). Phylum Fusobacteria and its genus Leptotrichia were associated with decreased pancreatic cancer risk (OR per per cent increase of relative abundance=0.94 and 95% CI 0.89 to 0.99; OR=0.87 and 95% CI 0.79 to 0.95, respectively). Risks related to these phylotypes remained after exclusion of cases that developed within 2 years of sample collection, reducing the likelihood of reverse causation in this prospective study. CONCLUSIONS: This study provides supportive evidence that oral microbiota may play a role in the aetiology of pancreatic cancer.

Knowledge Transfer Statement: This article discusses the proceedings of the conference organized by the Task Force on Design and Analysis in Oral Health Research on the new advances in host-microbiome interactions, analytical methods, and their implication in inflammatory periodontal disease management.

Background: A transmissible agent has long been suspected in the pathogenesis of SLE. We therefore investigated the potential contribution of the intestinal microbiome to LN.
Method(s): Blood and fecal samples from SLE patients were obtained, unless a patient had selective IgA deficiency, prior cytotoxic drugs, or antibiotics within four months. Fecal 16S rRNA NGS was performed. Sera samples were profiled for autoantibodies. Sera from two independent lupus cohorts were studied for validation.
Result(s): Compared to controls, the intestinal microbiome from SLE patients (N=61) showed decreased species richness diversity. The microbiomes of patients in clinical remission (based on SLEDAI) were most similar to healthy controls, while reductions in taxonomic complexity were most pronounced in those with high disease activity. Notably, SLE patients had an overall 5-fold greater representation of a particular species in the Blautia genus of the Lachnospiracaea family of obligate anaerobic Gram-positive cocci. Abundance of this species significantly correlated with serum IgG to a cell wall moiety from a strain of this species (P=0.002, N=61, Spearman) but not with 7 other strains. There was also a significant correlation between the distribution of SLEDAI scores and levels of these circulating anti-strain IgG antibodies (P=0.02, N=48). Using antigen treated with DNAse/proteinase K, levels of IgG anti-strain antibodies were significantly higher in those with active nephritis at time of sampling compared to SLE without renal activity (Cohort 1 P=0.01 N=48; Cohort 2 P=0.001, N=53, Mann-Whitney). Levels of anti-strain antibodies also significantly correlated with high-titer serum IgG to native DNA (P<0.0001, N=27), and inversely correlated with C3 and C4 (each P<0.01, N=61). High titers of these anti-bacterial antibodies were found in active Class III, IV and V LN.
Conclusion(s): These findings suggest a novel paradigm for the pathogenesis of LN: Specific strains of common intestinal commensal bacteria affect IgG-autoantibody responses in patients with LN. This is reminiscent of post-streptococcal GN, although the postulated intestinal bacterial bloom occurs without clinical infection