Charles B. Stephensen

Interactions among intestinal bacteria and the immune system contribute to the maintenance of a functional intestinal barrier in healthy individuals, and possibly to systemic immune activity. We hypothesized that intestinal bacteria would be associated with systemic biomarkers of innate and adaptive immune responses in healthy adults. 79 immune function markers were subjected to factor analysis resulting in 17 Immune Factors (IFs), each composed of 2–10 immune variables. Bacterial taxa from stool samples were identified at the family and genus levels by 16S rRNA amplicon sequence analysis and their read counts and relative abundances were utilized in a multiple linear regression model to identify microbial taxa associated with the IFs. A total of 10 significant associations were identified between bacterial taxa and IFs. The family Rikenellaceae showed a positive association with innate IF5 (including 5 chemokines, 2 cytokines, 2 adhesion molecules, and the macrophage metabolite neopterin) and a negative association with adaptive IF4 (including T-cells with activation marker HLA-DR). Additionally, Pseudomonadaceae and its genus Pseudomonas showed a negative relationship with innate IF5, and adaptive IF13 (including T-cell cytokines IL-10, IL-17, and IFN-γ) was negatively associated with Butyrivibrio and positively associated with Slackia . These associations suggest ongoing interactions between gut bacteria and the systemic immune system in healthy adults. The association of these taxa with the IFs may result from specific microbial-immune system interactions that play a role in maintenance of a healthy barrier integrity in our cohort of healthy adults. IMPORTANCE: Chronic inflammation may develop over time in healthy adults as a result of a variety of factors, such as poor diet directly affecting the composition of the intestinal microbiome, or by causing obesity, which may also affect the intestinal microbiome. These effects may trigger the activation of an immune response that could eventually lead to an inflammation-related disease, such as colon cancer. Before disease develops it may be possible to identify subclinical inflammation or immune activation attributable to specific intestinal bacteria normally found in the gut that could result in future adverse health impacts. In the present study, we examined a group of healthy men and women across a wide age range with and without obesity to determine which bacteria were associated with particular types of immune activation to identify potential preclinical markers of inflammatory disease risk. Several associations were found that may help develop dietary interventions to lower disease risk.

Dairy products are a good source of essential nutrients and past reviews have shown associations of dairy consumption with decreased systemic inflammation. Links between dairy intake and gastrointestinal (GI) inflammation are under-investigated. Therefore, we examined associations between reported dairy intake and markers of GI inflammation in healthy adults in a cross-sectional observational study, hypothesizing a negative association with yogurt intake, suggesting a protective effect, and no associations with total dairy, fluid milk, and cheese intake. Participants completed 24-h dietary recalls and a food frequency questionnaire (FFQ) to assess recent and habitual intake, respectively. Those who also provided a stool sample (n = 295), and plasma sample (n = 348) were included in analysis. Inflammation markers from stool, including calprotectin, neopterin, and myeloperoxidase, were measured along with LPS-binding protein (LBP) from plasma. Regression models tested associations between dairy intake variables and inflammation markers with covariates: age, sex, and body mass index (BMI). As yogurt is episodically consumed, we examined differences in inflammation levels between consumers (>0 cup equivalents/day reported in recalls) and non-consumers. We found no significant associations between dairy intake and markers of GI inflammation. In this cohort of healthy adults, dairy intake was not associated with GI inflammation.

Plasma trimethylamine n-oxide (TMAO) concentration increases in responses to feeding TMAO, choline, phosphatidylcholine, L-carnitine, and betaine but it is unknown whether concentrations change following a mixed macronutrient tolerance test (MMTT) with limited amounts of TMAO precursors. In this proof-of-concept study, we provided healthy female and male adults (n = 97) ranging in age (18–65 years) and BMI (18–44 kg/m²) a MMTT (60% fat, 25% sucrose; 42% of a standard 2000 kilo calorie diet) and recorded their metabolic response at fasting and at 30 min, 3 h, and 6 h postprandially. We quantified total exposure to TMAO (AUC-TMAO) and classified individuals by the blood draw at which they experienced their maximal TMAO concentration (TMAO-response groups). We related AUC-TMAO to the 16S rRNA microbiome, to two SNPs in the exons of the FMO3 gene (rs2266782, G>A, p.Glu158Lys; and rs2266780, A>G, p.Glu308Gly), and to a priori plasma metabolites. We observed varying TMAO responses (timing and magnitude) and identified a sex by age interaction such that AUC-TMAO increased with age in females but not in males (p-value = 0.0112). Few relationships between AUC-TMAO and the fecal microbiome and FMO3 genotype were identified. We observed a strong correlation between AUC-TMAO and TNF-α that depended on TMAO-response group. These findings promote precision nutrition and have important ramifications for the eating behavior of adults who could benefit from reducing TMAO exposure, and for understanding factors that generate plasma TMAO.

To evaluate the effect of a diet pattern based on Dietary Guidelines for Americans (DGA), in a controlled feeding setting, on plasma markers of inflammation and on cytokine production by peripheral blood mononuclear cells (PBMC). Women (n = 44) with one or more risk factors of metabolic syndrome (and BMI: 25.2-39.8 kg/m²) completed an 8-wk controlled feeding study. They were randomized to either a group following a diet based on DGA 2010 (DGA), or a group given a ‘typical American diet’ (TAD), based largely on a Western diet pattern. By design, women maintained their body weight. Fasting plasma and PBMC were collected at wk. 0 (baseline) and at wk. 8 (post-intervention). Sixteen plasma markers of inflammation and eight PBMC cytokines were measured at both time points, to evaluate if the diet had a significant effect on concentrations of these inflammatory markers. Data were analyzed using ANCOVA, followed by multiple-comparison adjustment using Benjamini-Hochberg method. Significant changes observed in Serum Amyloid A (SAA) and Matrix Metalloproteinase 3 (MMP3) in plasma did not retain significance upon multiple comparison adjustment. SAA: p = 0.044, adj p = 0.450; DGA mean change [95% CI] = − 12.6[− 32.3 to 7.04]; TAD mean change [95% CI] = − 2.24 [− 9.99 to 5.51]. MMP3: p = 0.014, adj p = 0.35; DGA mean change [95% CI] = 2.72[− 4.16 to 9.59]; TAD mean change [95% CI] = − 0.98[− 16.7 to 14.7]). Other inflammation markers were not differently altered by DGA relative to TAD. Effect size of change (Cohens d) indicated a large/medium-large effect of intervention on MMP3 and CRP, and medium effect on IL-6. No statistically significant changes were observed in the immune markers examined in this study. The biological roles and magnitude of the non-significant differences seen with two variables, CRP and MMP3, suggest that they be examined in future studies. Clinicaltrials.gov identifier NCT02298725.

Background: Tryptophan (Trp) metabolites from intestinal bacteria (indole, indole acetic acid [IAA] and indole propionic acid [IPA]), and the Trp metabolite kynurenine (Kyn) from the indoleamine 2,3-dioxygenase (IDO) pathway, are aryl hydrocarbon receptor (AhR) agonists and thus, can regulate immune activityviathe AhR pathway. We hypothesized that plasma concentrations of these metabolites would be associated with markers of immune activation in a cohort of healthy adults in a manner consistent with AhR-mediated immune-regulation. We also hypothesized that the plasma Kyn/Trp ratio, a marker of IDO activity, would be associated with immune markers reflecting IDO activation in innate immune cells. Finally, we hypothesized that some intestinal bacteria would be associated with plasma indole, IPA and IAA, and that these bacteria themselves would be associated with immune markers.Methods: A novel set of 88 immune markers, and plasma Trp metabolites, were measured in 362 healthy adults. Bacterial taxa from stool were identified by 16S rRNA gene analysis. Multiple linear regression analysis was used to identify significant associations with immune markers.Results: The sum of indole and IAA was positively associated with natural killer T-cells levels. Kyn and Kyn/Trp were positively associated with neopterin and IP-10, markers of type 1 immunity, and TNF-α and C-reactive protein (CRP), markers of the acute phase response, and the regulatory cytokine IL-10. Three bacteria negatively associated with Trp metabolites were associated with markers of immune activation: the family Lachnospiraceae with higher lymphocyte counts but lower level of activated CD4 T-cells, the genus Dorea with higher production of IFN-γ by T-cells in PBMC cultures, and the genus Ruminococcus with higher production IL-6 in PBMC cultures stimulated with bacterial lipopolysaccharide (LPS).Conclusions: In this cohort of healthy adults bacterial Trp metabolites were not strongly associated with immune markers. Conversely, the Kyn/Trp ratio was strongly associated with markers of systemic inflammation and the acute phase response, consistent with IDO activation in innate immune cells. Finally, commensal bacteria associated with lower plasma (and perhaps intestinal) levels of bacterial Trp metabolites were associated with greater immune activation, possibly reflecting decreased regulatory immune activity related to lower intestinal levels of bacterial indole metabolites.

Bifidobacterium species are beneficial and dominant members of the breastfed infant gut microbiome; however, their health benefits are partially species-dependent. Here, we characterize the species and subspecies of Bifidobacterium in breastfed infants around the world to consider the potential impact of a historic dietary shift on the disappearance of B. longum subsp. infantis in some populations. Across populations, three distinct patterns of Bifidobacterium colonization emerged: (1) The dominance of Bifidobacterium longum subspecies infantis, (2) prevalent Bifidobacterium of multiple species, and (3) the frequent absence of any Bifidobacterium. These patterns appear related to a country’s history of breastfeeding, with infants in countries with historically high rates of long-duration breastfeeding more likely to be colonized by B. longum subspecies infantis compared with infants in countries with histories of shorter-duration breastfeeding. In addition, the timing of infant colonization with B. longum subsp. infantis is consistent with horizontal transmission of this subspecies, rather than the vertical transmission previously reported for other Bifidobacterium species. These findings highlight the need to consider historical and cultural influences on the prevalence of gut commensals and the need to understand epidemiological transmission patterns of Bifidobacterium and other major commensals.

TMAO is elevated in individuals with cardiometabolic diseases, but it is unknown whether the metabolite is a biomarker of concern in healthy individuals. We conducted a cross-sectional study in metabolically healthy adults aged 18–66 years with BMI 18–44 kg/m² and assessed the relationship between TMAO and diet, the fecal microbiome, and cardiometabolic risk factors. TMAO was measured in fasted plasma samples by liquid chromatography mass spectrometry. The fecal microbiome was assessed by 16S ribosomal RNA sequencing and recent food intake was captured by multiple ASA24 dietary recalls. Endothelial function was assessed via EndoPAT. Descriptive statistics were computed by fasting plasma TMAO tertiles and evaluated by ANOVA and Tukey’s post-hoc test. Multiple linear regression was used to assess the relationship between plasma TMAO and dietary food intake and metabolic health parameters. TMAO concentrations were not associated with average intake of animal protein foods, fruits, vegetables, dairy, or grains. TMAO was related to the fecal microbiome and the genera Butyribrio, Roseburia, Coprobaciullus, and Catenibacterium were enriched in individuals in the lowest versus the highest TMAO tertile. TMAO was positively associated with α-diversity and compositional differences were identified between groups. TMAO was not associated with classic cardiovascular risk factors in the healthy cohort. Similarly, endothelial function was not related to fasting TMAO, whereas the inflammatory marker TNF-α was significantly associated. Fasting plasma TMAO may not be a metabolite of concern in generally healthy adults unmedicated for chronic disease. Prospective studies in healthy individuals are necessary.