UC Davis

ABSTRACTBackground The tryptophan metabolite kynurenine from the indoleamine 2,3-dioxygenase (IDO) pathway is an aryl hydrocarbon receptor (AhR) agonist and thus, can regulate immune activity via the AhR pathway. Hydroxylation of the vitamin D metabolite, 25-hydroxyvitamin D [25(OH)D] by 25-hydroxylase stimulates the synthesis of calcitriol, vitamin D receptor (VDR) agonist which regulates immune activity via the VDR pathway. In addition, direct interaction among the intestinal bacteria and the immune system through various mechanisms (e.g., recognition of microbial-associated molecular patterns [MAMPs] such as lipopolysaccharide [LPS] by pattern recognition receptors [PRRs]) as well as indirect interaction via tryptophan metabolites indole, indole acetic acid (IAA) and indole propionic acid (IPA), the AhR agonists from intestinal bacteria contribute to the regulation of intestinal and systemic immune activity. In this research, we hypothesized that intestinal bacteria would be associated with markers of innate and adaptive immune responses. Also, we hypothesized that microbial tryptophan metabolites indole, IAA and IPA as well as the host tryptophan metabolite kynurenine would be associated with immune markers in a manner consistent with AhR-mediated immune activity in healthy adults. Further, we hypothesized that kynurenine and the kynurenine-to-tryptophan ratio (markers of IDO activity) would be associated with immune markers reflecting IDO activation in immune cells. Finally, we hypothesized that 25(OH)D would be associated with markers of the innate and adaptive immune responses, and we sought to determine the 25(OH)D inflection points around which the association would differ. Methods Tryptophan metabolites (indole, IAA, IPA, Kynurenine), total 25(OH)D, and 88 immune markers were measured from peripheral blood of 362 fasting healthy adults. To reduce dimensionality in the large spectrum of the immune markers, highly correlated immune variables were grouped together by factor analysis of 79 immune markers, resulting in 17 Immune Factors (IFs). Bacterial taxa from stool were identified by 16S rRNA gene analysis and sequences were analyzed using Qiime2 version 2019.10. Multiple linear regression models adjusted for sex, age, body mass index (BMI), and cytomegalovirus (CMV) infection status were used to identify significant associations between tryptophan metabolites and immune markers and between intestinal bacteria and IFs as well as the components of the identified significant IFs. Also, linear and second- and third-degree polynomial regression models were used to identify the immune markers that were associated with 25(OH)D via the linear and curvilinear associations. Piecewise regression models were used to identify 25(OH)D breakpoint concentrations around which the slope of the polynomial associations would differ. Statistical analyses were conducted using SAS version 9.4 (SAS Institute, Cary, North Carolina, United States) and R version 3.6.3. Results Association of intestinal bacteria with markers of immune activity The family Rikenellaceae showed a positive association with innate IF5 and intracellular/vascular cell adhesion molecules ICAM-1, VCAM-1 and macrophage chemoattractant protein (MCP)-1, three components of IF5. Also, Rikenellaceae showed a negative association with adaptive IF4 and three of its T-cell components expressing the activation marker HLA-DR including Tregs, memory Tregs and central memory CD4 T-cells. Pseudomonadaceae and its genus Pseudomonas showed a negative association with innate IF5 and interferon (IFN)-γ-induced protein (IP)-10, macrophage-derived chemokine (MDC), eotaxin and ICAM-1, four components of IF5. The adaptive IF13 and T-cell cytokine interleukin (IL)-10, component of IF13 were negatively associated with Butyrivibrio. IF13 and T-cell cytokines IL-10 and IL-17, components of IF13 were positively associated with Slackia. There were no other associations between intestinal bacteria and IFs.

Association of tryptophan metabolites from intestinal bacteria with immune activity

The sum of indole and IAA was positively associated with natural killer T-cells levels. Family Lachnospiraceae was associated with lower IAA, higher lymphocyte counts and lower levels of activated CD4 T-cells. Genera Dorea and Ruminococcus were associated with lower IPA. Dorea was associated with higher production of IFN-γ by T-cells, and Ruminococcus with higher production of IL-6 in PBMC cultures stimulated with bacterial LPS.

Association of Kynurenine and 25(OH)D with immune activity

Kynurenine and the Kynurenine-to-Tryptophan ratio were positively associated with neopterin and IP-10, markers of type 1 immunity, and tumor necrosis factor (TNF)-α and C-reactive protein (CRP), markers of the acute phase response, and the regulatory cytokine IL-10.

25(OH)D across the full range was negatively associated with IL-6 and MDC and 25(OH)D < 38.9 nmol/L was associated with a higher blood concentration of platelets. The rest of the associations were between immune markers and 25(OH)D with inflection points in the range of 50-105 nmol/L including the association of 25(OH)D above 50 nmol/L with lower eotaxin, above 71.8 nmol/L with lower IP-10, and above 67.5 nmol/L with lower percentages of total and memory Tregs expressing HLA-DR. Also, 25(OH)D was associated with a higher percentage of memory Tregs up to the threshold of 102.3 nmol/L at which memory Tregs were suppressed. Further, 25(OH)D above 93.3 nmol/L was associated with a higher percentage of CD4 T-cells, and a lower percentage of CD8 T-cells, above 100 nmol/L was associated with lower myeloperoxidase (MPO), and above 104.7 nmol/L was associated with a lower percentage of Th2 cells, and above 104 nmol/L was associated with lower percentages of Th17 and CD4 T-cells expressing programmed cell death (PD)-1 protein, marker of T-cell exhaustion.

Conclusion The association of intestinal bacteria Rikenellaceae, Butyrivibrio and Slackia with IFs and their components including inflammatory cytokines and chemokines, and markers of systemic inflammation as well as the anti-inflammatory cytokine IL-10 indicates an ongoing interaction between gut bacteria and the intestinal and systemic immune system in healthy adults.

The association of the intestinal bacteria Lachnospiraceae, Dorea and Ruminococcus with lower plasma levels of bacterial tryptophan metabolites and greater immune activation suggests that low levels of indole metabolites might contribute to higher levels of immune activation induced by intestinal bacteria. The association of kynurenine and the Kynurenine-to-Tryptophan ratio with markers of systemic inflammation and the acute phase response is consistent with IDO activation in innate immune cells.

The association of 25(OH)D < 38.9 nmol/L with a higher platelet concentration indicates an association with an increased platelet release from bone marrow and higher levels of innate immune response and inflammation. This finding reflects the level below which vitamin D deficiency occurs consistent with the deficiency cutoff of < 50 nmol/L defined by the Endocrine Society (ES). The association of 25(OH)D across the full range with lower IL-6 and MDC concentrations, as well as the associations with inflection points in the range of 50-72 nmol/L including the association of 25(OH)D with lower eotaxin and IP-10 indicate an association of 25(OH)D with lower innate immune response. Also, lower percentages of total HLA-DR+ Tregs and HLA-DR+ memory Tregs associated with 25(OH)D above 67.5 nmol/L suggests a lower antigenic stimulation and lower activation of Tregs. The associations with inflection points in the range of 90-105 nmol/L including the association of 25(OH)D with lower MPO, marker of neutrophil activation, and lower percentages of Th17 and PD-1+ CD4 T-cells, and higher percentage of memory Tregs and CD4/CD8 T-cell ratio suggest an association of 25(OH)D with lower activation levels of innate and adaptive immune cells and support Treg development. The association of 25(OH)D in the range of 50-105 nmol/L with lower immune activation and higher immune regulation in our cohort of healthy adults reflects the levels above which vitamin D sufficiency occurs consistent with the sufficiency cutoff of ≥ 50 nmol/L defined by the Institute of Medicine (IOM).