Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/β-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3β-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

• Keywords
• anti-CD3, germ-free, gut microbiota, immune stress, mice, steroidogenic genes, steroids,
• MeSH
• Cholesterol Side-Chain Cleavage Enzyme genetics   metabolism   MeSH
• Corticosterone metabolism   MeSH
• Microbiota * MeSH
• Mice MeSH
• Steroids metabolism   MeSH
• Pituitary-Adrenal System metabolism   MeSH
• Hypothalamo-Hypophyseal System * metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol Side-Chain Cleavage Enzyme MeSH
• Corticosterone MeSH
• Steroids MeSH

The gut microbiota play an important role in shaping brain functions and behavior, including the activity of the hypothalamus-pituitary-adrenocortical (HPA) axis. However, little is known about the effect of the microbiota on the distinct structures (hypothalamus, pituitary, and adrenals) of the HPA axis. In the present study, we analyzed the influence of the microbiota on acute restraint stress (ARS) response in the pituitary, adrenal gland, and intestine, an organ of extra-adrenal glucocorticoid synthesis. Using specific pathogen-free (SPF) and germ-free (GF) male BALB/c mice, we showed that the plasma corticosterone response to ARS was higher in GF than in SPF mice. In the pituitary, stress downregulated the expression of the gene encoding CRH receptor type 1 (Crhr1), upregulated the expression of the Fkbp5 gene regulating glucocorticoid receptor sensitivity and did not affect the expression of the proopiomelanocortin (Pomc) and glucocorticoid receptor (Gr) genes. In contrast, the microbiota downregulated the expression of pituitary Pomc and Crhr1 but had no effect on Fkbp5 and Gr. In the adrenals, the steroidogenic pathway was strongly stimulated by ARS at the level of the steroidogenic transcriptional regulator Sf-1, cholesterol transporter Star and Cyp11a1, the first enzyme of steroidogenic pathway. In contrast, the effect of the microbiota was significantly detected at the level of genes encoding steroidogenic enzymes but not at the level of Sf-1 and Star. Unlike adrenal Sf-1, the expression of the gene Lrh-1, which encodes the crucial transcriptional regulator of intestinal steroidogenesis, was modulated by the microbiota and ARS and this effect differed between the ileum and colon. The findings demonstrate that gut microbiota have an impact on the response of the pituitary, adrenals and intestine to ARS and that the interaction between stress and the microbiota during activation of glucocorticoid steroidogenesis differs between organs. The results suggest that downregulated expression of pituitary Pomc and Crhr1 in SPF animals might be an important factor in the exaggerated HPA response of GF mice to stress.

• Keywords
• HPA axis, acute restraint stress, extra-adrenal glucocorticoid synthesis, germ-free, gut microbiota, intestine, mice,
• MeSH
• Cholesterol Side-Chain Cleavage Enzyme genetics   MeSH
• Phosphoproteins genetics   MeSH
• Restraint, Physical * MeSH
• Pituitary Gland metabolism   MeSH
• Ileum metabolism   microbiology   MeSH
• Colon metabolism   microbiology   MeSH
• Corticosterone blood   MeSH
• Mice, Inbred BALB C MeSH
• Adrenal Glands metabolism   MeSH
• Pro-Opiomelanocortin genetics   MeSH
• Stress, Psychological blood   microbiology   MeSH
• CRF Receptor, Type 1 MeSH
• Receptors, Corticotropin-Releasing Hormone genetics   MeSH
• Gene Expression Regulation MeSH
• Steroidogenic Acute Regulatory Protein MeSH
• Steroidogenic Factor 1 genetics   MeSH
• Gastrointestinal Microbiome * MeSH
• Pituitary-Adrenal System * MeSH
• Hypothalamo-Hypophyseal System * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol Side-Chain Cleavage Enzyme MeSH
• Phosphoproteins MeSH
• Corticosterone MeSH
• Pro-Opiomelanocortin MeSH
• CRF Receptor, Type 1 MeSH
• Receptors, Corticotropin-Releasing Hormone MeSH
• Steroidogenic Acute Regulatory Protein MeSH
• Steroidogenic Factor 1 MeSH