Supplemental materials for Glucocorticoid production in lymphoid organs: Acute effects of lipopolysaccharide in neonatal and adult mice

Supplemental materials for "Glucocorticoid production in lymphoid organs: Acute effects of lipopolysaccharide in neonatal and adult mice" submitted to Endocrinology. Abstract: Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of GCs, steroidogenic enzymes expression, and components of the HPA axis (e.g., CRH) expression. We administered LPS (50µg/kg i.p.) or vehicle control to male and female C57BL/6J neonatal (post-natal day (PND) 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hr later. We measured progesterone, 11-deoxycorticosterone (DOC), corticosterone, and 11-dehydrocorticosterone (DHC) via liquid chromatography tandem mass spectrometry (LC-MS/MS). We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via qPCR. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominately in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants (RGPIN-2014-04884 and RGPIN-2019-04837) and Discovery Accelerator Supplement (RGDAS-2019-00033) and a Canada Foundation for Innovation Grant (32631) to KKS, NSERC CGS-M, NSERC CGS-D and Endocrine Society Research Program fellowships to MS, a UBC Four-Year Doctoral Fellowship to JEH, and a Society for Neuroscience Trainee Professional Developmental Award to HB