Adrenal-Derived Hormones Differentially Modulate Intestinal Immunity in Experimental Colitis

Abstract

The adrenal glands are able to modulate immune responses through neuroimmunoendocrine interactions and cortisol secretion that could suppress exacerbated inflammation such as in inflammatory bowel disease (IBD). Therefore, here we evaluated the role of these glands in experimental colitis induced by 3% dextran sulfate sodium (DSS) in C57BL/6 mice subjected to adrenalectomy, with or without glucocorticoid (GC) replacement. Mice succumbed to colitis without adrenals with a higher clinical score and augmented systemic levels of IL-6 and lower LPS. Furthermore, adrenalectomy negatively modulated systemic regulatory markers. The absence of adrenals resulted in augmented tolerogenic lamina propria dendritic cells but no compensatory local production of corticosterone and decreased mucosal inflammation associated with increased IFN-γ and FasL in the intestine. To clarify the importance of GC in this scenario, GC replacement in adrenalectomized mice restored different markers to the same degree of that observed in DSS group. Finally, this is the first time that adrenal-derived hormones, especially GC, were associated with the differential local modulation of the gut infiltrate, also pointing to a relationship between adrenalectomy and the modulation of systemic regulatory markers. These findings may elucidate some neuroimmunoendocrine mechanisms that dictate colitis outcome.

Figure 1

Adrenal glands are important to control clinical outcome in experimental colitis. C57BL/6 mice were adrenalectomized and exposed to dextran sulfate sodium (DSS) 3%. On day 6 mice were euthanized to obtain samples of serum: (a) plasma corticosterone levels; (b) overall clinical score of the disease; (c) postmortem score; (d) serum IL-6 levels; (e) serum lipopolysaccharide (LPS) levels. DSS: C57BL/6 mice exposed to DSS; ADX + DSS: C57BL/6 adrenalectomized mice exposed to DSS. The dashed lines correspond to the control group of C57BL/6 mice not exposed to DSS. Continuous line in (a) corresponds to adrenalectomized mice without colitis. EU/mL = endotoxin units/mL. These results are representative of 3 independent experiments with 5 mice/group. ^∗ p < 0.05.

Figure 2

Reduced accumulation of regulatory T cells (Treg) in the absence of adrenal glands. C57BL/6 mice were adrenalectomized and exposed to dextran sulfate sodium (DSS) 3%. The spleen and MLN (mesenteric lymph nodes) were collected on the 6th day of colitis and processed for staining with specific antibodies for regulatory markers and flow cytometry. Analyses were performed according to the cell size (FSC) and granularity (SSC), followed by a selecting gate in the lymphocytes region and evaluation of dot plots for CD4⁺CD25⁺ and CD4⁺CD25⁻ cells (a), in which the frequency of Treg markers was quantified by histograms. (b) Percentage of CD4⁺CD25⁻PD1⁺ and CD4⁺CD25⁺PD1⁺ leukocytes from spleen and MLN. (c) Frequency of CD4⁺CD25⁻CD73⁺ and CD4⁺CD25⁺CD73⁺ cells from spleen and MLN. (d) Percentage of CD4⁺CD25⁻FR4⁺ and CD4⁺CD25⁺FR4⁺ leukocytes from spleen and MLN. (e) Frequency of CD4⁺CD25⁻FoxP3⁺ and CD4⁺CD25⁺FoxP3⁺ cells from spleen and MLN. (f) Mean fluorescence intensity (MFI) of FoxP3 in the CD4⁺CD25⁺ population of spleen and MLN. DSS: C57BL/6 mice exposed to DSS; ADX + DSS: C57BL/6 adrenalectomized mice exposed to DSS. The dotted lines correspond to the control group without colitis. Results are representative of two independent experiments, with 5 mice/group. ^∗ p < 0.05.

Figure 3

Adrenal-derived mediators modulate local tissue damage. C57BL/6 mice were adrenalectomized and exposed to dextran sulfate sodium (DSS) 3%. The colon was collected on the 6th day of colitis for histological analysis (a) in (b) immunophenotyping of CD11b⁺ cells, (c) dendritic proinflammatory cells (CD11b⁺CD11c⁺CD103⁻), and (d) tolerogenic dendritic cells (CD11b⁺CD11c⁺CD103⁺), evaluated by flow cytometry. (e) Myeloperoxidase (MPO) and (f) eosinophil peroxidase (EPO) assays. DSS: C57BL/6 mice exposed to DSS; ADX + DSS: C57BL/6 adrenalectomized mice exposed to DSS. The dotted lines correspond to the control group of C57BL/6 mice without colitis. Results are representative of two independent experiments, with 5 mice/group. ^∗ p < 0.05.

Figure 4

Adrenal-derived mediators altered the accumulation of lymphocytes in the intestine.C57BL/6 mice were adrenalectomized and exposed to dextran sulfate sodium (DSS) 3%. The colon was collected on the 6th day of colitis for phenotypic characterization of leukocytes from LP (lamina propria) (a) and IEL (intraepithelial cells) (b). After acquisition, samples were analyzed by FlowJo software. DSS: C57BL/6 mice exposed to DSS; ADX + DSS: C57BL/6 adrenalectomized mice exposed to DSS. The dotted lines correspond to the control group of C57BL/6 mice without colitis. Results are representative of two independent experiments, with 5 mice/group. ^∗ p < 0.05.

Figure 5

Adrenal-derived glucocorticoid is one of the key players in the control of intestinal inflammation. C57BL/6 mice were adrenalectomized and exposed to dextran sulfate sodium (DSS) 3%. After surgery, a group of mice was treated i.p. with 1 mg/Kg/day of dexamethasone from the 3rd to the 5th day of colitis, for replacement of GC, as described in Material and Methods. The colon was collected on the 6th day of colitis for histological analysis (a and b): (a) erosion lesions; (b) histological score; (c) intestinal corticosterone levels; (d) IFN-γ; and (e) FasL levels. DSS: C57BL/6 mice exposed to DSS; ADX + DSS: C57BL/6 adrenalectomized mice exposed to DSS. ADX + DSS + GC: C57BL/6 adrenalectomized mice exposed to DSS and treated with dexamethasone. The dotted lines correspond to the control group of C57BL/6 mice without colitis. Results are representative of two independent experiments, with 5 mice/group. ^∗ p < 0.05.