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. 2017 Nov 16;7:15730. doi: 10.1038/s41598-017-15897-2

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of 19 days of chronic subordinate colony housing (CSC) on the spleen, in the presence and absence of CD11b⁺-cells. Depicted is the absolute spleen weight [mg] (A) and the number of splenocytes per spleen (B) following 20 days of CSC (n = 48) and single-housing (single housed controls, SHC; n = 48). Splenocytes were cultured without (basal) and with lipopolysaccharide (LPS, 1 µg/ml) in presence of various corticosterone (CORT) concentrations. After 48 h of incubation, cell viability was measured. Depicted is the cell viability [optical density (OD)] under basal and LPS-stimulated conditions in the absence of CORT (C) and the basal cell viability (without LPS) in the presence of different CORT concentrations (CORT = 0 µM was set to 100%; D). Delta cell viability of LPS-stimulated minus respective basal wells at CORT = 0 µM is depicted in (E). Shown is further the delta cell viability [OD] (LPS-stimulated minus respective basal wells) at various CORT concentrations in percent of respective values at CORT = 0 µM (set to 100%; F). Depicted are further the absolute numbers of CD19⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD11b⁺ cells within total viable splenocytes (G) of another set of SHC (n = 8) and CSC (n = 8) mice. CD11b⁺ as well as CD11b⁻ cells from these mice were cultured without (basal) and with LPS (1 µg/µl) in absence or presence of various CORT concentrations. Depicted is the cell viability under basal and LPS-stimulated conditions (H) as well as delta cell viability of LPS-stimulated minus respective basal wells (I) of CD11b⁻ and CD11b⁺ splenocytes. Shown is further the delta cell viability (LPS-stimulated minus respective basal wells) of CD11b⁻ splenocytes at various CORT concentrations in percent of respective values at CORT = 0 µM (set to 100%; J). A third set of SHC (n = 11) and CSC (n = 10) mice was employed to determine relative protein expression of glucocorticoid receptor (GR; K) and FK506 binding protein 51 (FKBP51; L) [grey density] on day 20 of CSC using western blotting, all normalized to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Representative images of bands detected for GR (~86 kDa) and FKBP51 (~51 kDa) and respective loading control GAPDH (~36 kDa) are shown for SHC and CSC mice below each subfigure. SHC; CSC. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus respective SHC; ^# P ≤ 0.05, ^## P ≤ 0.01, ^### P ≤ 0.001 versus respective basal (CORT = 0 µM) values.

Inline graphic — Effects of 19 days of chronic subordinate colony housing (CSC) on the spleen, in the presence and absence of CD11b⁺-cells. Depicted is the absolute spleen weight [mg] (A) and the number of splenocytes per spleen (B) following 20 days of CSC (n = 48) and single-housing (single housed controls, SHC; n = 48). Splenocytes were cultured without (basal) and with lipopolysaccharide (LPS, 1 µg/ml) in presence of various corticosterone (CORT) concentrations. After 48 h of incubation, cell viability was measured. Depicted is the cell viability [optical density (OD)] under basal and LPS-stimulated conditions in the absence of CORT (C) and the basal cell viability (without LPS) in the presence of different CORT concentrations (CORT = 0 µM was set to 100%; D). Delta cell viability of LPS-stimulated minus respective basal wells at CORT = 0 µM is depicted in (E). Shown is further the delta cell viability [OD] (LPS-stimulated minus respective basal wells) at various CORT concentrations in percent of respective values at CORT = 0 µM (set to 100%; F). Depicted are further the absolute numbers of CD19⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD11b⁺ cells within total viable splenocytes (G) of another set of SHC (n = 8) and CSC (n = 8) mice. CD11b⁺ as well as CD11b⁻ cells from these mice were cultured without (basal) and with LPS (1 µg/µl) in absence or presence of various CORT concentrations. Depicted is the cell viability under basal and LPS-stimulated conditions (H) as well as delta cell viability of LPS-stimulated minus respective basal wells (I) of CD11b⁻ and CD11b⁺ splenocytes. Shown is further the delta cell viability (LPS-stimulated minus respective basal wells) of CD11b⁻ splenocytes at various CORT concentrations in percent of respective values at CORT = 0 µM (set to 100%; J). A third set of SHC (n = 11) and CSC (n = 10) mice was employed to determine relative protein expression of glucocorticoid receptor (GR; K) and FK506 binding protein 51 (FKBP51; L) [grey density] on day 20 of CSC using western blotting, all normalized to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Representative images of bands detected for GR (~86 kDa) and FKBP51 (~51 kDa) and respective loading control GAPDH (~36 kDa) are shown for SHC and CSC mice below each subfigure. SHC; CSC. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus respective SHC; ^# P ≤ 0.05, ^## P ≤ 0.01, ^### P ≤ 0.001 versus respective basal (CORT = 0 µM) values.