11β-HSD1 suppresses cardiac fibroblast CXCL2, CXCL5 and neutrophil recruitment to the heart post MI

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   Figure 1

   Increased neutrophil mobilization and recruitment to the heart in Hsd11b1^−/− mice post MI. (A) Representative images of Gr-1 IHC of day 2 infarcted hearts from both WT (left) and Hsd11b1^−/− mice (right). (B) Analysis of the % Gr-1+ immunostaining in infarct and border regions of heart sections from Hsd11b1^−/− vs WT animals (n = 7/5). (C) Neutrophil numbers in the blood of WT vs Hsd11b1^−/− mice measured by flow cytometry at 1 day post MI (n = 4/3). (D) Plasma corticosteroid levels were raised post MI and did not differ between WT and Hsd11b1^−/− mice (n = 7/6; dashed line represents level for naïve C57BL/6 mice, approx. 50–100 ng/mL). *P < 0.05.
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   Figure 2

   Absence of gene expression in host cells in bone marrow chimera causes increased neutrophil mobilization but targeted deletion of Hsd11b1^−/− in smooth muscle cells and cardiomyocytes does not affect neutrophil mobilization or recruitment post MI. Flow cytometric analysis confirms appropriate presence or absence of 11β-HSD1 in the neutrophils of chimeric mice (A). Blood neutrophil numbers were measured in chimeric mice (B; n = 7/6/6, P < 0.05). (C) Neutrophils present in the heart 2 days post MI in control (Hsd11b1^CVCre−) vs experimental animals (Hsd11b1^CVCre+). (D) Analysis of blood neutrophil numbers in control (Hsd11b1^CVCre−) vs experimental animals (Hsd11b1^CVCre+) at 1 day post MI. n = 4/4.
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   Figure 3

   Inflammatory cytokines and chemokines involved in neutrophil recruitment increase in the heart post MI. qPCR was carried out on infarct and border cardiac tissue from WT or Hsd11b1^−/− mice 24 h post MI and compared to naïve WT ventricular tissue. (A) Cxcl1, NLRP2, Ccl3 and the adhesion molecule, L-selectin, increased in infarcted tissue compared to naïve myocardium but did not differ between WT and Hsd11b1^−/− infarcts. (B) Expression of the neutrophil inhibitory peptide Gdf-15 was not modified in hearts from Hsd11b1^−/− mice. (C) Cxcl2, Cxcl5, Il1b and Il6 were all expressed to a greater extent in Hsd11b1^−/− than in WT infarct tissue. (C) *P < 0.05, **P < 0.01, ***P < 0.005. In representative experiments shown, n = 4/3, norm., normalized to GAPDH.
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   Figure 4

   Cardiac fibroblasts express Cxcl2 and Cxcl5 post MI, which increase in the absence of intracellular glucocorticoid regeneration. qPCR analysis of Cxcl2 (A) and Cxcl5 (B), IL1b (C) and IL-6 (D) performed on RNA isolated from the cellular fractions 1–3 (Supplementary methods) in WT vs Hsd11b1^−/− mice. *P < 0.05, **P < 0.01, n = 5–6 per group.
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   Figure 5

   Cultured cardiac fibroblast expression of Hsd11b1, Cxcl2, Cxcl5 and Il-6 in the presence or absence of IL-1α ± Cort or DHC. (A) qPCR analysis of Cxcl2 and Cxcl5 in cardiac fibroblasts cultured in the presence or absence of IL-1α for 24 h. (B) qPCR carried out on cardiac fibroblasts in the presence or absence of IL-1α ± corticosterone (Cort) or 11-DHC, expressed as fold change over IL-1α treatment alone. Protein production of CXCL2 (C) and CXCL5 (D) in the supernatants as measured by ELISA. (E) The influence of IL-1α ± corticosterone (Cort) or 11-DHC on expression of Il-6 by cultured mouse cardiac fibroblasts. *P < 0.05, **P < 0.01, ***P < 0.005. n = 3–5 per group.
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   Figure 6

   Schema for the regulation of neutrophil recruitment to the heart by 11β-HSD1. (A) Chemokines produced by fibroblasts in response to pro-inflammatory cytokines recruit neutrophils to the heart post MI (1). Thy1^high and Thy1^low cardiac fibroblasts preferentially produce CXCL2 and CXCL5, respectively (2). 11β-HSD1 catalyzes the regeneration of local glucocorticoid, dampening chemokine expression (3). Circulating corticosterone and IL-1α from necrotic cardiomyocytes increase 11β-HSD1 expression (4). (B) In the absence of 11β-HSD1, fibroblasts are driven to produce excess CXCL2 and CXCL5 (5), unleashed due to the lack of dampening local glucocorticoid (6), driving increased neutrophil recruitment to the infarcted heart (7).

• © 2017 The authors