Ablation of PC1/3 in POMC-Expressing Tissues but Not in Immune Cells Induces Sepsis Hypersensitivity

Abstract

Prohormone convertase 1/3 (PC1/3) is an endopeptidase required for the processing of neuropeptide and endocrine peptide precursors; it is expressed in neuroendocrine tissues as well as in immune cells. In response to endotoxemia, global PC1/3 knockout mice mount a cytokine storm and die rapidly. Further, immune cells isolated from these mice have a pro-inflammatory signature, suggesting that PC1/3 activates an unknown anti-inflammatory peptide precursor in immune cells. Here, we tested this hypothesis using tissue-specific PC1/3 ablation models. Knocking out PC1/3 in the myeloid or the hematopoietic compartment did not induce any phenotype. In contrast, proopiomelanocortin (POMC)-specific PC1/3 knockout mice phenocopied global PC1/3 knockout mice, including an enlarged spleen size and a hyperinflammatory sepsis phenotype in response to mild endotoxemia. This phenotype was prevented by steroid therapy and mimicked by blocking corticoid receptors in wild-type mice. Thus, our data suggest that sepsis hypersensitivity in PC1/3 deficiency is uncoupled from immune cell intrinsic PC1/3 expression and is driven by a lack of anti-inflammatory glucocorticoids due to an impairment in the hypothalamic-pituitary-adrenal axis.

Keywords: hypothalamus-pituitary-adrenal axis; inflammation; prohormone convertase 1/3; sepsis; steroids.

Figure 1.

High mortality and hyperinflammation in whole-body PC1/3 knockout mice. (A) Number of mice that reached or died prior to weaning (21 days of age) obtained from intercrossing Pcsk1^wt/ko mice (left) or breeding Pcsk1^wt/ko to wild-type mice (right) on a Swiss genetic background. (B) Genotype distribution in male (left) and female (right) pups that died between 0 and 21 days of age obtained from intercrossing Pcsk1^wt/ko mice. (C) Circulating IL-1β levels before and 2, 4, 8, and 24 hours after injection of 40 μg/kg LPS in a 12-week-old female Pcsk1 global knockout and a wild-type littermate mouse. (D-E) Body weight of male (D) and female (E) 24-week-old Pcsk1^wt/ko and Pcsk1^wt/wt littermate mice.

Figure 2.

Myeloid cell-specific PC1/3 knockout mice are not hyperinflammatory. A-B, Body weight development in male (A) and female (B) Lyz2-Cre Pcsk1^fl/fl mice. C-D, Spleen weight of 12-week-old male (C) and female (D) Lyz2-Cre Pcsk1^fl/fl mice. E, Schematic representation of endotoxemia experiment. F-G, Circulating levels of IL-1β (F) and respective AUC (G) in 12-week-old male Lyz2-Cre Pcsk1^fl/fl mice injected with 2 mg/kg body weight LPS at timepoint 0. H-I, Body weight (H) and glycemia (I) before and 24 hours after injection of 2 mg/kg body weight LPS in 12-week-old male Lyz2-Cre Pcsk1^fl/fl mice. (J) Blood glucose in 12-week-old male Lyz2-Cre Pcsk1^fl/fl mice obtained from an additional cohort of untreated mice. K-L, Circulating levels of IL-1β (K) and respective AUC (L) in 12-week-old female Lyz2-Cre Pcsk1^fl/fl mice injected with 2 mg/kg body weight LPS at timepoint 0. M-O, Body weight (M) and glycemia (N) before and 24 hours after injection of 2 mg/kg body weight LPS as well as in an additional untreated cohort of mice (O) in 12-week-old female Lyz2-Cre Pcsk1^fl/fl mice. Abbreviations: AUC, area under the curve; LPS, lipopolysaccharide.

Figure 3.

Hematopoietic cell-specific PC1/3 knockout mice are not hyperinflammatory. (A) Body weight development in male Vav-iCre Pcsk1^fl/fl mice. (B) Spleen weight of 12-week-old male Vav-iCre Pcsk1^fl/fl mice. C-D, Circulating levels of IL-1β (C) and respective AUC (D) in 12-week-old male Vav-iCre Pcsk1^fl/fl mice injected with 2 mg/kg body weight LPS at timepoint 0. E-F, Body weight (E) and glycemia (F) before and 24 hours after injection of 2 mg/kg body weight LPS in 12-week-old male Vav-iCre Pcsk1^fl/fl mice. (G) Body weight development in female Vav-iCre Pcsk1^fl/fl mice. (H) Spleen weight of 12-week-old female Vav-iCre Pcsk1^fl/fl mice. I-J, Circulating levels of IL-1β (I) and respective AUC (J) in 12-week-old female Vav-iCre Pcsk1^fl/fl mice injected with 2 mg/kg body weight LPS at timepoint 0. K-L, Body weight (K) and glycemia (L) before and 24 hours after injection of 2 mg/kg body weight LPS in 12-week-old female Vav-iCre Pcsk1^fl/fl mice. Abbreviation: AUC, area under the curve; LPS, lipopolysaccharide.

Figure 4.

Pomc-specific Pcsk1 ablation or blocking glucocorticoid receptors reproduces the pro-inflammatory phenotype of Pcsk1 null mice. (A) Circulating corticosterone levels before and after injection of 10 μg/kg body weight ACTH in 26- to 30-week-old male Pomc-Cre Pcsk1^fl/fl mice. B-C, Spleen (B) and adrenal (C) weight of 12- to 14-week-old male Pomc-Cre Pcsk1^fl/fl mice. (D) Circulating levels of IL-1β before and after injection of 2 mg/kg body weight LPS in 12-week-old male Pomc-Cre Pcsk1^fl/fl mice. # indicates that a mouse reached a humane endpoint. E-F, Circulating IL-1β (E) and respective AUC (F) before and after injection of 40 μg/kg body weight LPS in 12-week-old male Pomc-Cre Pcsk1^fl/fl mice. G-H, Body weight (G) and glycemia (H) before and 24 hours after injection of 40 μg/kg body weight LPS in 12-week-old male Pomc-Cre Pcsk1^fl/fl mice. I, Schematic overview of mifepristone experiment. J, Circulating levels of IL-1β before and after injection of 2 mg/kg body weight LPS in 12-week-old male wild-type mice preinjected with 500 mg/kg mifepristone or oil. # indicates that a mouse reached a humane endpoint. K-L, Circulating IL-1β (K) and respective AUC (L) before and after injection of 40 μg/kg body weight LPS in 14- to 15-week-old male wild-type mice preinjected with 500 mg/kg mifepristone or oil. M-N, Body weight (M) and glycemia (N) before and 24 hours after injection of 40 μg/kg body weight LPS in 14- to 15-week-old male wild-type mice preinjected with 500 mg/kg mifepristone or oil. Abbreviations: AUC, area under the curve; LPS, lipopolysaccharide.

Figure 5.

Steroid therapy prevents LPS-induced sepsis in Pomc-Cre Pcsk1^fl/fl mice. (A), Schematic presentation of dexamethasone experiment. B-C, Circulating IL-1β levels (B) and respective AUC (C) before and after injection of 80 μg/kg body weight LPS in 12-week-old male Pomc-Cre Pcsk1^fl/fl mice preinjected with 5 mg/kg dexamethasone or oil. D-F, Body weight (D), glycemia (E) and spleen weight (F) before and after injection of 80 μg/kg body weight LPS in 12-week-old male Pomc-Cre Pcsk1^fl/fl mice preinjected with 5 mg/kg dexamethasone or oil. Abbreviations: AUC, area under the curve; Dex, dexamethasone; LPS, lipopolysaccharide.

Figure 6.

Gating strategy of peritoneal and splenic immune cells. (A) Sorting of macrophages (CD45 + CD11b^highF4/80+), neutrophils (CD45 + CD11b^highLy6G+), and B cells (CD45 + CD11b^low) from peritoneal lavage. (B) Sorting of T cells (CD45 + CD3+) from splenocytes. Arrows pointing to specific populations indicate cells that were collected for downstream analysis.