Sex Hormone-Dependent Lipid Mediator Formation in Male and Female Mice During Peritonitis

Abstract

Introduction: Sex differences in inflammation are obvious and contribute to divergences in the incidence and severity of inflammation-related diseases that frequently preponderate in women. Lipid mediators (LMs), mainly produced by lipoxygenase (LOX) and cyclooxygenase (COX) pathways from polyunsaturated fatty acids (PUFAs), regulate all stages of inflammation. Experimental and clinical studies revealed sex divergences for selected LM pathways without covering the entire LM spectrum, and only few studies have addressed the respective role of sex hormones. Here, we performed the comprehensive LM profile analysis with inflammatory peritoneal exudates and plasma from male and female mice in zymosan-induced peritonitis to identify the potential sex differences in LM biosynthesis during the inflammatory response. We also addressed the impact of sex hormones by employing gonadectomy. Methods: Adult male and female CD1 mice received intraperitoneal injection of zymosan to induce peritonitis, a well-established experimental model of acute, self-resolving inflammation. Mice were gonadectomized 5 weeks prior to peritonitis induction. Peritoneal exudates and plasma were taken at 4 (peak of inflammation) and 24 h (onset of resolution) post zymosan and subjected to UPLC-MS-MS-based LM signature profiling; exudates were analyzed for LM biosynthetic proteins by Western blot; and plasma was analyzed for cytokines by ELISA. Results: Pro-inflammatory COX and 5-LOX products predominated in the peritoneum of males at 4 and 24 h post-zymosan, respectively, with slightly higher 12/15-LOX products in males after 24 h. Amounts of COX-2, 5-LOX/FLAP, and 15-LOX-1 were similar in exudates of males and females. In plasma of males, only moderate elevation of these LMs was apparent. At 4 h post-zymosan, gonadectomy strongly elevated 12/15-LOX products in the exudates of males, while in females, free PUFA and LOX products were rather impaired. In plasma, gonadectomy impaired most LMs in both sexes at 4 h with rather up-regulatory effects at 24 h. Finally, elevated 15-LOX-1 protein was evident in exudates of males at 24 h which was impaired by orchiectomy without the striking impact of gonadectomy on other enzymes in both sexes. Conclusions: Our results reveal obvious sex differences and roles of sex hormones in LM biosynthetic networks in acute self-resolving inflammation in mice, with several preponderances in males that appear under the control of androgens.

Keywords: inflammation; lipid mediator; lipoxygenase; peritonitis; sex differences; specialized pro-resolving mediators.

FIGURE 1

Lipid mediator biosynthetic pathway and lipid mediator levels in mouse peritoneal exudates. (A) Scheme of LM biosynthetic pathways yielding 12/15-LOX–, 5-LOX/FLAP–, and COX-1/2–derived products and respective enzymes involved. (B) Peritoneal lavages (exudates) from male and female mice were taken at different time points post-zymosan/vehicle i. p. injection. After solid phase extraction, LMs were analyzed by UPLC–MS-MS; time = 0 h: peritoneal lavages immediately taken from mice after treatment with vehicle (no zymosan); t = 4 h and t = 24 h: peritoneal lavages (exudates) post-zymosan. Amounts of relevant LM groups related to respective biosynthetic enzymes: COX products (sum of PGE₂, PGD₂, PGF_2α, and TXB₂), 5-LOX products (sum of LTB₄, t-LTB₄, 5-HETE, and 5-HEPE), 12/15-LOX products (sum of 12-HETE, 12-HEPE, 14-HDHA, 15-HETE, 15-HEPE, and 17-HDHA), and PUFA (sum of AA, EPA, and DHA). Results are shown in pg/mL exudate in scatter dot plots of individual data points and the mean in black lines; n = 5–6; ***p < 0.001 male vs. female; two-way ANOVA with Šídák’s multiple comparison test.

FIGURE 2

Effect of hormone depletion on the LM content in peritoneal exudates from male and female mice upon peritonitis induction. (A) Experimental setup and time line. (B) Amounts of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in peritoneal exudates of gonadectomized and non-gonadectomized (sham-operated) male and female mice after treatment with vehicle (no zymosan = 0 h) and 4 h after zymosan injection. Results are shown in pg/mL exudate in scatter dot plots as individual data points and means as black line; n = 4–6; ordinary one-way ANOVA with Tukey’s multiple comparison test. (C) Principal component analysis of LM profiles in the exudates from gonadectomized and non-gonadectomized (sham-operated) male and female mice, 4 h after zymosan injection (corresponding raw data are shown in Table 2). (D,E) Amounts of 12/15-LOX products in peritoneal exudates of gonadectomized and non-gonadectomized (sham-operated) male and female mice 4 h (D) and 24 h (E) after zymosan injection. Results are shown in pg/mL exudate in scatter dot plots as individual data points and the means as black lines; n = 5–6; *p < 0.05, **p < 0.01 non-gonadectomized vs. gonadectomized; unpaired Student’s t test; ns = not significant.

FIGURE 3

Lipid mediator levels in plasma of male and female mice during peritonitis. Amounts of COX products (sum of PGE₂, PGD₂, PGF_2α, and TXB₂), 5-LOX products (sum of LTB₄, t-LTB₄, 5-HETE, and 5-HEPE), 12/15-LOX products (sum of 12-HETE, 12-HEPE, 14-HDHA, 15-HETE, 15-HEPE, and 17-HDHA), and PUFA (sum of AA, EPA, and DHA) in plasma of male and female mice 4 h after zymosan injection. Results are shown in pg/mL plasma in scatter dot plots as individual data points and the means as black lines; n = 5–6; *p < 0.05, male vs. female; unpaired Student’s t test; ns = not significant.

FIGURE 4

Effect of sex hormone depletion on the expression of LM biosynthetic proteins in peritoneal exudates upon peritonitis induction. Protein levels and the densitometric analysis of 5-LOX, FLAP, platelet-type 12-LOX, 15-LOX, and COX-2 in lysed peritoneal exudates of gonadectomized and non-gonadectomized (sham-operated) male and female mice 4 h (A) and 24 h (B) after zymosan injection, normalized to β-actin. Western blots are shown as representatives from three different mice and given as mean values of n = 4–6; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey’s multiple comparison test for statistical analysis.