Paternal sepsis induces alterations of the sperm methylome and dampens offspring immune responses-an animal study

Abstract

Background: Sepsis represents the utmost severe consequence of infection, involving a dysregulated and self-damaging immune response of the host. While different environmental exposures like chronic stress or malnutrition have been well described to reprogram the germline and subsequently offspring attributes, the intergenerational impact of sepsis as a tremendous immunological stressor has not been examined yet.

Methods: Polymicrobial sepsis in 12-week-old male C57BL/6 mice was induced by cecal ligation and puncture (CLP), followed by a mating of the male survivors (or appropriate sham control animals) 6 weeks later with healthy females. Alveolar macrophages of offspring animals were isolated and stimulated with either LPS or Zymosan, and supernatant levels of TNF-α were quantified by ELISA. Furthermore, systemic cytokine response to intraperitoneally injected LPS was assessed after 24 h. Also, morphology, motility, and global DNA methylation of the sepsis survivors' sperm was examined.

Results: Comparative reduced reduction bisulfite sequencing (RRBS) of sperm revealed changes of DNA methylation (n = 381), most pronounced in the intergenic genome as well as within introns of developmentally relevant genes. Offspring of sepsis fathers exhibited a slight decrease in body weight, with a more pronounced weight difference in male animals (CLP vs. sham). Male descendants of sepsis fathers, but not female descendants, exhibited lower plasma concentrations of IL-6, TNF-alpha, and IL-10 24 h after injection of LPS. In line, only alveolar macrophages of male descendants of sepsis fathers produced less TNF-alpha upon Zymosan stimulation compared to sham descendants, while LPS responses kept unchanged.

Conclusion: We can prove that male-but surprisingly not female-descendants of post-sepsis fathers show a dampened systemic as well as pulmonary immune response. Based on this observation of an immune hypo-responsivity, we propose that male descendants of sepsis fathers are at risk to develop fungal and bacterial infections and might benefit from therapeutic immune modulation.

Keywords: Epigenetic; Germline; Intergenerational inheritance; Methylation.

Fig. 1

Offspring of sepsis fathers exhibit a higher mortality and altered development. a Experimental design of the study. b Survival, c body weight, and d clinical severity of CLP (n = 20) vs. sham (n = 10) operated male C57BL/6 mice of the paternal generation. e Survival of offspring mice (both sexes) grouped for paternal exposure (98 CLP vs. 110 sham). Group comparison was performed by Gehan-Breslow-Wilcoxon test. f Litter size and successful breedings (numbers). g Sex distribution of all litters according to paternal exposure. Body weight development of female (h) and male (i) offspring animals according to paternal exposure over 57 days after weaning, data points represent mean with 95% confidence interval. j Body weight on day 57 after weaning grouped for paternal exposure and sex (column height represents mean, numbers indicate sample size). Group comparison was performed by Mann-Whitney U test

Fig. 2

Post-sepsis animals possess more, but immobile and defect sperm. a Sperm cell count of individual animals assessed by swim-out from both Caudae epidydimis in animals after CLP (n = 9) or sham (n = 7). b Classification of sperm motility in both groups. c Ratio of morphologically normal to defect sperm in both groups. Group comparison was performed by Mann-Whitney U test

Fig. 3

Distinct methylome alterations occur in sperm of post-sepsis animals. a Circos plot depicting the distribution of differentially methylated cytosines (n = 381) over the chromosomes (orange: hypermethylated CLP vs. sham; blue: hypomethylated CLP vs. sham). b Distribution of differentially methylated cytosines according to genomic region. c Principal component analysis using the 381 cytosines for clustering. d Heat map representation of differential cytosine methylation after unsupervised hierarchical clustering separated into genomic regions (promoter, exons, intron); bottom annotation represents individual animal (C = CLP, S = sham). e–g Corresponding gene lists were separately subjected to gene ontology term analysis. Top five overrepresented biological processes are shown. Dashed red line depicts statistical threshold of p = 0.01. Data is derived from n = 5 animals of each experimental condition (sham or CLP)

Fig. 4

Male offspring of sepsis fathers show a reduced cytokine response after systemic LPS administration. Plasma cytokines were measured by multiplex cytometric bead array 24 h after intraperitoneal injection of saline (open squares) respectively LPS (1 mg/kg, black squares) into male (a–d) and female (e–h) offspring of fathers subjected to CLP or sham. N = 8 animals of each condition were used with exception of the groups of females with saline injection (n = 7). Horizontal line depicts the median; group comparison was performed by Mann-Whitney U test

Fig. 5

Selectively reduced response of alveolar macrophages from male offspring of sepsis fathers. TNF-α cytokine levels in the supernatant of alveolar macrophages either untreated (open squares), after stimulation with LPS (200 ng/ml; light gray squares) or zymosan (250 μg/ml; dark gray squares) of male (a) or female (b) offspring. Cells were yielded by bronchoalveolar lavage of the lungs after euthanasia; n = 12 for each group, horizontal line depicts median. Group comparisons were performed by Mann-Whitney U test