Gender differences in expression of the human caspase-12 long variant determines susceptibility to Listeria monocytogenes infection

Abstract

Inflammatory caspases are important effectors of innate immunity. Caspase-12, of the inflammatory caspase subfamily, is expressed in all mammals tested to date, but has acquired deleterious mutation in humans. A single-nucleotide polymorphism introduces a premature stop codon in caspase-12 in the majority of the population. However, in 20% of African descendants, caspase-12 is expressed and sensitizes to infections and sepsis. Here, we examined the modalities by which human caspase-12 confers susceptibility to infection. We have generated a fully humanized mouse that expresses the human caspase-12 rare variant (Csp-12L) in a mouse casp-12(-/-) background. Characterization of the humanized mouse uncovered sex differences in Csp-12L expression and gender disparity in innate immunity to Listeria monocytogenes infection. The Csp-12L transgene completely reversed the knockout resistance-to-infection phenotype in casp-12(-/-) males. In contrast, it had a marginal effect on the response of female mice. We found that estrogen levels modulated the expression of caspase-12. Csp-12L was expressed in male mice but its expression was repressed in female mice. Administration of 17-beta-estradiol (E2) to humanized male mice had a direct suppressive effect on Csp-12L expression and conferred relative resistance to infection. Chromatin immunoprecipitation experiments revealed that caspase-12 is a direct transcriptional target of the estrogen receptor alpha (ERalpha) and mapped the estrogen response element (ERE) to intron 7 of the gene. We propose that estrogen-mediated inhibition of Csp-12L expression is a built-in mechanism that has evolved to protect females from infection.

Fig. 1.

NMD targets the Csp-12S message for degradation. (A) Exon–intron organization of human and mouse caspase-12 genes. Numbers above the human Csp12 gene structure correspond to exon lengths in base pairs and the asterisk indicates the SNP. Domain structures of the predicted Csp-12S, Csp-12L, and mouse caspase-12 are depicted. (B) Kinetics of Csp-12S mRNA accumulation following treatment with cycloheximide. mRNA levels were analyzed by RT-PCR using the primers depicted in A. β2-microglobulin (β2m) mRNA was used as a standardizing control. (C) RT-PCR analysis of the levels of UPF1 mRNA (Left) and Csp-12 mRNA (Right) in HeLa cells following knockdown of UPF1. mRNA from HEK293T cells expressing a fusion protein between Csp-12L and GFP was used as a positive control.

Fig. 2.

Generation of a humanized mouse expressing the human Csp-12L variant. (A) G-banding to FISH on a primary fibroblast culture derived from a female casp-12^−/−/Csp-12L mouse. The human Csp-12L BAC was labeled with Spectrum Orange (red signal) and a control BAC probe for mouse chromosome 6 was labeled with Spectrum Green (green signal). The Csp-12L transgene expression pattern in mouse tissues was examined by RT-PCR (B) and Western blot (C). The right arrowhead in C indicates an in vitro transcribed and translated Csp-12L.

Fig. 3.

Csp-12L compensates for loss of mouse caspase-12 in male but not female mice. (A) Casp-12^+/+, casp-12^−/−, and Csp-12L/casp-12^−/− (n = 4/genotype) were challenged intravenously with 5 × 10⁴ CFU of L. monocytogenes, and pathogen clearance was determined in livers and spleens of male and female mice on different days pi. (B and C) RNA extracted from spleens of male and female mice on different days pi with L. monocytogenes was analyzed by qPCR for IL-10 (B) and IFNγ (C) expression. Data represent average expression ± SEM from 4 different mice.

Fig. 4.

Csp-12L dampens cytokine production. (A) Splenocytes from male and female mice (n = 4/genotype/sex) were primed overnight with 50 ng/mL of pure LPS and then infected for 3 h with L. monocytogenes. Supernatants were collected and IL-1β levels were determined by ELISA. (B) Splenocytes were treated for 1 h with 1 μg/mL of crude LPS. mRNA was extracted and pro-IL-1β, TNFα, and IL-12 levels were measured by qPCR.

Fig. 5.

Estrogen levels control human caspase-12 expression. (A) Csp-12L expression levels were quantified by qPCR in splenocytes derived from male and female humanized mice on days 1 and 3 pi. (B) E2 was injected i.p. 4 h before infection and was injected daily for 3 days pi. On day 3 pi, bacterial loads were determined in mice treated or not with E2. Data represent average expression ± SEM from 4 mice. (C) The Csp-12L transgene is expressed in the spleen of male mice infected with L. monocytogenes but its expression is repressed in females and males injected with E2. (D) ChIP was performed using anti-mouse ERα or IgG control antibodies in splenocytes derived from humanized mice (males, males treated with E2, or females), and pull-down of human casp-12 was determined by qPCR. Data represent average expression ± SEM from 2 different mice.