NLRP3 in somatic non-immune cells of rodent and primate testes

Abstract

NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate (marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3. NLRP3 and associated genes (PYCARD, CASP1, IL1B) were also found in isolated human testicular peritubular cells and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase-expressing transgenic mice (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to WT controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation.

Figure 1. NLRP3 is expressed in Sertoli cells of mouse and primate testes and in peritubular cells of the human testis.

Immunohistochemistry for NLRP3 revealed staining in mouse (n = 14; A: adult, 3 months) rhesus macaque (n = 4; B: adult, 18 years), common marmoset (n = 7; C: adult, 11 years; G: new-born) and human testes (n = 8; D: adult, 48 years). Sertoli cells expressed NLRP3 in all samples examined. Expression in peritubular cells (arrows) of the seminiferous tubule was solely detected in human samples. Higher magnifications of adult marmoset (E) and human (F) sections confirmed these findings. Inlays: negative controls. Bars = 25 μm.

Figure 2. Cellular expression of NLRP3 in human and mouse testes.

(A) In human (h), mouse (m), marmoset monkey (cj) and rhesus macaque (rh) NLRP3 transcripts indicated expression in whole testis lysates. (B) NLRP3 transcripts were detected in cultured human testicular peritubular cells (HTPCs; n = 2). Samples stem from men exhibiting normal spermatogenesis. Inflammasome-associated gene expression of ASC, CASP1 and IL1B was identified in HTPCs as well. (C) Mouse Sertoli cell line TM4 exhibited expression of Nlrp3, Pycard, Casp1 and Il1b transcripts. Negative controls consisted of non-reverse transcribed RNA as template (-RT) and a non-template reaction (–). Labels indicate amplicon lengths.

Figure 3. NLRP3 expression in human testes is associated with phenotypic characteristics in subfertility patients.

(A1) In patients suffering from mixed atrophy (MA) syndrome (n = 5) staining in Sertoli cells remained prominent, especially in tubules with impaired spermatogenesis (arrow). (B1) Staining of peritubular cells and the tubular wall (arrowheads) intensified corresponding to thickened sectors of the tubular wall, which are associated with MA pathology. Tubular walls stained most intensely in seminiferous tubules, which lacked Sertoli cell staining. (A2, B2) Negative controls corresponding to A1 and B1, respectively. Bars = 25 μm.

Figure 4. Testicular Nlrp3 levels are elevated in an infertility mouse model with increasing age.

(A-C) Nlrp3 transcript expression was elevated ~3-fold in 2 months old AROM+ mice (n = 4) compared to WT mice (n = 3), ~4-fold in 5 months old AROM+ mice (n = 5) compared to WT mice (n = 3) and ~6-fold in 10 months old AROM+ mice (n = 8) compared to WT mice (n = 7). Data represent means ± SEM normalized to WT control. Asterisks (*) denote statistical significance, p < 0.05 (unpaired t-test).

Figure 5. Elevated testicular Nlrp3 levels can be reduced by an intervention strategy.

(A) When treated with an aromatase inhibitor (AI) Nlrp3 transcript levels in 2.5 months old AROM+ (n = 7) decreased from significant ~3-fold elevation in comparison to WT (n = 8) almost by half (n = 6). AI treatment of WT mice (n = 8) did not show any differences from WT. Data represent means ± SEM normalized to WT control. Asterisks (*) denote statistical significance, p < 0.05 (ANOVA with Newman-Keuls post-test). (B) Staining for NLRP3 in WT and AROM+ testicular sections (2.5 months) depicted positively stained Sertoli cells and revealed alterations of the testicular phenotype in AROM+ compared to WT mice, e.g. vacuolization (crosses). Inlays: negative controls. Bars = 25 μm.