Uropathogenic Escherichia coli Infection Compromises the Blood-Testis Barrier by Disturbing mTORC1-mTORC2 Balance

Abstract

The structural and functional destruction of the blood-testis barrier (BTB) following uropathogenic E. coli (UPEC) infection may be a critical component of the pathologic progress of orchitis. Recent findings indicate that the mammalian target of the rapamycin (mTOR)-signaling pathway is implicated in the regulation of BTB assembly and restructuring. To explore the mechanisms underlying BTB damage induced by UPEC infection, we analyzed BTB integrity and the involvement of the mTOR-signaling pathway using in vivo and in vitro UPEC-infection models. We initially confirmed that soluble virulent factors secreted from UPEC trigger a stress response in Sertoli cells and disturb adjacent cell junctions via down-regulation of junctional proteins, including occludin, zonula occludens-1 (ZO-1), F-actin, connexin-43 (CX-43), β-catenin, and N-cadherin. The BTB was ultimately disrupted in UPEC-infected rat testes, and blood samples from UPEC-induced orchitis in these animals were positive for anti-sperm antibodies. Furthermore, we herein also demonstrated that mTOR complex 1 (mTORC1) over-activation and mTORC2 suppression contributed to the disturbance in the balance between BTB "opening" and "closing." More importantly, rapamycin (a specific mTORC1 inhibitor) significantly restored the expression of cell-junction proteins and exerted a protective effect on the BTB during UPEC infection. We further confirmed that short-term treatment with rapamycin did not aggravate spermatogenic degeneration in infected rats. Collectively, this study showed an association between abnormal activation of the mTOR-signaling pathway and BTB impairment during UPEC-induced orchitis, which may provide new insights into a potential treatment strategy for testicular infection.

Keywords: blood-testis barrier; male infertility; mammalian target of rapamycin; orchitis; uropathogenic E. coli.

Figure 1

UPEC infection disrupts BTB structure and results in an elevation of serum anti-sperm antibodies in an orchitis rat model. A bacterial suspension (0.5×10⁶ CFU/50 μl) or 50 μl of saline was bilaterally injected into testes of 10–11-week-old SD rats. (A) Animals were sacrificed at the indicated time-points and BTB structure in the testis was observed using TEM. Scale bar, 1 μm. White arrow, TJ (tight junction); black arrow, ER (endoplasmic reticulum); black arrowhead, F-actin bundles. The photographs in the right column are the enlarged images of the white box regions. (B) Serum samples were collected from rats after three months, and anti-sperm antibodies in serum were detected with ELISA kits. Histograms show quantitative results, and p value was determined with ANOVA; ***p<0.001.

Figure 2

UPEC infection down-regulates the expression of cell-junction proteins in the testes of orchitis-model rats. In the orchitis model, rats were sacrificed at the indicated times, and we evaluated the expression of cell-junction proteins in seminiferous epithelium using immunofluorescence staining under an inverted laser scanning confocal microscope. Red, cell-junction proteins; blue, Hoechst 33342 nuclear staining. The images in the insets are the same testes cross-sections at lower magnification (scale bar, 100 μm). The white broken lines indicate the relative location of the basement membrane adjacent to the BTB of the seminiferous tubules.

Figure 3

UPEC-secreted virulence factors in cell culture supernatant disturb Sertoli cell connections and down-regulate the expression of cell-junction proteins. Sertoli cells treated with the indicated amounts of supernatant from UPEC bacterial cultures for 24 or 40 hours. (A) TM4 cellular connections were observed using SEM. Scale bar, 2 μm. (B) Expression of cell-junction proteins in primary Sertoli cells were evaluated using immunofluorescence and observed under confocal microscopy. Scale bar, 20 μm. (C) The expression levels of occludin in primary Sertoli cells were evaluated using immunoblotting analysis, and representative blots are depicted. Data are presented as mean ± SD from triplicate experiments. Histograms shows quantitative results and p values were determined by ANOVA; ***p < 0.001.

Figure 4

UPEC-derived soluble virulence factors activate the mTORC1-signaling pathway and disturb mTORC1-mTORC2 balance. Primary Sertoli cells were treated with UPEC culture supernatant for 40 hours, with either vehicle solution (UPEC) or with 0.1 μM rapamycin as supplement (UPEC + rapamycin). We extracted proteins in RIPA buffer and determined expression levels using western blotting analysis. Representative blots are shown. Data are presented as mean ± SD from triplicate experiments. Histograms show quantitative results, and p value was determined by ANOVA; *p < 0.05, ***p < 0.001.

Figure 5

Rapamycin restores mTORC1-mTORC2 balance and rescues UPEC-induced down-regulation of cell-junction proteins in Sertoli cells. Primary Sertoli cells were treated with UPEC culture supernatant for 40 hours with vehicle solution (UPEC) or with 0.1 μM rapamycin supplement (UPEC+Rapamycin). Expression of Raptor and Rictor (A), as well as cell-junction proteins (B), was evaluated using immunofluorescence staining. Scale bars: (A) 50 μm, (B) 20 μm. (C) The occludin expression levels in Sertoli cells were further confirmed using western blotting, and representative blots are shown. Data are presented as mean ± SD from triplicate experiments. Histograms show quantitative results, and p value was determined by ANOVA; ***p < 0.001.

Figure 6

Rapamycin (5 mg/kg/day) treatment for seven days partially rescues down-regulation of cell-junction proteins in UPEC-infected testes. (A) We evaluated cell-junction protein expression in all three groups using immunofluorescence staining and confocal microscopy. The images in the insets are the same testes cross-sections at lower magnification (scale bar, 100 μm). The white broken lines indicate the relative location of the basement membrane adjacent to the BTB of the seminiferous tubules. (B) Representative blots show the expression patterns of selected junctional proteins in the testes from all three groups animals (n=4 in each group). Histograms show quantitative results, and p value was determined by ANOVA; **p < 0.01, ***p < 0.001.

Figure 7

Rapamycin treatment reduces biotin infiltration through the BTB and does not perturb spermatogenesis in testes from UPEC-infected rats. (A) BTB integrity was functionally evaluated using a sulfo-NHS-LC-biotin diffusion assay, and the representative images of biotin diffusion in the testes of different groups are shown. The testes of CdCl₂-treated rats served as positive controls. The white brackets indicate the distance penetrated by biotin. The images in the insets are the same testes cross sections at lower magnification (scale bar, 200 μm). (B) Semi-quantified data from the sulfo-NHS-LC-biotin diffusion assay are demonstrated as a bar graph. For an oval-shaped tubule from an oblique cross-section, we calculated the radius using the mean of the longest and shortest radii. Approximately 50 tubules were randomly scored per testis. D_Biotin, distance penetrated by biotin in seminiferous tubule; D_radius, the radius of the corresponding seminiferous tubule. p value was determined with ANOVA, ***p<0.001. (C) Paraffin sections of testes (4 μm) were stained with hematoxylin and eosin. Histopathologic evaluation was performed on the testes of control (n=4), UPEC + vehicle-treated (n=6), and rapamycin-treated animals (n= 6) using light microscopy. The images were captured using a NanoZoomer S210 system and representative figures are depicted (a-c: scale bar, 100 μm; e-m: scale bar, 20 μm). Seminiferous tubules marked with asterisks indicate Sertoli cell-only tubules (b, c, h); those marked with arrowheads indicate hypospermatogenic tubules (b and c). e–g: higher-magnification images showing seminiferous epithelium at stages II-III, VIII, and XIII in control rat testes, respectively; h–j: higher-magnification images demonstrating typical pathologic changes in UPEC-infected rats testes (h, extensive germ-cell loss, and only Sertoli cells remained in the seminiferous epithelium; I, infiltration of inflammatory cells in the interstitial compartment; j, the presence of multinucleated, round spermatids are indicated by green arrowheads); k–m, higher-magnification images show pathologic changes in testes from orchitis-model animals treated with rapamycin (k, disordered seminiferous epithelium with germ cell exfoliation; l, the presence of multinucleated round spermatids are indicated by green arrowheads; m, elongated spermatids with defects in polarity are indicated by blue arrowheads, in which the spermatid heads were misoriented by pointing 45°–90° away from the basement membrane). (D) The percentage of abnormal seminiferous tubules in each testicular cross-section was calculated by assessing over 200 tubules in every sample. The data are presented as mean ± SD and shown in the histogram. p value was determined by ANOVA; ***p<0.001. (E) The ratios of testis to body weight in the three groups are presented as mean ± SD and shown in the histogram. p value was determined using ANOVA.