Uropathogenic E. coli induce different immune response in testicular and peritoneal macrophages: implications for testicular immune privilege

Abstract

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells.

Figure 1. UPEC do not induce cytokine secretion in PM.

PM were infected with UPEC CFT073, UPEC 536, pathogenic island mutants of UPEC 536 (ΔPAI I-V) as well as a TIR domain deletion mutant of UPEC CFT073 (UPEC CFT073 ΔTIR) and UPEC 536 (UPEC 536 ΔTIR) for 5 h (MOI = 0.1). Incubation with wild type UPEC strains (CFT073, 536) and their mutants did not cause secretion of (A) TNF-α or (B) IL-6 from cultured PM. As positive control PM were treated with 10 µg/ml LPS to induce IL-6 and TNF-α secretion. ND = not detectable. Significance levels for ELISA were determined using Tukey-Kramer multiple comparison test and p≤0.05 was considered significant.

Figure 2. Hierarchical clustering of significantly regulated genes in PM and TM clearly distinguished between PM and TM pretreatment, 30 min and 60 min after infection with UPEC CFT073.

(A) Blue indicates downregulation and red upregulation of genes. A time-dependent pattern of gene regulation can be observed within the macrophage populations. The picture clearly demonstrates how different PM and TM respond on the gene expression level upon infection with the same UPEC strain. (B) Venn diagram of significantly regulated genes in PM and TM. A total of 1710 genes (PM) and 400 genes (TM) were significantly regulated (FDR<0.05) at both time points 30 and 60 min. Both PM and TM showed an overlap of 125 genes with PM having 1585 unique genes and TM 275 unique genes.

Figure 3. Ca²⁺ influx is dependent on presence of hemolysin genes in UPEC.

UPEC strains CFT073 and 536 induced a Ca²⁺ influx in rat TM (A, C) and PM (B, D). After recording the baseline ratio of Fura-2 AM fluorescence after excitation at 340 nm versus excitation at 380 nm for 1 min, cells were treated with bacteria or vehicle as indicated by arrows. (A, B) PM and TM stimulated with UPEC CFT073 reveal a rapid and sustained increase in [Ca²⁺]_i, while cells treated with vehicle (HEPES) or NPEC 470 do not respond with Ca²⁺ mobilization. (C, D) UPEC 536 deficient for HlyA (UPEC 536 HDM) were not effective in triggering a Ca²⁺ rise, whilst wildtype UPEC 536 elicited a rapid cytoplasmic Ca²⁺ influx in both PM and TM. Numbers of measured cells are given in brackets. (E) Soluble factor(s) present in the supernatants induced a rise in [Ca²⁺]_i. UPEC bacteria were added as a positive control at the end of the experiment and caused further elevation of [Ca²⁺]_i levels. Intracellular Ca²⁺ was monitored by the Fura-2 method. Areas under the curve were calculated by summing up values obtained for each cell. Non-parametric rank based Kruskal-Wallis test was used to compare multiple groups and if significant differences were detected, it was followed by Mann-Whitney test to compare between two experimental groups.

Figure 4. UPEC virulence factor hemolysin inhibits pro-inflammatory cytokine secretion.

(A, C) PM were infected with NPEC mutants expressing hemolysin (FOS 2, FOS 9 and FOS 22), UPEC 536 hemolysin double mutant and wild type NPEC EPI-T1^R strain for 5 h with or without concomitant LPS stimulation. (B, D) PM and TM were treated with 20 ng/ml hemolysin in the presence of LPS or without for 5 h. IL-6 and TNF-α concentrations in culture supernatants were measured by sandwich ELISA. Values are means ± SD of triplicates. MOI = 0.1. Tukey-Kramer multiple comparisons test was used to analyze significance of data. Values with different letters superscript differ significantly (p<0.001) compared to NPEC and HDM. ND = not detectable.

Figure 5. UPEC activates NFATC2 signaling pathway.

(A, B) PM and TM were treated with UPEC CFT073 (MOI = 20) for 1 h. (A) TM and PM were pretreated for 15 min with 2 µM cyclosporine A (CSA) or left unstimulated, followed by incubation with UPEC CFT073 or 40 ng/ml alpha-hemolysin (H) for 30 min, respectively. For Western blot analysis 40 µg of protein were separated on a 7.5% SDS-PAGE and immunoblots were probed using an anti-NFATC2 antibody (NFAT). (B) UPEC CFT073 and alpha-hemolysin (HlyA) induced calcineurin-dependent NFATC2 nuclear translocation. TM and PM were pretreated with 2 µM of the calcineurin inhibitor CSA for 15 min or left unstimulated with subsequent challenge by UPEC (MOI = 20) or 40 ng/ml HlyA for 30 min. In support of the data shown in (B), nuclear translocation of NFATC2 (red) after UPEC CFT073 and HlyA treatment was observed in both TM and PM. Pretreatment of PM and TM with CSA blocked NFATC2 nuclear translocation. Nuclei were counterstained with Cy5-conjugated TO-PRO-3.

Figure 6. qRT-PCR analyses of anti-inflammatory cytokines (IL-3, IL-4, IL-10, IL-13, upper row) and pro-inflammatory cytokines (IL1-α, IL1-β, IL-6, TNF-α, lower row) after challenge of TM and PM with UPEC CFT073 (MOI = 20) or hemolysin A (HlyA, 40 ng/ml) for 1 h.

Results were normalized using β-microglobulin and β-actin as endogenous controls and are shown as fold changes relative to uninfected controls. HlyA was preincubated with polymyxin B (50 µg/ml) at 4°C for 30 min to remove any possible LPS contamination. Values are means ± SD of triplicates. Mann-Whitney U test was used to analyze data and p≤0.05 was considered significant.

Figure 7. HlyA suppresses MAP kinase activation.

(A) PM and (B) TM were treated with 10 µg/ml LPS and 40 ng/ml of alpha-hemolysin (HlyA) for 30 min as indicated. For Western blot analysis 20 µg of protein were separated by 10% SDS-PAGE and immunoblots were probed with antibodies specific for the phosphorylated forms of MAP kinases p38 (p-P38), JNK (p-JNK) and ERK 1/2 (p-ERK1/2), respectively. Changes in the total amount of kinases following treatment and equal loading of samples were assessed by detecting total levels of p38, JNK, and ERK1/2. β-actin levels served as general loading control. AP-1 signaling pathway activation in TM and PM was verified by assessing phospho-c-JUN (p-c-JUN) levels after treatment with (C) UPEC CFT073 (MOI = 20) for 30 min. (D) HlyA treatment strongly reduces p-c-JUN levels in LPS stimulated and unstimulated PM, but not in TM. (E) HlyA caused IκBα degradation in PM and TM. Cells were treated with LPS and/or HlyA for 30 min as indicated above and blots were probed with an anti-IκBα antibody. (C-E) β-actin detection served as loading control. Membranes in A-D were stripped and reprobed to test for the phosphorylated or unphosphorylated form or loading control, respectively. Antibodies against p-ERK and ERK were probed on split samples run on parallel gels. (F) HlyA induces apoptosis in PM, but not in TM. TM and PM were incubated with HlyA (40 ng/ml) for 1 h and DNA fragmentation was examined by the TUNEL assay as an index for apoptosis. HlyA was preincubated with polymyxin B (50 µg/ml) at 4°C for 30 min to remove any possible LPS contamination.

Figure 8. Bacterial orchitis elicited by in vivo infection with UPEC CFT073.

Infection resulted in an increased number of (A, B) ED1+ED2+ total testicular macrophages and (C) ED1+ ‘inflammatory’ macrophages. Immunolabeled macrophages were counted in whole cross-sections and related to the number of cross-sectioned seminiferous tubules in the same section visualized by DAPI stain. (D) Testosterone concentrations were measured by RIA in testicular homogenates of testis obtained from animals 7 days post UPEC infection into the vas deferens and PBS injected sham operated rats (Ctrl). Testosterone concentrations are significantly lower in orchitis compared to sham-operated animals. (E) In TM isolated from testis 7 days post UPEC infection in the vas deferens, NFATC2 showed a nuclear localization. In contrast, NFATC2 is found in the cytoplasm of TM obtained from testis of PBS injected sham operated rats (Ctrl). Nuclei are counterstained with DAPI (blue). Inserts show a higher magnification of a cell seen in the respective overview. Macrophage quantification: values are means ± SD of n =  2 (Ctrl), n = 4 (UPEC). Testosterone measurements: values are means ± SD of Ctrl (n = 8); UPEC (n = 10). Statistical analysis was performed using student t-test. * = p< 0.05, ** = p<0.01.