Activation Of The NLRP3 Inflammasome Pathway By Prokineticin 2 In Testicular Macrophages Of Uropathogenic Escherichia Coli-Induced Orchitis

Part 1 PK2 is upregulated in testicular macrophages of UPEC-induced rat orchitis[Purpose] To explore the expression of PK2 in UPEC-induced orchitis.[Methods] UPEC saline suspension was separately injected into the vas deferens proximal to the cauda epididymis,which reached the testis via a retrograde infection.Sham operated rats injected with the same volume of saline served as the control.The model establishment was confirmed by the changes of testicular structure and function.The location of UPEC in testis was detected by electron microscope.The location of PK2 in testicular macrophages was detected by immunofluorescence.PK2 expression in testicular macrophages was detected by q RT-PCR and Western blot.The PK2 protein expression in the supernatants of testicular macrophages and the testicular interstitial fluid isolated from rats was analyzed by ELISA.[Results] Seven days after the treatment,the testes exhibited characteristics including a smaller size,various degrees of swelling and a softer texture compared with the control rats.The total sperm count and forward motility were decreased in the UPEC-infected group.UPEC was detected in testicular interstitium.The location of PK2 in the testicular macrophage was identified by the expression of the rat macrophage surface receptor marker CD68 in the different groups.PK2 was observed only in the nucleus in the control group.However,UPEC infection induced PK2 to predominantly diffuse throughout the cytoplasm,while a relatively lower PK2 signal was detected in the nucleus.PK2 m RNA expression was upregulated in testicular macrophages isolated from the UPEC-infected rats.In addition,PK2 protein expression was obviously elevated in cell lysates,as well as in testicular macrophages supernatants of the UPEC-infected group.The level of PK2 in the supernatants of the UPECinfected group was remarkably higher compared with the control group,according to the quantitative analysis.The expression of PK2 was not found in normal interstitial fluid,whereas it was dramatically increased in the UPEC-infected group.Subsequently,normal testicular macropages secreted little PK2,while UPEC promoted the release of PK2.[Conclusions]Testicular macrophages were induced to express pro-inflammatory PK2 in UPEC-infected rats,which abundantly accumulated in the cytoplasm and was released into the extracellular fluid to a greater extent compared with the control.Part 2 PKR-A alleviates UPEC-induced testicular inflammatory impairment[Purpose] To investigate PK2 effects on the progression of the inflammatory response and male fertility.[Methods] On day one post-infection,the UPEC-infected rats were injected with either 20 mg/kg of PKR-A or 5% DMSO into the testes.The sham operated rats received 5% DMSO as a control.The total sperm count,forward motility and testosterone production in the serum were measured.The portion of CD68+-CD163+ and CD68+-CD163-macrophages was detected by flow cytometric analysis.[Results] The spermatogenic epithelium was thinner,vacuoles appeared in the germ cell cytoplasm,and the infiltration of inflammatory cells was detected in the UPEC-infected group.However,after PKR-A treatment,inflammation was alleviated and the damage to germ cells damage was milder.Furthermore,UPEC exerted a negative influence on the total sperm count,forward motility and testosterone production in the serum,while the PKR-A invention group exhibited a partial recovery of these factors.In the UPEC-infected group,the proportion of CD68+-CD163+ macrophages with immunosuppressive effects was decreased compared to the control group.However,this change was reversed after PKA-R treatment.[Conclusions] PK2 promoted inflammation in the testis,which impaired male reproduction,however,the PK2 antagonist PKA-R alleviated the inflammation and facilitated the recovery of sperm count and forward motility.Part 3 PK2 promotes the activation of the NLRP3 inflammasome pathway[Purpose] To explore the potential involvement of PK2 in IL-1β secretion.[Methods] In vivo experiments,animals were grouped according to Part 2.IL-1β secretion in testicular interstitial fluid was measured by ELISA.Caspase-1 activity of testicular macrophages was detected by caspase-1 activity kit.The expression level of NLRP3 pathways associated proteins was evaluated by Western blot.For the in vitro study,testicular macrophages were primed with LPS or untreated,and then infected with UPEC with or without PK2 for 2 h.In the inhibitory experiments,LPS-primed testicular macrophages were pretreated with MCC950 or VX-765,and then infected with UPEC with or without PK2 for 2 h.Detection indicators and methods were the same as in vivo experiments.[Results] UPEC triggered robust IL-1β release into the interstitial fluid,however,this response was greatly reduced after PKR-A treatment.A similar trend was observed for cleaved caspase-1 activity: caspase-1 cleavage was elicited in testicular macrophage from rats infected with UPEC,but this effect was weakened in the PKR-A-treated group.The Western blot showed that UPEC induced a significant increase of cleaved IL-1β and cleaved caspase-1 release from the testicular macrophages into the supernatants compared with the uninfected rats,but this release was decreased by PKR-A treatment.In testicular macrophages,the protein expression of NLRP3,cleaved caspase-1 and cleaved IL-1β were markedly increased in the UPEC-infected group,but this increase was attenuated in the PKRA group.UPEC promoted the cleavage of pro-IL-1β to form mature IL-1β to be released,thus the level of the pro-IL-1β protein was decreased in the infected group.Interestingly,after the PKR-A treatment,pro-IL-1β expression remained at a high level in the testicular macrophages.IL-1β secretion in the supernatants was increased by UPEC with LPS priming.The additional PK2 induced IL-1β secretion in a dose-dependent manner.As illustrated in the Western blot results,cleaved IL-1β in the supernatants was higher in the treated group compared to the untreated group.Correspondingly,a significant increase in cleaved caspase-1 activity occurred after UPEC infection,and higher cleaved caspae-1 production was further promoted by the additional PK2.Cleaved caspase-1 in the supernatants and NLRP3 expression in testicular macrophages were strongly upregulated when the cells were challenged with UPEC with or without PK2,compared unstimulated cells.In LPS-primed testicular macrophages,MCC950 and VX-765 significantly suppressed the UPEC-induced IL-1β release in the supernatants,and additional PK2 did not reverse this trend.Similarly,in LPS-primed testicular macrophages,cleaved IL-1β was detected in the supernatants after UPEC infection with or without PK2 treatment,but the presence of cleaved IL-1β in the supernatants was eliminated by the inhibitors.Cleaved caspase-1 activity was stimulated by UPEC with or without PK2,but this activation was substantially depressed by MCC950 and VX-765.After the intervention with inhibitors,cleaved caspae-1 was not observed in the supernatants after UPEC infection with or without PK2[Conclusions] NLRP3 inflammasome pathway was activated in testicular macrophages after UPEC infection in vivo,leading to the cleavage of pro-IL-1β to IL-1β.PK2 augments the effects of UPEC on the activation of the NLRP3 inflammasome pathway in vitro.NLRP3 inflammasome pathway is involved in PK2-mediated IL-1β secretion in testicular macrophages.Part 4 IL-1β secretion from testicular macrophages inhibits testosterone synthesis[Purpose] To explore the effect of excessive IL-1β secretion from testicular macrophages on testosterone synthesis in Leydig cells.[Methods] Recombinant IL-1β protein was added at various concentrations to the conditioned medium in which Leydig cells were growing for 24 h.Moreover,the conditioned medium from testicular macrophages with various treatments was collected and filtered and then co-cultured with Leydig cells for 24 h.An anti-IL-1β antibody was used to block IL-1β activity.CCK8 cell proliferation assay was performed to evaluate the cell viability.The levels of testosterone were measured using a chemiluminescent immunoassay kit.The m RNA expression of testosterone synthesis-related enzymes was detected by using q RTPCR.[Results] IL-1β directly inhibited testosterone biosynthesis in a dose-dependent manner.Testosterone production was shown to be decreased in Leydig cells after they were cocultured with testicular macrophages supernatants.When Leydig cells was stimulated with IL-1β in various concentrations,the m RNA expression of steroidogenic acute regulatory protein(St AR)was increased,whereas cholesterol side-chain cleavage P450(P450scc)and 17α-hydroxylase/C17-20 lyase(P450c17)were decreased,and no significant differences were found in the expression of 3β-hydroxysteroid dehydrogenase-Δ4-Δ5 isomerase(3β-HSD)or 17β-hydroxysteroid dehydrogenase(17β-HSD).Then,the expression of genes encoding for the key enzymes were detected in Leydig cells that were co-cultured with various supernatants.The results closely resembled those of the IL-1β treatment;upregulation of St AR and downregulation of P450 scc and P450c17 were observed.These alterations were repressed by the anti-IL-1β antibody[Conclusions] IL-1β secretion from testicular macrophages triggers an acute compensatory response of testosterone synthesis via the upregulation of St AR,and then testosterone production is repressed by inhibiting P450 scc and P450c17...