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. 2008 Sep;23(9):2095-103.
doi: 10.1093/humrep/den207. Epub 2008 Jun 20.

"V体育2025版" Antiviral responses of human Leydig cells to mumps virus infection or poly I:C stimulation

A Le Tortorec  1 , H Denis, A-P Satie, J-J Patard, A Ruffault, B Jégou, N Dejucq-Rainsford
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Antiviral responses of human Leydig cells to mumps virus infection or poly I:C stimulation

"VSports最新版本" A Le Tortorec et al. Hum Reprod. 2008 Sep.

Abstract

Background: The immuno-privileged status of the testis is essential to the maintenance of its functions, and innate immunity is likely to play a key role in limiting harmful viral infections, as demonstrated in the rat VSports手机版. In men mumps virus infects Leydig cells and has deleterious effects on testosterone production and spermatogenesis. The aim of this study was to test whether mumps virus infection of isolated human Leydig cells was associated with an inhibition of their innate antiviral defences. .

Methods: Leydig cell production of mRNA and protein for interferons (IFNs) and of three antiviral proteins-2'5' oligoadenylate synthetase (2'5'OAS), double-stranded RNA-activated protein kinase (PKR) and MxA-was investigated, in the absence or presence of mumps virus or viral stimuli including poly I:C, a mimetic of RNA viruses replication product. V体育安卓版.

Results: Stimulated or not, human Leydig cells appeared unable to produce routinely detectable IFNs alpha, beta and gamma. Although the level of PKR remained unchanged after stimulation, the expression of 2'5'OAS and MxA was enhanced following either mumps virus or poly I:C exposure (P < 0. 05 versus control) V体育ios版. .

Conclusions: Overall, our results demonstrate that mumps virus replication in human Leydig cells is not associated with a specific inhibition of IFNs or 2'5'OAS, MxA and PKR production and that these cells display relatively weak endogenous antiviral abilities, as opposed to their rat counterparts VSports最新版本. .

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Figures

Figure 1:
Figure 1:
IFNs α, β, γ and cell marker transcripts detection in Leydig cells. RT–PCR was performed to detect mRNAs encoding: a consensus region for the 12 human IFNs α, IFN β, IFN γ, CD45 (haematopoietic cell marker), 3β-HSD (Leydig cell marker), IL-6 (produced by Leydig cells) (all 35 cycles of PCR) and β-actin (30 cycles) in human Leydig cell cultures stimulated or not (control) for 5–10 h by poly I:C. For each mRNA sample, both a reverse transcriptase (RT) reaction (+) and a negative control reaction without RT (−) were performed (C+: positive control represented by PBMC stimulated 24 h by poly I:C; C−: negative control).
Figure 2:
Figure 2:
TLRs expression by human Leydig cells. RT–PCR was performed to detect mRNAs encoding TLR3, TLR7 and TLR8 in independent Leydig cell cultures from three donors (Leydig C1–3) (C+: positive control represented by PBMC stimulated for 24 h by poly I:C; C−: negative control).
Figure 3:
Figure 3:
Transduction of human Leydig cells with an exogenous IFN β promoter and poly I:C stimulation. 293T cells (A and B) and human Leydig cells (C and D) were transduced for 48 h with a VSV pseudotyped vector construct containing the enhancer region of the ubiquitous spleen focus forming virus (SFFV) promoter inserted upstream of the eGFP open reading frame (positive control). The efficacy of transduction was assessed by checking eGFP fluorescence which appeared at 3–4 days post-transduction, in both 293T cells (A and B) and Leydig cells (C and D). At this time point, 293T cells (E and F) and Leydig cells (G–I) transduced with the IFNβ promoter were stimulated with poly I:C. eGFP expression was then checked regularly (every 6–12 h) for up to 5 days. Leydig cells were identified on morphological criteria (C and G) and by immunostaining for LH receptor (I). Although fluorescence was consistently detected in IFN β promoter-transduced 293T cells stimulated with poly I:C, this stimulus had no effect on the transduced Leydig cells. The results presented are representative of three independent Leydig cell cultures. ×200 magnification.
Figure 4:
Figure 4:
Quantification of transcripts encoding antiviral proteins in human Leydig cells exposed to mumps virus, poly I:C or IFNa. Real-time quantitative RT-PCR analysis of double-stranded RNA- activated protein kinase (PKR), MxA and the 40 kD isoform of 2′5′ oligoadenylate synthetase (p40 2′5′OAS) transcript expression in human Leydig cells stimulated by poly I:C (25 µg/ml), IFN α (1000 U/ml) or infected by the mumps virus (multiplicity of infection 0.1 PFU/cell) for the indicated durations. The results of independent Leydig cell cultures from four donors were pooled (±SEM). MRC-5 cells stimulated by poly I:C were used as a positive control. Stars indicate statistical difference between controls and stimulated cultures (Kruskal–Wallis test, **P < 0.05, *P < 0.15).
Figure 5:
Figure 5:
Antiviral proteins expression in human Leydig cells exposed to mumps virus, poly I:C or IFNa. Western-blot analysis of PKR and MxA protein expression in Leydig cells in the absence (−) or presence (+) of IFN α (MxA and PKR), poly I:C (MxA) (24–72 h) or mumps virus (MxA) (24–96 h). C+: positive control represented by PBMC stimulated for 24 h with poly I:C. Annexin V represents the loading control. The results presented are representative of independent Leydig cell cultures from three donors.
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