CLEC5A mediates Zika virus-induced testicular damage

Virus maintenance

ZIKV virus strain PRVABC59 was obtained from the Taiwan Center for Disease Control (provided by Dr. Guann-Yi Yu, NHRI, Taiwan). Virus stocks were propagated in mycoplasma-free Vero cells (also provided by Dr. Guann-Yi Yu), and virus titers were titrated by PFU assay as described previously [55].

Mosquito maintenance

Aedes aegypti (Higgs strain) mosquitoes were fed a 10% sugar solution and raised at 28 °C and 70% humidity. For ZIKV infection, one-week-old female mosquitoes were anesthetized on a cold tray and inoculated with 200 PFU of ZIKV via thoracic injection. Inoculated mosquitoes were maintained under normal rearing conditions for 7 days before conducting the mouse infection experiments.

Mouse maintenance

WT C57BL/6, c1ec5a−/− knockout mice, stat1−/− knockout mice, and stat1−/−clec5a−/− double-knockout mice (all from the C57BL/6 background) were provided by Dr. Shie-Liang Hsieh (GRC, Academia Sinica, Taiwan). dap12−/− knockout mice and stat1−/−dap12−/− double-knockout mice (both on the C57BL/6 background) were ordered from the National Laboratory Animal Center in Taiwan. All experimental mice were males aged 8–12 weeks.

Mosquito-to-mouse infection experiments

ZIKV-infected mosquitoes were starved for 10 h prior biting WT C57BL/6, c1ec5a−/−, stat1−/−, stat1−/−clec5a−/−, dap12−/−, and stat1−/−dap12−/− mice. All mice were anesthetized prior to exposure using a mixture of Ketalar (100 mg/kg mouse; Pfizer, Taiwan) and Rompun (16 mg/kg mouse; Bayer Animal Health, Monheim, Germany) administrated intraperitoneally. The mice were then shaved on the ventral side and put in a mosquito-housing cage (10 mosquitoes/cage) for mosquito feeding. Each mouse was bitten by 5–10 mosquitoes. Mouse serum was collected at 2 dpi to test for infectious virus particles and to conduct a serum cytokine expression assay. 9 week old mice (clec5a-related experiments) or 12 week old mice (dap12-related experiments) were monitored daily for survival, weight loss, and disease symptoms for 7–21 d, depending on the experiment. For hematoxylin and eosin staining, immunohistochemistry, immunofluorescence, and viral titer analysis, the mice were sacrificed, and the testes and epididymis were collected and processed at 7 dpi.

Infectious virus titer of saliva or whole body in mosquitoes

At 7 dpi, the whole body or saliva collected from 5-day-old female Aedes aegypti mosquitoes following thoracic infection with ZIKV (Asian strain PRVABC59 from Taiwan CDC; 200 PFU/mosquito) were examined for ZIKV titer as measured by plaque forming units (PFU) using Vero cells. The saliva was collected into 5% serum-containing DMEM medium and stored at – 80 °C until assayed.

Histology and immunohistochemistry

Tissues from the ZIKV-infected mice (stat1−/−, stat1−/−clec5a−/−, dap12−/−, and stat1−/−dap12−/−) and uninfected WT mice were collected immediately after perfusion with phosphate-buffered saline (PBS) and fixed overnight in 4% paraformaldehyde. The samples were then stored in 70% ethanol and delivered to the NHRI pathology core lab for paraffin embedding. After deparaffinization, slides were stained with hematoxylin and eosin for histological analyses.

For immunohistochemistry, tissue sections from the ZIKV-infected stat1−/−, stat1−/−clec5a−/−, dap12−/−, and stat1−/−dap12−/− mice and the noninfected WT mice were boiled in target retrieval solution (K800521, Dako). After the sections had been washed with PBS, 3% H2O2 was added to quench endogenous peroxidase activity, and PBS containing 10% FBS and 5% BSA was used as a blocking buffer. Tissue sections were incubated overnight at 4 °C with primary antibodies ZIKV NS1 (172–351) antiserum [40], anti-Ly6G (BE0075-1, BioXcell; provided by Dr Li-Rung Huang, NHRI, Taiwan), anti-TRA98 (ab82527, Abcam), anti-CLEC5A, and anti-flavivirus group antigen [D1-4G2-4–15] (AB00230-23.0, Absolute Antibody). The sections were washed with PBS and then incubated with secondary antibodies for 1 h at room temperature. Section staining was performed using a DAB substrate kit (SK-4105, Vector), and the sections were then counterstained with hematoxylin.

Protein binding assay

To determine the binding ability of CLEC5A to ZIKV, 3 μg of recombinant human CLEC5A protein (Cat: 8544-CL, R&D SYSTEMS) or GFP protein was pre-incubated with ZIKV (5 × 106 PFU) in a reaction volume of 500 μl RIPA buffer at 4 °C for 1 h. The protein-virus mixture was then incubated with 2 μl anti-CLEC5A (Cat No. GTX127349, GeneTex) and a negative control was conducted with 3 μl anti-GFP (Cat No. GTX113617, GeneTex) antibody overnight at 4 °C, followed by immunoprecipitation with 20 µL protein A/G magnetic beads for 3 h at 4 °C. The samples were boiled and resolved by 15% SDS-PAGE. Primary antibodies anti-4G2 (1:2000; Cat: GTX57154, GeneTex), anti-CLEC5A (1:1000), or anti-GFP (1:2000) were used for western blot analysis.

Detection of DAP12 expression in mouse tissues

Following perfusion with normal saline, tissues from the testis or epididymis of stat1−/−, stat1−/−clec5a−/− and stat1−/−dap12−/− male mice with or without ZIKV infection were collected at 7 dpi, homogenized, and prepared for western blot analysis with anti-DAP12 antibody (1:2000; Cat: ab283679, Abcam). Spleen tissues (without saline perfusion) from C57BL/6 mice were used as a positive control for DAP12 expression.

Sperm examination and ZIKV detection

Mature sperm were isolated from the cauda epididymis as previously described [56], with some modifications. The cauda epididymis was harvested and lysed, then centrifuged for 10 min at 4 °C prior to sperm characterization. The sperm was collected by incubating the harvested tissue in Vitro Fert medium (K-RVFE 50, Cook Medical) for 30 min at 37 °C to allow the sperm to swim out. Sperm suspensions were analyzed for sperm count and sperm motility, conducted within 1 h of the cauda epididymis dissection.

To examine sperm morphology, air-dried sperm smears were prepared, and Papanicolaou staining was performed according to the guidelines of the World Health Organization [57]. In brief, slides were placed in 95% ethanol for at least 15 min for fixation, and then sequentially immersed in 80% ethanol, 50% ethanol, and purified water and were finally stained with Harris’ hematoxylin. After acid ethanol had been used to remove nonspecific staining, slides were sequentially immersed in 50% ethanol, 80% ethanol, and 95% ethanol and were stained with G-6 orange and EA-50 green stains. The slides were dehydrated in 100% ethanol and immersed in xylene for 1 min. Mounting medium (Micromount, Leica) was used to seal the slides.

For ZIKV detection, sperm smears were blocked with PBS (10% FBS, 5% BSA, 0.5% Triton X-100) for 1 h. The sections were incubated overnight at 4 °C with mouse anti-NS1 antibody. Confocal analysis was performed with a TCS SP5 II laser scanning microscope (Leica; Optical Biology Core Lab, NHRI).

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis

Total RNA was extracted from homogenized mouse tissues using TRI Reagent (Sigma–Aldrich). Complementary DNA (cDNA) was synthesized from 1 μg of RNA using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative RT-PCR was performed on a ViiA 7 RT-PCR System (Thermo Fisher Scientific) using a KAPA SYBR FACT qPCR Kit, and gene expression levels were normalized using 18S rRNA. The following primers were used: TNF-α (5′-TCCCAGGTTCTCTTCAAGGGA-3′ forward; 5′-GGTGAGGAGCACGTAGTCGG-3′ reverse), MCP-1 (5′-AGCTGTAGTTTTTGTCACC-3′ forward; 5′-GTGCTGAAGACCTTAGGGC-3′ reverse), CLEC5A (5′-CCAGAAACTGGGATTTTCACCA-3′ forward; 5′-GCCAGTGTGGATCCTTGTGT-3′ reverse), and 18S rRNA (5′-GTAACCCGTTGAACCCCATT-3′ forward; 5′-CCATCCAATCGGTAGTAGCG-3′ reverse).

Detection of testosterone and cytokines

Serum and testis tissues were harvested from ZIKV-infected stat1−/−, stat1−/−clec5a−/−, dap12−/−, and stat1−/−dap12−/− mice at 2 or 7 dpi, as well as from uninfected WT mice. Analysis of the serum samples collected at 2 dpi or from uninfected mice was outsourced (SUU-FLOWER, Taiwan), and the concentration of testosterone and cytokines was determined according to standard protocols. Quantitative RT-PCR analysis was used to examine the mRNA expression of TNF and MCP-1 in the testes collected from uninfected mice or at 7 dpi from infected mice.

Analysis of testicular apoptosis

Testicular tissues from the testes or epididymis of ZIKV-infected (7 dpi) or uninfected mice were removed and then fixed in 4% paraformaldehyde prior to dehydration and paraffin embedding. The 5-µm-thick testis sections were prepared and subjected to TUNEL staining using an In Situ Cell Death Detection Kit (12,156,792,910, Roche) according to the manufacturer’s protocol. The sections were embedded in paraffin and the TUNEL assay was conducted. Briefly, sections were dewaxed and rehydrated in graded concentrations of xylene and ethanol. They were treated with proteinase K working solution for 10 min at room temperature (RT). Detection of TUNEL-positive cells was based on the fixed and permeabilized tissue sections treated with DNase I for 10 min at RT. The sections were washed and then incubated with the TUNEL reaction mixture for 60 min at 37 °C in a humidified atmosphere in the dark. All labeled sections were viewed and imaged using a Leica SP5 confocal microscope.

Data analysis

Data were analyzed using Prism v6.0 (GraphPad Software). For the immunohistochemistry and immunofluorescence analyses, tissues were collected from at least three mice from each strain. The intensity of staining signals was determined using ImageJ, and the results were analyzed using the Mann–Whitney U test. Statistical significance was assumed at p < 0.05.

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