目的:趋化因子/趋化因子受体轴,参与免疫细胞贩运,
结论:睾丸趋化因子及其受体(尤其是单核细胞运输必需的)在睾丸炎中升高,和激活素A调节趋化因子/趋化因子受体网络的表达,以促进单核细胞/巨噬细胞和T细胞渗入睾丸,造成广泛的组织损伤.
背景:与健康睾丸相比,实验性自身免疫性睾丸炎(EAO)中CC基序趋化因子受体(CCR)2及其配体CC基序趋化因子配体(CCL)2的水平升高,和缺乏CCR2的小鼠被保护免受EAO诱导的组织损伤。激活素A诱导巨噬细胞CCR2表达,促进他们的移民。此外,睾丸激活素A浓度与自身免疫性睾丸炎的严重程度呈正相关。通过过表达卵泡抑素(FST)抑制激活素A活性可减少EAO诱导的睾丸损伤。
方法:在10-12周龄雄性C57BL/6J(野生型;WT)和B6.129P2-Ccr2tm1Mae/tm1Mae(Ccr2-/-)小鼠(n=6)中诱导EAO。还包括佐剂(n=6)和未处理(n=6)年龄匹配的对照小鼠。用完全弗氏佐剂中的睾丸匀浆在第一次免疫后50天收集睾丸。在另一个实验装置中,在EAO诱导前30天,向WT小鼠注射携带FST315表达基因盒的非复制重组腺相关病毒载体(rAAV-FST315;n=7-9)或空对照载体(n=5)。还检查了适当的佐剂(n=4-5)和未处理的(n=4-6)对照。此外,研究了人睾丸活检显示局灶性白细胞浸润和精子发生受损(n=17)。包括显示完整精子发生的活检作为对照(n=9)。从WT小鼠产生的骨髓来源的巨噬细胞(BMDM)用激活素A(50ng/ml)处理6天。然后将活化素A处理或未处理的BMDM与纯化的小鼠脾T细胞共培养两天以评估趋化因子和细胞因子的产生。
方法:使用定量实时PCR(qRT-PCR)分析从小鼠收集的总睾丸RNA中趋化因子的表达。免疫荧光染色检测激活素A,小鼠睾丸中的F4/80和CD3表达。通过qRT-PCR在人睾丸活检中检查趋化因子/趋化因子受体编码基因的表达。分析趋化因子表达水平与免疫细胞浸润密度或平均精子发生评分之间的相关性。免疫荧光染色用于评估人睾丸活检中CD68和CCR2的表达。从鼠BMDM分离的RNA用于在它们的趋化因子/趋化因子受体表达水平方面表征这些细胞。收集来自BMDM和T细胞的共培养物的条件培养基以确定趋化因子水平和T细胞的促炎细胞因子肿瘤坏死因子(TNF)和干扰素(IFN)-γ的产生。
结果:在WT小鼠睾丸中诱导EAO可增加趋化因子受体如Ccr1的表达(P<0.001),Ccr2(P<0.0001),Ccr3(P<0.0001),Ccr5(P<0.0001),CXC基序趋化因子受体(Cxcr)3(P<0.01),和CX3C基序趋化因子受体(Cx3cr)1(P<0.001),以及他们的大多数配体。Ccr2缺乏通过减少Ccr1的表达逆转了一些与EAO相关的变化(P<0.0001),Ccr3(P<0.0001),Ccr5(P<0.01),Cxcr3(P<0.001),和Cx3cr1(P<0.0001)。重要的是,显示精子发生受损和伴有局灶性白细胞浸润的活检显示CCL2表达较高(P<0.01),CCR1(P<0.05),CCR2(P<0.001),和CCR5(P<0.001)比对照活检没有炎症和完整精子发生的迹象。CCR2及其配体CCL2的基因表达与免疫细胞浸润密度呈正相关(P<0.05),与平均精子发生评分呈负相关(P<0.001)。此外,表达CCR2的CD68+巨噬细胞存在于具有白细胞浸润的人睾丸中,具有肾小管损伤的证据。BMDM的治疗,作为睾丸巨噬细胞的替代品,与激活素A一起增加了Ccr1,Ccr2和Ccr5的表达,同时减少了Ccl2,Ccl3,Ccl4,Ccl6,Ccl7Ccl8和Ccl12的表达。这些发现在体内得到了验证,通过显示在EAO小鼠中通过过表达FST抑制激活素A活性降低了睾丸中Ccr2(P<0.05)和Ccr5(P<0.001)的表达。有趣的是,共培养活化素A处理的BMDMs和T细胞降低CCL2水平(P<0.05),CCL3/4(P<0.01),和CCL12(P<0.05)在培养基中降低了T细胞对TNF的产生(P<0.05)。EAO睾丸中分泌活化素A的大多数细胞被鉴定为巨噬细胞。
方法:不适用。
结论:BMDMs被用作睾丸巨噬细胞的替代物。因此,从体外实验获得的结果可能不能完全代表体内睾丸的情况。此外,因为从睾丸组织中提取总RNA来检测趋化因子的表达,单个细胞类型作为特定趋化因子生产者的贡献可能被忽视了.
结论:我们的数据表明,巨噬细胞通过表达CCR2和激活素A参与睾丸炎症的发展和进展,最终重塑趋化因子/趋化因子受体网络并招募其他免疫细胞到炎症部位。因此,抑制CCR2或激活素A可作为减少睾丸炎症的潜在治疗策略.
背景:这项工作得到了国际研究培训小组在“男性生殖障碍的分子发病机理”中的支持,JustusLiebig大学(吉森)和莫纳什大学(墨尔本)(GRK1871/1-2)之间的合作,由德意志大学和莫纳什大学资助,澳大利亚国家健康与医学研究委员会的想法赠款(1184867),以及维多利亚州政府的运营基础设施支持计划。作者宣布没有竞争的经济利益。
OBJECTIVE: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network?
CONCLUSIONS: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage.
BACKGROUND: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage.
METHODS: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund\'s adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production.
METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells.
RESULTS: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages.
METHODS: N/A.
CONCLUSIONS: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked.
CONCLUSIONS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation.
BACKGROUND: This work was supported by the International Research Training Group in \'Molecular pathogenesis on male reproductive disorders\', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government\'s Operational Infrastructure Support Programme. The authors declare no competing financial interests.