研究问题 参与免疫细胞运输的趋化因子/趋化因子受体轴是否有助于睾丸炎症的病理学以及激活素 A 如何调节该网络？摘要答案 睾丸趋化因子及其受体（特别是那些对单核细胞运输至关重要的受体）在睾丸炎中升高，激活素 A 调节趋化因子/趋化因子受体网络的表达，促进单核细胞/巨噬细胞和 T 细胞浸润到睾丸中，引起广泛的组织损伤损害。已知的情况与健康睾丸相比，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 的水平增加，并且 CCR2 缺陷的小鼠可以免受 EAO 侵害。诱发组织损伤。激活素 A 诱导巨噬细胞表达 CCR2，促进其迁移。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。研究设计、大小、持续时间 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) 和未治疗 (n = 6) 年龄匹配的对照小鼠。用弗氏完全佐剂中的睾丸匀浆第一次免疫后50天收集睾丸。在另一个实验设置中，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)，持续 30 天EAO 诱导前。 还检查了适当的佐剂（n = 4-5）和未经处理的（n = 4-6）对照。此外，对显示局灶性白细胞浸润和精子发生受损的人类睾丸活检（n = 17）进行了研究。显示完整精子发生的活组织检查被纳入作为对照（n = 9）。用激活素 A (50 ng/ml) 处理 WT 小鼠产生的骨髓源性巨噬细胞 (BMDM) 6 天。然后将经激活素 A 处理或未经处理的 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。参与者/材料、环境、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。免疫荧光染色用于检测小鼠睾丸中激活素A、F4/80和CD3的表达。通过 qRT-PCR 检查人类睾丸活检中趋化因子/趋化因子受体编码基因的表达。分析了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 中分离的 RNA 用于表征这些细胞的趋化因子/趋化因子受体表达水平。收集来自 BMDM 和 T 细胞共培养物的条件培养基，以确定 T 细胞的趋化因子水平以及促炎细胞因子肿瘤坏死因子 (TNF) 和干扰素 (IFN)-γ 的产生。主要结果和机会的作用 WT 小鼠睾丸中 EAO 的诱导增加了趋化因子受体的表达，例如 Ccr1 (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) 以及它们的大多数配体。 Ccr2 缺陷通过降低 Ccr1 (P < 0.0001)、Ccr3 (P < 0.0001)、Ccr5 (P < 0.01)、Cxcr3 (P < 0.001) 和 Cx3cr1 的表达，逆转了与 EAO 相关的一些变化。 P< 0.0001）。重要的是，活检显示精子发生受损并伴有局灶性白细胞浸润，其 CCL2 (P < 0.01)、CCR1 (P < 0.05)、CCR2 (P < 0.001) 和 CCR5 (P < 0.001) 表达高于对照。活检没有炎症迹象和完整的精子发生。 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+ 巨噬细胞存在于人类睾丸中，有白细胞浸润，并有肾小管损伤的证据。用激活素 A 处理作为睾丸巨噬细胞替代物的 BMDM，可增加其 Ccr1、Ccr2 和 Ccr5 的表达，同时减少其 Ccl2、Ccl3、Ccl4、Ccl6、Ccl7、Ccl8 和 Ccl12 的表达。这些发现在体内得到了验证，结果显示，通过在 EAO 小鼠中过度表达 FST 来抑制激活素 A 活性，会降低睾丸中 Ccr2 (P < 0.05) 和 Ccr5 (P < 0.001) 的表达。有趣的是，共培养经激活素 A 处理的 BMDM 和 T 细胞降低了培养基中 CCL2 (P < 0.05)、CCL3/4 (P < 0.01) 和 CCL12 (P < 0.05) 的水平，并减弱了T 细胞产生 TNF (P < 0.05)。 EAO 睾丸中分泌激活素 A 的大多数细胞被鉴定为巨噬细胞。大规模数据不适用。 局限性和注意事项 BMDM 被用作睾丸巨噬细胞的替代物。因此，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，由于从睾丸组织中提取总RNA来检查趋化因子的表达，个体细胞类型作为特定趋化因子产生者的贡献可能被忽视了。研究结果的更广泛意义我们的数据表明，巨噬细胞通过表达 CCR2 和激活素 A 参与睾丸炎症的发生和进展，最终重塑趋化因子/趋化因子受体网络并将其他免疫细胞招募到炎症部位。因此，抑制 CCR2 或激活素 A 可以作为减少睾丸炎症的潜在治疗策略。研究经费/竞争利益 这项工作得到了贾斯图斯·李比希大学（吉森）和莫纳什大学（墨尔本）合作的“男性生殖障碍的分子发病机制”国际研究培训小组的支持（GRK1871/1-2）由德国研究协会和莫纳什大学、澳大利亚国家健康和医学研究委员会创意拨款 (1184867) 以及维多利亚州政府的运营基础设施支持计划资助。作者声明不存在竞争的经济利益。
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Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration
STUDY QUESTION 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? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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.
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