Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration
Authors: Hasan Hiba , Peng Wei , Wijayarathna Rukmali , Wahle Eva , Fietz Daniela , Bhushan Sudhanshu , Pleuger Christiane , Planinić Ana , Günther Stefan , Loveland Kate L , Pilatz Adrian , Ježek Davor , Schuppe Hans-Christian , Meinhardt Andreas , Hedger Mark P , Fijak Monika 
Published: May 2024

Abstract

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 &lt; 0.001), Ccr2 (P &lt; 0.0001), Ccr3 (P &lt; 0.0001), Ccr5 (P &lt; 0.0001), CXC motif chemokine receptor (Cxcr)3 (P &lt; 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P &lt; 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 &lt; 0.0001), Ccr3 (P &lt; 0.0001), Ccr5 (P &lt; 0.01), Cxcr3 (P &lt; 0.001), and Cx3cr1 (P &lt; 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P &lt; 0.01), CCR1 (P &lt; 0.05), CCR2 (P &lt; 0.001), and CCR5 (P &lt; 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 &lt; 0.05) and negatively with the mean spermatogenesis score (P &lt; 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 &lt; 0.05) and Ccr5 (P &lt; 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P &lt; 0.05), CCL3/4 (P &lt; 0.01), and CCL12 (P &lt; 0.05) in the medium and attenuated the production of TNF (P &lt; 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.