S100A9 Activates the Immunosuppressive Switch Through the PI3K/Akt Pathway to Maintain the Immune Suppression Function of Testicular Macrophages

Macrophages are functionally plastic and can thus play different roles in various microenvironments. Testis is an immune privileged organ, and testicular macrophages (TMs) show special immunosuppressive phenotype and low response to various inflammatory stimuli. However, the underlying mechanism to maintain the immunosuppressive function of TMs remains unclear. S100A9, a small molecular Ca2+ binding protein, is associated with the immunosuppressive function of macrophages. However, no related research is available about S100A9 in mouse testis. In the present study, we explored the role of S100A9 in TMs. We found that S100A9 was expressed in TMs from postnatal to adulthood and contributed to maintaining the immunosuppressive phenotype of TMs, which is associated with the activation of PI3K/Akt pathway. S100A9 treatment promotes the polarization of bone marrow-derived macrophages from M0 to M2 in vitro. S100A9 was significantly increased in TMs following UPEC-infection and elevated S100A9 contributed to maintain the M2 polarization of TMs. Treatment with S100A9 and PI3K inhibitor decreased the proportion of M2-type TMs in control and UPEC-infected mouse. Our findings reveal a crucial role of S100A9 in maintaining the immunosuppressive function of TMs through the activation of PI3K/Akt pathway, and provide a reference for further understanding the mechanism of immunosuppressive function of TMs.

Abstract 16: Altered Testicular Macrophage Polarization Is Associated With Reproductive Dysfunction In Hypertensive Mice

Elevated circulating proinflammatory (M1) and decreased anti-inflammatory (M2) macrophages contribute to hypertension (HTN) and end-organ damage. HTN is associated with reproductive dysfunction in men. However, the impact of HTN on testicular macrophages and inflammation is unknown. We hypothesized that HTN increases M1 and decreases M2 testicular macrophages, which is associated with inflammation and reproductive dysfunction. Male mice were made hypertensive by either providing L-arginine methyl ester hydrochloride (L-NAME) (0.5 mg/mL) in the drinking water for 3 weeks [L-NAME-induced HTN (LHTN)] or L-NAME water for 2 weeks, followed by a 2-week washout period and a subsequent 3-week 4% high salt diet [salt-sensitive hypertension (SSHTN)]. Control (C) mice received tap water and normal diet. Flow cytometry analysis revealed a significant increase in both M1 (C: 15%±1, LHTN: 22%±2; p<0.05) and M2 (C: 10%±1, LHTN: 21%±2; p<0.05) macrophages in testes from LHTN mice. Similarly, testes from SSHTN mice had a significant increase in M1 (C: 17%±1, SSHTN: 28%±2; p<0.05) but had a significant decrease in M2 (C: 14%±1, SSHTN: 7%±1; p<0.05) macrophages. Testes from both hypertension models had a significant increase in gene expression of the proinflammatory cytokines TNFa, IFNg, IL-1b, IL-6, and IL-17. Sperm concentration (C: 8.5±0.7, LHTN: 6.5±0.2, SSHTN: 4.7±0.5; both p<0.05) and the percentage of sperm mitochondrial activity (C: 88%±3, LHTN: 71±5, SSHTN: 64%±3; both p<0.05) were decreased significantly in both hypertension groups. Hypertensive mice presented a significantly increased percentage of sperm with abnormal morphology (C: 5%±1, LHTN: 8%±1, SSHTN: 13%±2; both p<0.05) and damaged acrosome (C: 1.4%±0.2, LHTN: 2.8%±0.2, SSHTN: 4%±0.5; both p<0.05). There was a significant decrease in gene expression of the hormone receptors AR, ERa, and LHR, and the steroidogenic enzymes StAR, 3bHSD, 17bHSD, and CYP17a1 in the testes of LHTN and SSHTN mice. These data demonstrate that HTN alters testicular macrophage polarization which is associated with inflammation and impaired reproductive health. Therapeutic strategies may be developed to improve reproductive health in male hypertensive patients by targeting testicular macrophage imbalance.

Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM+ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM+ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206+ and peritubular MHC II+ macrophages, with higher levels in CD206+ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2+ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM+ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206+MHC II+ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2+ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM+ mice. The changes in testis are distinct from the described alterations in other organs of AROM+, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM+ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM+ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.

The Presence of Colony-Stimulating Factor-1 and Its Receptor in Different Cells of the Testis; It Involved in the Development of Spermatogenesis In Vitro

Spermatogenesis is a complex process, in which spermatogonial cells proliferate and differentiate in the seminiferous tubules of the testis to generate sperm. This process is under the regulation of endocrine and testicular paracrine/autocrine factors. In the present study, we demonstrated that colony stimulating factor-1 (CSF-1) is produced by mouse testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells (such as CDH1-positively stained cells; a marker of spermatogonial cells). In addition, we demonstrated the presence of CSF-1 and its receptor (CSF-1R) in testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells of human testis. We also show that the protein levels of CSF-1 were the highest in testis of 1-week-old mice and significantly decreased with age (2–12-week-old). However, the transcriptome levels of CSF-1 significantly increased in 2–3-week-old compared to 1-week-old, and thereafter significantly decreased with age. On the other hand, the transcriptome levels of CSF-1R was significantly higher in mouse testicular tissue of all examined ages (2–12-week-old) compared to 1-week-old. Our results demonstrate the involvement of CSF-1 in the induction the proliferation and differentiation of spermatogonial cells to meiotic and postmeiotic stages (BOULE- and ACROSIN-positive cells) under in vitro culture conditions, using methylcellulose culture system (MCS). Thus, it is possible to suggest that CSF-1 system, as a testicular paracrine/autocrine system, is involved in the development of different stages of spermatogenesis and may be used in the development of future therapeutic strategies for treatment of male infertility.

Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis

Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.

Corticosterone Enhances the AMPK-Mediated Immunosuppressive Phenotype of Testicular Macrophages During Uropathogenic Escherichia coli Induced Orchitis

Testicular macrophages (TM) play a central role in maintaining testicular immune privilege and protecting spermatogenesis. Recent studies showed that their immunosuppressive properties are maintained by corticosterone in the testicular interstitial fluid, but the underlying molecular mechanisms are unknown. In this study, we treated mouse bone marrow-derived macrophages (BMDM) with corticosterone (50 ng/ml) and uncovered AMP-activated protein kinase (AMPK) activation as a critical event in M2 polarization at the phenotypic, metabolic, and cytokine production level. Primary TM exhibited remarkably similar metabolic and phenotypic features to corticosterone-treated BMDM, which were partially reversed by AMPK-inhibition. In a murine model of uropathogenic E. coli-elicited orchitis, intraperitoneal injection with corticosterone (0.1mg/day) increased the percentage of M2 TM in vivo, in a partially AMPK-dependent manner. This study integrates the influence of corticosterone on M2 macrophage metabolic pathways, phenotype, and function, and highlights a promising new avenue for the development of innovative therapeutics for orchitis patients.