Regulation and functions of macrophages in the adult mouse testis
Macrophages, which regulate inflammation and dictate the course of infection, are the most important immune cell type in the testis. It has been established that the majority of macrophages in the rodent testis are ‘alternatively activated’ (M2, or anti-inflammatory), consistent with the immune privilege of the organ. However, there is still much ... to be discovered about the regulation of this testicular environment. Significantly, activins produced by Sertoli cells, pachytene spermatocytes and round spermatids, and spermatogonia in the seminiferous tubules, myoid cells in the peritubular region and Leydig cells and macrophages in the interstitium are implicated in regulating inflammatory responses by macrophages (Hedger et al., 2011). Activin A is an important regulator of macrophage and dendritic cell development, and its endogenous binding protein, follistatin, has been found to be therapeutic in a number of inflammatory and immunological disease models (Hedger and de Kretser DM, 2013). The aim of this research was to investigate the effect of activin A on the number, distribution and functions of macrophages in different compartments of the adult mouse testis, specifically the parenchyma and compartments like the rete testis, subcapsular region and tunica albuginea, using mice with reduced levels of either activin A or follistatin, by immunohistochemical studies. Additionally, macrophage subsets were studied in order to understand their functions in normal, infected and activin A-deficient mouse testes by immunofluorescence studies. Lastly, the functional regulation of the testicular macrophages was investigated by studying their gene expression profile compared to bone-marrow-derived macrophages in vitro. There was a significant decrease in the total number of F4/80+ interstitial macrophages (31% compared with litter-mate controls) and peritubular macrophages (49%) per testis in activin-deficient Inhba+/- mice. The volume densities of the peritubular macrophages and subcapsular macrophages were also significantly reduced (53% and 36%, respectively). When F4/80+ macrophage subsets were studied further using expression of CX3CR1 (a chemokine receptor involved in leukocyte migration), MHC class II molecules (involved in antigen presentation and a marker for activated macrophages), and CD206 (a mannose-specific scavenger receptor and anti-inflammatory marker), it was observed that the volume density of interstitial macrophages was 8-9-fold higher in the interstitial and peri-epithelial regions of the rete testis as compared to their corresponding populations in the parenchyma. The proportions of F4/80+CD206+ interstitial and peri-epithelial macrophages in the rete testis were 71% lower than interstitial macrophages in the parenchyma and 47% lower than peritubular macrophages in the parenchyma, respectively. Additionally, there was a 70% increase in rete testis interstitial macrophages expressing MHCII as compared to the parenchyma and a 10% increase in MHCII+ interstitial macrophages in the rete testis of activin A-deficient mice. When infected by uropathogenic bacteria, there was considerable increase of MHCII+ macrophages in the rete testis at day 10 post-infection, which later resolved by day 28. When highly-purified testicular macrophages were investigated in vitro, using multiplex RNAseq analysis, several key pro-inflammatory pathways, including PI3K-AKT, toll-like receptor 4 signaling, and some anti-viral responses were differentially expressed compared to bone marrow-derived macrophages. Testicular macrophages were unresponsive to stimulation by bacterial lipopolysaccharide (LPS), which correlated with low expression of genes involved in the LPS-regulated toll-like receptor (TLR4) signaling pathway (Tlr4, Cd14, Ly96, Tirap, Tram1), and higher expression of genes encoding proteins that inhibit TLR signaling via NF-κB (Chuk, Ptpn6, Sigirr, Ikbkb). Some transcripts encoding proteins involved in regulating anti-viral responses, including Irf5 and several interferon-stimulated gene transcripts (Ifitm2, Ifitm3, Ifi35, Oas1a, Oas1g, Oas3), also displayed lower expression in TMs, but other crucial anti-viral genes (Tlr3, Tlr7, Irf1, Irf3, Mx, Ddx58) were higher. Additionally, key regulators of mTOR/PI3K/AKT pathway that promote the anti-inflammatory/M2 phenotype (Akt1, Pten, Inpp5d, Tsc1, Pik3r2) were elevated in expression. Moreover, testicular macrophages expressed significantly higher transcripts of genes encoding proteins involved in antigen-presentation (MHC class II antigens, Cd80, Cd86, Ciita), anti-inflammatory genes (Il10, Socs1, Nfkbiz) and markers of alternatively activated, or M2, macrophages (Mrc1/CD206, Stat3, Stat6, Gata3, Egfr), relative to the BMMs. Overall, this analysis thereby showed several novel aspects of the anti-inflammatory nature of the testicular macrophages and their immunoregulatory roles. In conclusion, these studies have demonstrated that activin A regulates macrophage number and function in the adult mouse testis, and have identified distinct macrophage subsets in the rete testis that appear to be both immunoregulatory and tolerogenic. Additionally, testicular macrophages have a predominantly anti-inflammatory phenotype, and may lack the capacity to respond to some bacterial and viral infections. These studies considerably expand our understanding of the different macrophage subsets in the mouse testis, and their potential roles in regulating tolerance to sperm antigens and responses to infection.
Joint PhD from Monash University in Melbourne, Australia and Justus-Liebig University (JLU) in Giessen, Germany