Tissue resident macrophages are the sentinel cells of most organs and play an essential role in normal organ homeostasis, inflammatory responses and various pathological diseases (Bleriot et al., 2020). Mouse tissue-resident macrophages arise during embryogenesis and are maintained by self-renewal in organs such as the brain, lung, and liver (Hoeffel et al., 2015, Ginhoux and Guilliams, 2016, Liu et al., 2019). In contrast, a proportion of embryonically-derived macrophages are replaced by bone marrow-derived monocytes in the intestine, heart and dermis (Epelman et al., 2014, Shaw et al., 2018, Liu et al., 2019). Once they have colonized a particular organ, macrophages are governed by tissue-specific molecular cues that regulate their phenotype. Like other organ-resident macrophages, mouse testicular macrophages (TM) arise from fetal or neonatal monocytes and form a heterogeneous population, maintaining themselves in adult life with minimal contribution from blood monocytes (Lokka et al., 2020, Wang et al., 2021, Gu et al., 2023). In addition, murine TM play a critical role in maintaining the immune privilege of the testis by exhibiting hyporesponsiveness to inflammatory cues (Winnall et al., 2011, Bhushan et al., 2015, Wang et al., 2017). Stimulation with lipopolysaccharide (LPS) skews murine TM towards an alternative macrophage phenotype, characterized by secretion of low levels of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) with concomitant high levels of the anti-inflammatory cytokine interleukin (IL)–10 (Bhushan et al., 2015, Wang et al., 2017).

Although murine TM show a suppressed inflammatory response to protect testicular integrity, it is not known how local testicular factors influence the TM phenotype. Numerous immunomodulatory molecules including corticosterone, testosterone, prostaglandins, 25-hydroxycholesterol (25HC), and activins are present in the mouse testis, all of which may putatively influence the TM phenotype by inducing a unique set of transcription factors (TF) (Wang et al., 2017, Meinhardt et al., 2018, Zhang et al., 2020). Our previous study demonstrated that corticosterone, testosterone, and prostaglandins polarized granulocyte macrophage colony stimulating factor (GM-CSF)-derived macrophages towards an alternative macrophage phenotype (Wang et al., 2017, Meinhardt et al., 2018). However, a putative role for 25HC in shaping the murine TM phenotype is not known. 25HC was initially found to serve critical antiviral functions and to modulate inflammatory responses (Canfran-Duque et al., 2023). Moreover, 25HC has been shown to regulate macrophage functions in a context-dependent manner, with both pro-inflammatory and anti-inflammatory effects observed in mouse species (Cao et al., 2020, Wong et al., 2020, Canfran-Duque et al., 2023). In this regard, 25HC inhibited the production of IL-1β in vivo, whereas at high concentrations in vitro 25HC increased the production of the pro-inflammatory cytokines IL-6 and IL-8 (Wong et al., 2020).

Our previous study comparing the transcriptomic profiles of mouse TM and brain microglia showed that the mRNA level of the transcription factor interferon regulatory factor 7 (Irf7) was highly differentially expressed in TM (Wang et al., 2021). Irf7 belongs to a group of type I IFN-induced genes that control the progression of diseases by modulating the production of type I IFNs (Wu et al., 2019). Previous reports identified Irf7 as critical for maintaining the alternative macrophage phenotype in mice. Correspondingly, silencing of Irf7 by small interfering RNA in monocytes resulted in the production of high levels of pro-inflammatory cytokines and concomitant low levels of anti-inflammatory cytokines after LPS stimulation (Cohen et al., 2014). Thus, according to our previous work and other studies, we hypothesized that the oxysterol 25HC may play a critical role in the polarization of murine TM through the regulation of Irf7.