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Macrophages are the major type of immune cell in the testis of both humans and rodents. Testicular macrophages (TMs) play critical roles in maintaining the testicular microenvironment, such as Leydig cell-dependent hormone production, spermatogenesis, and immune balance. A substantial number of studies have used rodent models to investigate the functions of TMs with various methods and harvest macrophages from the testis. Studies have demonstrated that enzyme digestion, an essential part of these methods, can improve the number and purity of TMs while unavoidably altering the immunoprofile of macrophages, which is detrimental for further study in terms of immune investigation. Here, we modified the existing method of microglia isolation and set up a novel method without the enzyme digestion step to isolate TMs. According to the characteristics of testicular tissue looseness and the physical and biological characteristics of macrophages, by combining mechanical separation, gradient centrifugation, and immuno-magnetic bead selection, we can effectively avoid the enzymatic digestion of testis tissue and maintain the immune characteristics of macrophages. Additionally, we verified the purity of TM with flow cytometry (FC) at approximately 91-95%, and the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was lower than that isolated with enzyme digestion. In contrast to the traditional method, this novel protocol can assist those who have no convenient access to fluorescence-activated cell sorting (FACS) to isolate a sufficient number of TMs and, most importantly, avoid altering the immunoprofile of TMs without enzyme digestion., Competing Interests: Declaration of Competing Interest We declare that we have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 CD64 hi MHCII lo and CD64 lo MHCII hi 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., Competing Interests: The authors declare no competing interest.

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti-inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti-inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine-1-phosphate (S1P) in a S1P lyase (SPL)-dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro-inflammatory cytokines by suppressing nuclear factor-κB and mitogen-activated protein kinases signalling pathways. Furthermore, vitamin B6-reduced accumulation of S1P by promoting SPL activity. The anti-inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti-inflammatory mechanism of vitamin B6 and provided guidance on its clinical use., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Innate immune signaling in macrophages during viral infection is regulated by ISGylation, the covalent attachment of the ubiquitin-like protein interferon-stimulated gene 15 (ISG15) to protein targets. Here, we explored the role of ISGylation in the macrophage response to infection with Mycobacterium tuberculosis. In human and mouse macrophages, the E3 ubiquitin ligases HERC5 and mHERC6, respectively, mediated the ISGylation of the phosphatase PTEN, which promoted its degradation. The decreased abundance of PTEN led to an increase in the activity of the PI3K-AKT signaling pathway, which stimulated the synthesis of proinflammatory cytokines. Bacterial growth was increased in culture and in vivo when human or mouse macrophages were deficient in the major E3 ISG15 ligase. The findings expand the role of ISGylation in macrophages to antibacterial immunity and suggest that HERC5 signaling may be a candidate target for adjunct host-directed therapy in patients with tuberculosis. Editor's summary: The antimicrobial response in macrophages is regulated in part by a protein modification called ISGylation. Du et al. found that ISGylation mediated by the protein HERC5 (or the functional equivalent in mice, HERC6) enabled macrophages to respond to infection with the bacterium that causes tuberculosis. HERC5- or HERC6-mediated ISGylation of the phosphatase PTEN induced its degradation, thus alleviating its suppression of the PI3K-AKT signaling pathway and promoting the production of cytokines that facilitated clearance of the bacteria. The findings reveal a host-response mechanism against tuberculosis that might inform the development of improved treatments for patients. —Leslie K. Ferrarelli [ABSTRACT FROM AUTHOR]

Background: As deubiquitinases (DUBs), ubiquitin C-terminal hydrolase (UCH)-L1 has been shown to play a crucial role in regulating diverse biological processes. However, its function in macrophage polarization remains unclear. Methods: We performed in vivo and in vitro experiments to investigate the role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a kind of DUBs, in macrophage differentiation by using UCHL1-deficiency mice. Results: We demonstrated that LPS stimulation induced UCHL1 expression in macrophages. The deficiency of UCHL1 expression decreased the expression of CD80 and CD86 but increased the expression of CD206. The expression of TNF-α, IL-6, iNOS, and IL-10 was downregulated, while that of Arg1, Ym1, and Fizz1 was upregulated in UCHL1 deficient macrophages. Moreover, we observed that UCHL1 promoted the degradation of p110α through autophagy, but paradoxically increased the activity of AKT, thereby promoting polarization of macrophages into pro-inflammatory states. Conclusion: In this study, we identified UCHL1 as a positive regulator of M1 macrophage polarization. Our findings may help in developing therapeutic interventions for the treatment of inflammatory diseases and pathogenic infections. [ABSTRACT FROM AUTHOR]

Testicular macrophages (TM) are critical for the function of the testis by regulating homeostasis and inflammatory responses. However, the mechanisms by which TM fulfil these roles remain elusive. In this study, we explored the impact of two key testicular microenvironmental factors, namely 25-hydroxycholesterol (25HC), an oxysterol related to sex hormones and macrophage colony-stimulating factor (M-CSF), a factor crucial for macrophage survival and differentiation, on the regulation of the TM phenotype. Specifically, we examined their role in controlling the expression of the transcription factor interferon regulatory factor 7 (Irf7), a factor critical for maintaining the alternative macrophage phenotype. To achieve this, we used an in vitro bone marrow-derived macrophage (BMDM) model as a surrogate for TM to investigate the roles of 25HC and M-CSF in regulating the expression of Irf7 during the polarization of murine TM. M-CSF was identified as the main regulator of Irf7 expression, while 25HC production is a consequence of Irf7 activation in BMDM. In turn, 25HC plays a role in a negative feedback loop on the expression levels of Irf7 in BMDM. Using flow cytometry in Irf7 -/- mouse testis the CD64loMHChi TM subpopulation was found to be decreased. Together with lower IL-10 protein levels in Irf7 -/- TM this indicates a shift towards an M1-like macrophage profile. In summary, our data indicates that M-CSF could act as an inducer of high Irf7 expression levels in the mouse testis. However, the exact role of the high 25HC concentration in the testis in maintaining the local immune milieu still needs further study. • M-CSF-1regulates immunomodulatory profiles TM by modulating the expression of IRF7. • The phenotype of testicular macrophages is regulated by local factors. • Irf7 is essential for the immunomodulatory phenotype of macrophages. [ABSTRACT FROM AUTHOR]

NLRC3, a member of the NLR family, has been reported as a negative regulator of inflammatory signaling pathways in innate immune cells. However, the direct role of NLRC3 in modulation of CD4+ T-cell responses in infectious diseases has not been studied. In the present study, we showed that NLRC3 plays an intrinsic role by suppressing the CD4+ T cell phenotype in lung and spleen, including differentiation, activation, and proliferation. NLRC3 deficiency in CD4+ T cells enhanced the protective immune response against Mycobacterium tuberculosis infection. Finally, we demonstrated that NLRC3 deficiency promoted the activation, proliferation, and cytokine production of CD4+ T cells via negatively regulating the NF-κB and MEK-ERK signaling pathways. This study reveals a critical role of NLRC3 as a direct regulator of the adaptive immune response and its protective effects on immunity during M. tuberculosis infection. Our findings also suggested that NLRC3 serves as a potential target for therapeutic intervention against tuberculosis. [ABSTRACT FROM AUTHOR]

• Breast cancer has distinct immunoprofiles among different molecular types. • Tumor-associated macrophages display various and even opposite functions. • Fetal-derived tissue resident macrophages are versatile and re-polarized. • Recruited macrophages are polarized by tissue microenvironments. • Fetal-derived resident and recruited macrophages are two contrary populations in tumor. Macrophage is the important sentinel cell type of innate immune system, and bridge with the adaptive immune response via antigen presentation. Tissue-resident macrophages are universal in almost all organs and play essential roles in maintaining specific organ homeostasis, inflammation responses, and disease genesis, including tumorigenesis. Macrophage is generally divided into two extreme statuses, M1 and M2, with sophisticated continuous subtypes due to different stimuli and microenvironments. Tumor-associated macrophage (TAM) is regarded as the key factor related to the prognosis, staging, classification, and treatment strategy of various cancers. However, emerging evidence indicated potential opposite functions of TAM in different tumor models. Recent studies found that different originated resident macrophages show notably different profiles in the same tissue niche. More evidence pointed out that the strategies to repolarize the subtypes of TAM or resident macrophages are valuable in carcinoma treatments. In the breast cancer model, studies pointed that macrophages located differently in histology show obvious different cell markers and functions. In this review, we will illustrate the profiles of resident macrophages in breast cancer with various aspects, including origination, polarization, tumoricidal activity, tumorigenesis, and the factors that could regulate the functions of macrophages. [ABSTRACT FROM AUTHOR]