Monocytes and macrophages comprise a variety of subsets with diverse functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1Msr1Ly6CF4/80Mac1 monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein β (C/EBPβ), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb haematopoietic cells. Adoptive transfer of SatM into Cebpb mice resulted in fibrosis. Notably, SatM are derived from Ly6CFcεRI granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6CFcεRI granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPβ licenses differentiation of SatM from their committed progenitor.

With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b⁺ monocytic cells (F4/80⁺, Gr-1⁻, Ly6C⁻, I-A/E(-/lo), CD11c⁻, CD115⁺, CXCR4⁺, CCR2⁺, CX₃CR1⁻) when cultured with a Lin⁻c-kit⁺ population from mouse spleen cells. The developed CD11b⁺ cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

The development of effective treatments against cancers is urgently needed, and the accumulation of CD8 T cells within tumors is especially important for cancer prognosis. Although their mechanisms are still largely unknown, growing evidence has indicated that innate immune cells have important effects on cancer progression through the production of various cytokines. Here, we found that () has an antitumor effect. An s.c. inoculated tumor model produced fewer IL-12 p40 macrophages and activated CD8 T cells within the tumors of mice compared with WT mice. In vitro studies also revealed that the IL-12 p40 expression was significantly lower in macrophages following their stimulation by toll-like receptor ligands, such as R848. Additionally, we found that BATF2 interacts with p50/p65 and promotes IL-12 p40 expression. In conclusion, has an antitumor effect through the up-regulation of IL-12 p40 in tumor-associated macrophages, which eventually induces CD8 T-cell activation and accumulation within the tumor.

Interleukin 34 (IL-34) constitutes a cytokine that shares a common receptor, colony-stimulating factor-1 receptor (CSF-1R), with CSF-1. We recently identified a novel type of monocytic cell termed follicular dendritic cell-induced monocytic cells (FDMCs), whose differentiation depended on CSF-1R signaling through the IL-34 produced from a follicular dendritic cell line, FLY. Here, we report the functional mechanisms of the IL-34-mediated CSF-1R signaling underlying FDMC differentiation. CRIPSR/Cas9-mediated knockout of the Il34 gene confirmed that the ability of FLY cells to induce FDMCs completely depends on the IL-34 expressed by FLY cells. Transwell culture experiments revealed that FDMC differentiation requires a signal from a membrane-anchored form of IL-34 on the FLY cell surface, but not from a secreted form, in a direct interaction between FDMC precursor cells and FLY cells. Furthermore, flow cytometric analysis using an anti-IL-34 antibody indicated that IL-34 was also expressed on the FLY cell surface. Thus, we explored proteins interacting with IL-34 in FLY cells. Mass spectrometry analysis and pulldown assay identified that IL-34 was associated with the molecular chaperone 78-kDa glucoseregulated protein (GRP78) in the plasma membrane fraction of FLY cells. Consistent with this finding, GRP78-heterozygous FLY cells expressed a lower level of IL-34 protein on their cell surface and exhibited a reduced competency to induce FDMC differentiation compared with the original FLY cells. These results indicated a novel GRP78-dependent localization and specific function of IL-34 in FLY cells related to monocytic cell differentiation. Colony-stimulating factor-1 receptor (CSF-1R) 2 activation promotes the proliferation, survival, and differentiation of mononuclear phagocytes, such as macrophages, Langerhans cells, and osteoclasts (1-4). Specifically, binding of CSF-1 to CSF-1R can induce the activation of the tyrosine kinase activity of CSF-1R through the phosphorylation of seven tyrosine residues (Tyr 559 , Tyr 697 , Tyr 706 , Tyr 721 , Tyr 807 , Tyr 921 , and Tyr 974) of the cytoplasmic domain, followed by activation of signal transduction cascades including Src/Pyk2 and PI3K pathways (5, 6). Accordingly, CSF1 op/op mice bearing a null mutation in the Csf1 gene exhibit osteoperitrotic phenotypes including toothlessness, skeletal defects, and impaired development of macrophages and osteoclasts (7). However, the phenotype of CSF-1Rdeficient mice is more severe than that of the CSF1 op/op mice; for example, Langerhans cells and microglia are completely absent in CSF-1R-deficient mice (8, 9). Interleukin 34 (IL-34) has been identified as an alternative ligand for CSF-1R (10). IL-34 exhibits a similar stimulating activity for CSF-1R as the primary identified ligand, CSF-1 (11), although these two proteins share no sequence homology. Moreover, Il34 gene expression driven by the Csf1 promoter was shown to rescue the bone, osteoclast, tissue macrophage, and fertility defects of CSF1 op/op mice; thus, IL-34 is consider...

Growing evidence has revealed that the transcription factor basic leucine zipper transcription factor ATF-like 2 (BATF2) has unique transcriptional activities, including regulating cytokines via TLR signals in macrophages, which affect mortality due to infection and cancer. On the basis of genome-wide analyses using the chromatin immunoprecipitation-sequencing technique, we found that dual-specificity phosphatase 2 (Dusp2) had a significantly lower acetyl-histone status in Batf2-/- bone marrow-derived macrophages (BMDMs) compared with wild-type (WT) BMDMs. The phosphatase DUSP2 has been reported to play a critical role in inflammatory responses. Therefore, we evaluated the BATF2 transcriptional activities on the Dusp2 promoter. We found that the DUSP2 and IL-12 p40 expression levels were significantly lower in Batf2-/- BMDMs than in WT controls following their stimulation with TLR7 ligands. Further in vitro studies revealed that phospho-STAT3 was up-regulated and NF-κB p50/p65 were down-regulated in Batf2-/- BMDMs compared with their levels in WT controls. Additionally, Th1 immunity was impaired in Batf2-/- mice following their stimulation with TLR7 ligands. We also found that BATF2 interacts with NF-κB p65 and promotes DUSP2 expression through the NF-κB-binding site in the Dusp2 promoter at -203 to -121. Collectively, our findings suggest that BATF2 activates DUSP2 gene expression and up-regulates NF-κB activity via phospho-STAT3 dephosphorylation.

Schlafen-8 (Slfn8) is a member of the Schlafen family of proteins, which harbor helicase domains and are induced by LPS and interferons. It has been reported that the Schlafen family are involved in various cellular functions, including proliferation, differentiation and regulation of virus replication. Slfn8 has been implicated in T-cell differentiation in the thymus. However, the roles of Slfn8 in the immune system remains unclear. In this study, we generated Slfn8 knockout mice (Slfn8-/-) and investigated the immunological role of Slfn8 using the T-cell-mediated autoimmune model experimental autoimmune encephalomyelitis (EAE). We found that the clinical score was reduced in Slfn8-/- mice. IL-6 and IL-17A cytokine production, which are associated with EAE onset and progression, were decreased in the lymph nodes of Slfn8-/- mice. Immune cell populations in Slfn8-/- mice, including macrophages, neutrophils, T cells and B cells, did not reveal significant differences compared with wild-type mice. In vitro activation of Slfn8-/- T cells in response to TCR stimulation also did not reveal significant differences. To confirm the involvement of non-hematopoietic cells, we isolated CD45- CD31+ endothelial cells and CD45-CD31- gp38+ fibroblastic reticular cells by FACS sorting. We showed that the levels of IL-6 and Slfn8 mRNA in CD45- CD31+ endothelial cells were increased after EAE induction. In contrast, the level of IL-6 mRNA after EAE induction was markedly decreased in CD31+ endothelial cells from Slfn8-/- mice. These results indicate that Slfn8 may play a role in EAE by regulating inflammation in endothelial cells.