Regulatory Macrophages Inhibit Alternative Macrophage Activation and Attenuate Pathology Associated with Fibrosis This information is current as Prabha Chandrasekaran, Salman Izadjoo, Jessica Stimely, of September 25, 2021. Senthilkumar Palaniyandi, Xiaoping Zhu, Wagner Tafuri and David M. Mosser J Immunol published online 20 September 2019 http://www.jimmunol.org/content/early/2019/09/19/jimmun ol.1900270 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/09/20/jimmunol.190027 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published September 20, 2019, doi:10.4049/jimmunol.1900270 The Journal of Immunology Regulatory Macrophages Inhibit Alternative Macrophage Activation and Attenuate Pathology Associated with Fibrosis Prabha Chandrasekaran,*,† Salman Izadjoo,*,† Jessica Stimely,*,† Senthilkumar Palaniyandi,‡ Xiaoping Zhu,‡ Wagner Tafuri,x and David M. Mosser*,† Diversity and plasticity are the hallmarks of macrophages. The two most well-defined macrophage subsets are the classically activated macrophages (CAMfs) and the IL-4–derived alternatively activated macrophages (AAMfs). Through a series of studies, we previously identified and characterized a distinct population of macrophages with immunoregulatory functions, collectively termed regulatory macrophages (RMfs). Although considerable advances have been made in understanding these various macrophage subsets, it is not known whether macrophages of one activation state can influence the other. In this study, we examined whether RMfs capable of inhibiting inflammatory responses of CAMfs could also inhibit AAMfs and their profi- brotic responses. Our results demonstrated that RMfs significantly dampened the alternate activation phenotype of AAMfs Downloaded from generated in vitro and intrinsically occurring AAMfs from TACI2/2 macrophages. Further, RMfs inhibited AAMf-promoted arginase activity and fibroblast proliferation in vitro. This inhibition occurred regardless of the strength, duration, and mode of alternative activation and was only partially dependent on IL-10. In the chlorhexidine gluconate–induced peritoneal fibrosis model, AAMfs worsened the fibrosis, but RMfs rescued mice from AAMf-mediated pathological conditions. Taken together, our study demonstrates that RMfs are a specialized subset of macrophages with a nonredundant role in limiting overt pro- regenerative functions of AAMfs, a role distinct from their well-defined role of suppression of inflammatory responses by http://www.jimmunol.org/ CAMfs. The Journal of Immunology, 2019, 203: 000–000. acrophages (Mfs) are heterogeneous cells with varied exposure of murine Mfs to IL-4, IL-13, or helminth infections functions and transcriptional signatures (1–3). All Mfs, results in Mfs with a well-defined phenotype (7) that is profibrotic M regardless of their origin, can respond to danger signals in character and includes the expression of reliable biomarkers and assume an inflammatory phenotype. The (so-called M1) Mfs Ym1 and Relma (8). The M2 subset of Mfs has been loosely produce a vast armamentarium of inflammatory cytokines, che- expanded to encompass a wide range of Mfs with noninflam- mokines, and mediators. Classically activated Mfs(CAMfs) matory roles, including tissue remodeling, immunoregulation, and arise in response to a combination of danger signals and host angiogenesis (9). The expansion of this M2 designation has led by guest on September 25, 2021 IFNs, which are typically generated during a cell-mediated im- to considerable ambiguity in understanding the phenotype and mune response (4, 5). In 1992, Stein et al. (6) described a pop- functions of the variety of existing and emerging Mf subsets, ulation of “alternatively activated” Mfs (AAMfs), which arose in including tumor-associated Mfs (10), Mox Mfs that are associ- response to the TH2 cytokine IL-4. Mfs exposed to IL-4 failed to ated with the oxidized low-density lipoprotein–rich environment induce inflammatory cytokine production as CAMfsdid,but of atherosclerosis (11), and Mfs involved in the resolution of upregulated the expression of the mannose receptor and MHC immune responses (12). class II and were therefore termed alternatively activated (6). We had proposed a scheme to group Mfs based on their ho- Subsequent studies by many other groups have demonstrated that meostatic activities of host defense, wound healing, and immune regulation (3). This wider grouping followed our description of a population of Mfs with potent anti-inflammatory and immuno- *Department of Cell Biology and Molecular Genetics, University of Maryland, College † f f Park, MD 20742; Maryland Pathogen Research Institute, University of Maryland, regulatory activity that we termed regulatory M s(RM s) (3, 13–15). College Park, MD 20740; ‡Virginia-Maryland Regional College of Veterinary Med- x We subsequently demonstrated that there were many ways to icine, University of Maryland, College Park, MD 20470; and Departamento de generate Mfs with immunoregulatory and growth-promoting ac- Patologia Geral, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil tivity. The transcriptional programs of Mfs stimulated with three ORCIDs: 0000-0002-2916-280X (J.S.); 0000-0002-1672-9504 (X.Z.); 0000-0002- different “reprogramming” signals, namely, high-density immune 9503-4187 (D.M.M.). complexes (IC), PGE2, and Adenosine (Ade), were compared Received for publication March 7, 2019. Accepted for publication August 14, 2019. (16). All three of these populations of Mfs secreted lower levels This work was supported by grants from the National Institutes of Health (R01 of inflammatory mediators but higher levels of anti-inflammatory GM102589-01 and U01 AI088650). cytokines and growth and angiogenic factors. All three populations Address correspondence and reprint requests to Dr. David M. Mosser, University of of Mfs could confer some level of protection against lethal Maryland, Department of Cell Biology and Molecular Genetics, 3102 Bioscience endotoxemia (7, 16, 17). We, therefore, grouped them together as Research Building, College Park, MD 20742. E-mail address: [email protected] RMfs and conceded that each of these RMfs should be named The online version of this article contains supplemental material. by the stimulus that was used to generate it (1). Importantly, all Abbreviations used in this article: AAMf, alternatively activated Mf; Ade, adeno- f sine; BMDM, bone marrow–derived Mf; CAMf, classically activated Mf; CG, three of these RM s were transcriptionally quite distinct from chlorhexidine gluconate; IC, immune complex; LAde, LPS + Ade; LIC, LPS + IC; IL-4–treated AAMfs. f f f LPGE, LPS + PGE2;M , macrophage; PF, peritoneal fibrosis; RM , regulatory M ; Although there are several studies pertaining to Mf polariza- WT, wild-type. tion, only a very few have attempted to explore how Mfs of one Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 polarized state interact and influence the other under basal or www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900270 2 INHIBITION OF ALTERNATE ACTIVATION BY REGULATORY MACROPHAGES pathological conditions. In the current study, we investigated Fig. 1A), and the expression of these housekeeping genes did not differ how RMfs affect the activation status, phenotype, and function under stimulation conditions (Supplemental Fig. 1B). of AAMfs. The results demonstrate that RMfs are capable of ELISA and Western blot inhibiting the alternate activation state of AAMfsandcanres- cuemicefromAAMf-mediated pathologic fibrosis. Mouse IL-10 and IL-12/23p40 were measured by Duoset ELISA kits (R&D Systems). Mfs were lysed with RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling, Danvers, MA). Anti-OVA Abs Materials and Methods added as IC during stimulation were cleared from protein lysates by in- cubating with Protein A Agarose beads (Santa Cruz Biotechnologies, Mice Dallas, TX) for 2 h at 4˚C. HRP-conjugated actin Ab, anti-Arg1, and rabbit In vitro experiments were performed using cells isolated from 6- to 8-wk- polyclonal Ab against mouse Ym1 were purchased from Santa Cruz old C57BL/6 female mice, and the fibrosis experiments were conducted Biotechnologies, Abcam, and Stemcell Technologies (Vancouver, Canada), using 8-wk-old C57BL/6 female mice, purchased from Taconic Biosciences respectively. 2/2 (Germantown, MD). TACI knockout mice (TACI )onaC57BL/6back- Arginase and urea assays ground were obtained from Dr. Mustafa Akkoyunlu (U.S. Federal Food and Drug Administration, Silver Spring, MD), as previously described (18). Arginase activity and urea levels were measured