A Novel Subset of Anti-Inflammatory CD138 + Macrophages Is Deficient in Mice with Experimental Lupus This information is current as Shuhong Han, Haoyang Zhuang, Stepan Shumyak, Jingfan of October 2, 2021. Wu, Hui Li, Li-Jun Yang and Westley H. Reeves J Immunol 2017; 199:1261-1274; Prepublished online 10 July 2017; doi: 10.4049/jimmunol.1700099 http://www.jimmunol.org/content/199/4/1261 Downloaded from References This article cites 71 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/199/4/1261.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology A Novel Subset of Anti-Inflammatory CD138+ Macrophages Is Deficient in Mice with Experimental Lupus Shuhong Han,* Haoyang Zhuang,* Stepan Shumyak,* Jingfan Wu,* Hui Li,† Li-Jun Yang,† and Westley H. Reeves* Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MF). Impaired apoptotic cell uptake by MF also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MF. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predom- inantly of a novel subset of highly phagocytic MF resembling small peritoneal MF (SPM) that expressed CD138+ and the Downloaded from scavenger receptor Marco. Treatment with anti-Marco–neutralizing Abs and the class A scavenger receptor antagonist polyino- sinic acid inhibited phagocytosis of apoptotic cells by CD138+ MF. CD138+ MF expressed IL-10R, CD206, and CCR2 but little TNF-a or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alterna- tively activated MF. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear http://www.jimmunol.org/ apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation. The Journal of Immunology, 2017, 199: 1261–1274. acrophages (Mf) play a key role in the noninflamma- receptors mediating uptake, and factors regulating the sorting of tory disposal of apoptotic cells (1). Monocyte-derived apoptotic cells after phagocytosis or the coupling of phagocytosis M Mf from systemic lupus erythematosus (SLE) patients to anti-inflammatory pathways (11–14). By overwhelming normal are poorly phagocytic (2) and patients accumulate apoptotic cells in clearance mechanisms, an increased rate of cell death also may their tissues (3–6). Dead cells also accumulate in tissues of mice promote lupus (15–19). with pristane-induced lupus (6), but not in mice treated with min- We show impaired clearance of dead cells by lupus bone marrow by guest on October 2, 2021 eral oil (MO), an inflammatory hydrocarbon that does not cause (BM) Mf and report a novel subset of peritoneal syndecan-1 lupus. Impaired phagocytosis of apoptotic cells promotes murine (CD138)+ Mf with an anti-inflammatory phenotype that efficiently lupus (7–9). Although phagocytosis is usually noninflammatory takes up apoptotic cells in the peritoneum. This subset is deficient (8, 9), impaired phagocytosis of dead cells in lupus facilitates in mice with pristane-induced lupus, resulting in impaired apo- endosomal recognition of self-nucleic acids by TLR7 and TLR9, ptotic cell clearance and inflammation. resulting in proinflammatory cytokine production (10). The out- come of phagocytosis (pro- versus anti-inflammatory) depends on Materials and Methods the release of damage-associated molecular patterns by dying cells, Patients whether the cells are apoptotic or necrotic, the type of phagocyte, BM core biopsies were identified from the University of Florida (UF) Department of Pathology archives. SLE was classified using American *Division of Rheumatology and Clinical Immunology, Department of Medicine, College of Rheumatology criteria (20, 21). Biopsies from adults with acute University of Florida, Gainesville, FL 32610; and †Department of Pathology, Immu- myelogenous leukemia (AML) undergoing myeloablation with cytarabine nology and Laboratory Medicine, University of Florida, Gainesville, FL 32610 plus daunorubicin 14-d earlier and children with B cell acute lymphocytic leukemia (B-ALL) treated with vincristine, prednisone, anthracycline, plus ORCID: 0000-0001-5182-5369 (J.W.). cyclophosphamide and/or L-asparaginase 8-d earlier were de-identified and Received for publication January 23, 2017. Accepted for publication June 12, 2017. examined by H&E staining and immunohistochemistry (IHC). The patients This work was supported by National Institute of Arthritis and Musculoskeletal and were not treated with radiation and did not receive cytokines or growth Skin Diseases/National Institutes of Health Research Grant R01-AR44731 and a factors in the week before BM biopsy. Biopsies in which marrow cellu- grant from the Lupus Research Institute. larity dropped from 100% to ,5% following myeloablation were selected Address correspondence and reprint requests to Dr. Westley H. Reeves, Division of for further study (n = 4). BM biopsies from patients undergoing myeloa- Rheumatology and Clinical Immunology, University of Florida, PO Box 100221, blation were compared with biopsies from SLE patients (n = 6) and con- Gainesville, FL 32610-0221. E-mail address: whreeves@ufl.edu trols undergoing BM biopsy for staging of lymphoma who had no evidence Abbreviations used in this article: AML, acute myelogenous leukemia; BAL, bron- of BM involvement (n = 6). The UF Institutional Review Board approved choalveolar lavage; B-ALL, B cell acute lymphocytic leukemia; BM, bone marrow; these studies. CD138, syndecan-1; CD138+ Mf, CD11b+F4/80intCD138+Tim42 subset; IHC, im- munohistochemistry; LPM, large peritoneal MF;Mf, macrophage; MFI, mean fluo- Immunohistochemistry rescence intensity; MHCII, MHC class II; MO, mineral oil; PDE4, phosphodiesterase type 4; PEC, peritoneal exudate cell; PKA, protein kinase A; Poly-I, polyinosinic BM core biopsies were fixed in 10% neutral buffered formalin and decalcified acid; Q-PCR, quantitative PCR; R, region; SLE, systemic lupus erythematosus; SPM, (6). Four-micrometer sections were deparaffinized and underwent heat- small peritoneal MF; UF, University of Florida. induced epitope retrieval before staining with anti–cleaved-caspase-3 (Cell Signaling), anti–TNF-a (Abcam), and anti-CD68 (Dako) Abs followed by Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 peroxidase- or alkaline phosphatase–conjugated goat secondary Abs (6). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700099 1262 CD138+ MF IN LUPUS Reaction product was visualized using ultraView DAB (brown) or Alkaline anti-CD11b, anti-Ly6C, anti-CD138, and anti-Ly6G Abs; washed; and Phosphatase Red Kits (Ventana). Slides were counterstained with hematox- fixed as above. ylin. Numbers of activated caspase-3+ cells (red) that did not colocalize with To assess the role of Marco in apoptotic cell uptake, IgG anti-Marco MF (brown) were determined as the mean number of red+brown2 cells per neutralizing Ab (ED-31; AbD Serotec) or isotype control were injected 1003 field (4 fields per patient). i.p. into MO-treated mice (100 mg/mouse). Thirty minutes later, pHrodo Red–labeled apoptotic cells were injected and uptake was determined 1.5 h Mice later as above. The class A scavenger receptor inhibitor polyinosinic acid (Poly-I) (Sigma) was used to further confirm the role of Marco. Poly-I (200 Mice were maintained under specific pathogen-free conditions at the UF mg/mouse) or PBS was injected i.p and 30 min later, pHrodo Red–labeled Animal Facility. C57BL/6 (B6) mice (Jackson Laboratory) received 0.5 ml apoptotic BW5147 cells were injected. Uptake was measured by flow of pristane (Sigma), MO (C.B. Fleet), 100 ng LPS from Salmonella enterica cytometry 1.5 h later. serotype Minnesota (Sigma), or PBS i.p. At indicated times, peritoneal In vitro phagocytosis of unopsonized polystyrene beads (3.2-mm di- exudate cells (PEC) were collected by lavage. Cells were analyzed within ameter) was measured by flow cytometry. PEC were obtained 1 wk after 1 h. Bronchoalveolar lavage (BAL) was performed after euthanizing the MO treatment and cultured for 1 h with PE-labeled polystyrene beads at a mice. A small incision was cut in the trachea and 1-ml PBS was injected 10:1 bead/cell ratio (DakoCytomation). Cells then were stained with anti- using a 20-gauge plastic feeding tube (Instech Laboratories). Lung CD11b, -Ly6C, -Ly6G, and -CD138 Abs. The percentage of CD11b+ washings were analyzed within 1 h. Animal studies were approved by the 2 2 2 2 2 Ly6G Ly6C CD138+ and CD11b+Ly6G Ly6C CD138 cells that had UF Institutional Animal Care and Use Committee. taken up PE-labeled beads was determined by flow cytometry.