Reticulon-1 and Reduced Migration Toward Chemoattractants By

Reticulon-1 and Reduced Migration toward Chemoattractants by Macrophages Differentiated from the Bone Marrow of Ultraviolet-Irradiated and This information is current as Ultraviolet-Chimeric Mice of October 1, 2021. Terence A. McGonigle, Amy R. Dwyer, Eloise L. Greenland, Naomi M. Scott, Kim W. Carter, Kevin N. Keane, Philip Newsholme, Helen S. Goodridge, Fiona J. Pixley and Prue H. Hart Downloaded from J Immunol published online 22 November 2017 http://www.jimmunol.org/content/early/2017/11/22/jimmun ol.1700760 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2017/11/22/jimmunol.170076 Material 0.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 22, 2017, doi:10.4049/jimmunol.1700760 The Journal of Immunology

Reticulon-1 and Reduced Migration toward Chemoattractants by Macrophages Differentiated from the Bone Marrow of Ultraviolet-Irradiated and Ultraviolet-Chimeric Mice

Terence A. McGonigle,* Amy R. Dwyer,† Eloise L. Greenland,† Naomi M. Scott,* Kim W. Carter,* Kevin N. Keane,‡ Philip Newsholme,‡ Helen S. Goodridge,x Fiona J. Pixley,† and Prue H. Hart*

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited Downloaded from monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV- chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to http://www.jimmunol.org/ chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity. The Journal of Immunology, 2018, 200: 000–000.

n both humans and experimental animals, exposure of the skin activated by UV irradiation, resulting in the transit of cells and to erythemal or chronic low-dose UV radiation (UVR) causes mediators to the draining lymph nodes. Published reviews have I a systemic immunosuppression such that responses to ex- extensively covered the numerous molecular and cellular changes perimental Ags applied to nonirradiated sites are reduced (1, 2). in UV-irradiated skin that have been implicated in downstream

UVR-induced systemic immunosuppression has been implicated suppression of both local and systemic immunity (1, 5). UVR- by guest on October 1, 2021 in the positive latitude gradients (higher latitude, less solar UVR, induced suppression of a response to a contact sensitizer applied more disease) reported for several chronic immune diseases such to a distant skin site can still be detected 1 mo after UVR exposure as multiple sclerosis and type 1 diabetes (3, 4), as well as growth (6). The long-lasting nature of UVR-induced immunosuppression of tumors at nonirradiated sites (1, 5). Many pathways in skin are suggests bone marrow (BM) stem and progenitor cell involvement, as does the season-of-birth effect reported for many autoimmune diseases (6, 7). In light of the suppressed immune *Telethon Kids Institute, University of Western Australia, West Perth, Western Aus- tralia 6872, Australia; †School of Biomedical Sciences, University of Western Aus- responses in UV-irradiated mice, analyses have concentrated on tralia, Western Australia 6009, Australia; ‡School of Biomedical Sciences, Curtin the development and activation of tolerogenic dendritic cells Health Innovation Research Institute, Curtin University, Perth, Western Australia x (DCs) and regulatory T and B cells (1, 5, 8–11), with little em- 6845, Australia; and Board of Governors Regenerative Medicine Institute, Cedars- Sinai Medical Center, Los Angeles, CA 90048 phasis on the effect of UV irradiation of skin on macrophages, a ORCIDs: 0000-0002-4422-1433 (A.R.D.); 0000-0002-4904-2872 (K.W.C.); 0000- cell type with diverse functions in tissue homeostasis and in- 0001-6248-7705 (K.N.K.); 0000-0002-1571-2532 (F.J.P.); 0000-0001-7207- flammation (12, 13). Previous studies have noted that there is a 6467 (P.H.H.). large influx of macrophages into UV-irradiated human and mouse Received for publication May 25, 2017. Accepted for publication October 18, 2017. skin (14, 15). It is proposed that the macrophages respond to This work was supported by the Cancer Council Western Australia (to P.H.H. and F.J.P.) UVB-induced ligands via their chemokine receptor CCR2, and in and the National Health and Medical Research Council, Australia (Grants 572660 and 1067209 to P.H.H.). neonatal skin, the macrophages accumulating in response to the The raw CEL files and processed data in this article have been submitted to the CCR2 ligand produce IFN-g (16) but are less efficient at pre- National Center for Biotechnology Information Gene Expression Omnibus (https:// senting skin Ags associated with UV damage (17). Two decades www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE98840) under accession number GSE98840. ago, it was reported that UV irradiation of skin reduced the phagocytosis and intracellular killing of mycobacteria by macro- Address correspondence and reprint requests to Prof. Prue H. Hart, Telethon Kids Institute, 100 Roberts Road, Subiaco, WA 6008, Australia. E-mail address: prue.hart@ phages isolated from the spleen and peritoneal cavity, and their telethonkids.org.au ability to produce NO (18). Thus, there is some evidence that The online version of this article contains supplemental material. macrophage function is altered by UV-irradiation of skin. Abbreviations used in this article: BM, bone marrow; DC, dendritic cell; ECAR, The ability of macrophages to respond to chemoattractants and extracellular acidification rate; EGF, epidermal growth factor; EGFR, EGF receptor; inflammatory signals is important for their attraction to inflam- UVR, UV radiation. matory sites and localization to sites of immune activity. Most Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 tissue macrophages have their origin prenatally in yolk sac and fetal

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700760 2 BASIS OF POOR MIGRATION BY MACROPHAGES FROM UV MICE liver, but monocyte-derived macrophages that respond during in- on BM-differentiated macrophages, a mouse monoclonal IgG (MON162) to flammation in postnatal life differentiate from the BM (19). This reticulon-1A (ab9274) (Abcam, Cambridge, MA) was used. After washing, links with studies in our laboratory of chimeric mice; to create these cells were incubated with an AF647 goat anti-mouse IgG (Ab1505115; Abcam). For intracellular staining of reticulon-1, BM-differentiated macro- them, BM cells are transplanted into BM-ablated naive, recipient phages were incubated in fixation/permeabilization buffer and fixed for mice. The chimeric model allows the study of changes to BM cells 30 min at room temperature. The cells were then washed, and Ab staining without the ongoing influence of UVR-induced changes to cells in was performed as above in permeabilization buffer (eBioscience, San Diego, the periphery of donor mice. In the recipient mice, after ∼12 wk, CA). Data were collected on a BD LSR-II flow cytometer (BD Biosciences), and flow cytometric analyses were performed using FlowJo software (ver- the progeny of the transferred BM cells have populated all tissues. sion 10.0; Tree Star, Ashland, OR). We have previously shown that, in both UV-irradiated mice and UV-chimeric mice, DCs differentiating from the BM are poorly Macrophage chemotaxis assay to CSF-1 and CCL2 immunogenic. Further investigations suggested that DCs differ- Replicate cultures of BM-differentiated macrophages (2.5 3 105) were entiating from the BM of UV-chimeric mice had a reduced ability seeded into transwell inserts with 8-mm pores (BD Biosciences) in 200 ml upon activation to migrate to draining lymph nodes, resulting in of RPMI 10 (CSF-1– or CCL2-free), and inserts were placed into a 24-well dampened immune responses (6, 20). companion plate with RPMI 10 containing 120 ng/ml CSF-1 or 20 ng/ml CCL2, respectively. Where indicated, BM-macrophages were resuspended Because macrophages and DCs differentiate from a common in RPMI 10 containing the anti-mouse reticulon-1A IgG (5 mg/ml) or, as a hematopoietic precursor in the BM (21), we used a similar ex- control, an anti-mouse IgG (5 mg/ml) prior to seeding into transwell in- perimental model to examine the effect of UVR on macrophages serts. The cells were allowed to migrate for 5 h before fixation in 4% differentiating from the BM of both UV-irradiated mice and UV- paraformaldehyde. The cells were then stained for 5 min with NucBlue chimeric mice. A reduced chemotactic response to CSF-1 and fixed cell stain (Life Technologies, VIC, Australia), as per manufacturer’s

instructions, and imaged using a Nikon C2 Plus confocal microscope Downloaded from CCL-2 was measured. Furthermore, the poor mobility was de- (Nikon, Tokyo, Japan). The number of migrated cells was counted for 10 tected even if the macrophages were activated with LPS. representative fields per sample at 203 magnification, and results were Monocytes/macrophages differentiated in vivo from the BM of normalized to the number of migrated control cells. Where indicated, BM- UV-chimeric mice were also inefficient in responding to an in- macrophages were stimulated for 24 h with 1 mg/ml LPS from Escherichia coli 0111:B4 (Sigma-Aldrich, St. Louis, MO). flammatory signal; significantly fewer accumulated in the peritoneal cavity following injection of thioglycollate or alum. In these Generating UV-chimeric mice studies, the basis for the functional changes in the macrophages http://www.jimmunol.org/ Donor BM cells were isolated from congenic B6.SJL-Ptprca (CD45.1 al- was sought by microarray, flow cytometry, Western blotting, im- loantigen) mice that had been administered 8 kJ/m2 UVR to their shaved munofluorescence microscopy, and use of a blocking Ab to backs 3 d previously (6). BM cells from tibias and femurs were dis- reticulon-1 in assays of macrophage migration. aggregated and passed through sterile cotton wool to remove bone debris. RBCs were lysed with ammonium chloride. The recipient mice were 8-wk-old C57BL/6J mice (CD45.2 alloantigen) that were gamma- Materials and Methods irradiated (2 3 550 rad) using a 137Cs source (Gammacell 3000 Elan; Mice and ethics statement MDS Nordion, Ottawa, ON, Canada) prior to injection of 2 3 106 donor BM cells. Control chimeric mice were gamma-irradiated and injected with All experiments were performed with the approval of the Telethon Kids In- 2 3 106 BM cells from naive congenic B6.SJL-Ptprca mice (shaved but stitute Animal Ethics Committee (approvals 278, 296), and standard operating not UV-irradiated). The engraftment of cells in the chimeric mice was by guest on October 1, 2021 procedures for euthanasia and anesthesia defined according to guidelines of the tracked for 12 wk in the BM, spleen, and lymph nodes (6, 20). National Health and Medical Research Council of Australia. Female C57BL/6J (CD45.2 alloantigen) and B6.SJL-Ptprca (CD45.1 alloantigen) mice were Assay of monocyte/macrophage migration into the peritoneal obtained from the Animal Resources Centre (Murdoch, WA). cavity UV irradiation of mice The migration capabilities of monocytes/macrophages into the peritoneal A bank of TL40W/12RS lamps (Philips, Amsterdam, the Netherlands) cavity of control- and UV-chimeric mice (12–15 wk since injection of emitting broadband UVR with 65% UVB (280–320 nm) and peak emission donor cells for their establishment) were examined by i.p. injection of 1 ml at 313 nm was used as previously described (22). UVR (8 kJ/m2) was thioglycollate (3.8% Medium Brewer Modified; BD Biosciences), 0.2 ml delivered; this is equivalent to three to four minimal erythemal doses and is alum (aluminum hydroxide; 2 mg/mouse) (SERVA Electrophoresis, measured by skin edema. Heidelberg, Germany) or 1 ml 0.9% saline. After 3 d, the peritoneal cavity was washed out with saline. The harvested cells were counted and ob- Culture of BM cells for differentiation of macrophages jectively identified (26) by staining with fluorescently labeled Abs directed against F4/80, CD11b, CD11c, and Gr1. BM-derived macrophages were isolated as previously described with some modifications (23). In brief, BM cells were collected from tibias and fe- Measurement of cytokine and chemokine production murs of mice and cultured in RPMI 1640 (HyClone; GE Health Care Life Sciences, Logan, UT) supplemented with 10% FCS (HyClone) (RPMI 10) Cytokines in culture supernatants were measured using Bio-Plex Pro Mouse and 0.6 ng/ml (50 IU/ml) CSF-1 (kind gift of Dr. E.R. Stanley) for 24 h. Cytokine 23-plex panel (Bio-Rad Laboratories, Hercules, CA) as per the Nonadherent cells were then collected and cultured in RPMI 10 supple- manufacturer’s instructions. Before analysis, samples were diluted 1:2, mented with 12 ng/ml CSF-1 (1000 IU/ml) for 3 d in 10-cm Petri dishes to 1:5, or 1:25 using Bio-Plex mouse serum diluent (Bio-Rad Laboratories) as induce differentiation (some cells were frozen at day 2). To induce further recommended by the manufacturer. Prepared plates were run on a Luminex spreading and adhesion as they continue to differentiate into mature 100 instrument (Luminex, Austin, TX). The cytokines analyzed included macrophages, at day 4 the immature adherent macrophages were lifted IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40, IL-12 using 2 mM EDTA in PBS, reseeded at 105 cells/ml in 10 ml RPMI 10 p70, IL-13, IL-17, Eotaxin (CCL11), G-CSF, GM-CSF, IFN-g, KC, MCP- supplemented with 120 ng/ml CSF-1 (10,000 IU/ml), and cultured unless 1, MIP-1a, MIP-1b, RANTES, and TNF-a. stated otherwise for a further 5 d prior to use (total of 9 d in culture) (24, 25). Assays of macrophage lactate production and responses to Macrophage phenotyping glycolytic stress

Prior to staining of cultured BM cells, or cells isolated from the peritoneal Concentrations of secreted L-lactate (subsequently referred to as lactate) in cavity, they were incubated for 5 min with rat anti-mouse CD16/CD32 Fc culture supernatants were quantified using the Glycolysis Cell-Based As- receptor Ab to prevent nonspecific binding. Cells were then stained for 30 say Kit (catalog no. 600450; Cayman Chemical, Ann Arbor, MI). In brief, min with fluorescently labeled rat anti-mouse Abs: FITC anti-F4/80, PE or BM cells after 8 d in culture were lifted using 2 mM EDTA in PBS and allophycocyanin.Cy7 anti-CD11b, PE anti-CD11c, allophycocyanin anti- then recultured, in triplicate, in flat-bottom 96-well plates at a density of Gr1, allophycocyanin anti-CCR2 (CD192), or allophycocyanin anti–CSF-1R 0.8 3 106/ml in 120 ml RPMI 1640 medium containing 0.5% FCS and (CD115) (BD Biosciences, San Jose, CA). For surface staining of reticulon-1 120 ng/ml CSF-1. Samples were cultured with or without 1 mg/ml LPS, The Journal of Immunology 3 and supernatants were collected after 24 h. Supernatants were immediately previously described (31). Anti–reticulon-1A Ab was used at 1:50 dilution; centrifuged (1500 rpm, 5 min) to ensure that they were cell-free and frozen the secondary Ab was an Alexa 488–conjugated goat anti-mouse F(ab9)2 immediately at 280˚C. fragment Ab from Life Technologies (Invitrogen). Staining with DAPI Bioenergetics was measured using a Seahorse XFe96 Extracellular Flux (part of the mounting step) was performed using Prolong Diamond +DAPI Analyzer (Seahorse Bioscience, North Billerica, MA) and the Seahorse (Invitrogen). Mounted coverslips were examined under an Olympus 1X-81 Glycolytic Stress Test as previously reported (27). The extracellular inverted microscope with images recorded using a cooled FluoView II acidification rate (ECAR) along with the seeding density and reagents were CCD digital monochrome camera. optimized according to manufacturer’s specification. In short, 1.6 3 105 cells/well were cultured in RPMI 10 supplemented with 120 ng/ml CSF-1 for 24 h in poly-D-lysine–coated (Sigma-Aldrich) culture microplates Results (Seahorse Bioscience). After 24 h, the medium was replaced with serum- Yield and phenotype of cells differentiating from the BM of free, DMEM Base medium (1 mM sodium pyruvate, 2 mM glutamine, and UV-irradiated mice 0 mM glucose). The following were injected (at final concentration): 25 mM glucose, 1 mM oligomycin, and 100 mM 2-deoxyglucose. Three BM cells were harvested from nonirradiated mice or mice 3 d after measurements were taken after each drug addition (6 min apart); the last administration of 8 kJ/m2 UVR to their shaved backs, and cultured measurement prior to subsequent injection was used for the statistical with increasing concentrations of CSF-1. There were no signifi- calculations. ECARs were normalized according to protein levels in each cant differences in the number of BM cells harvested per mouse, well using radioimmunoprecipitation buffer and the bicinchoninic acid protein assay (Thermo Fisher). Data were analyzed as published previously or the rate of growth and differentiation of macrophages from BM (28), with ECAR initial measurements standardized to 100% and all sub- progenitors (Table I). There was also no difference in cell yields sequent changes calculated as a percentage change relative to that 100%. from the BM of control- and UV-chimeric mice (12 wk after their establishment), and from these cells cultured with increasing Microarray of BM-macrophages after differentiation for 9 d

concentrations of CSF-1 (Table I). After 9 d in culture, the cells Downloaded from Total RNA was extracted from macrophages differentiated for 9 d from BM were .98% F4/80+CD11b+. The expression of CD11c by har- using TRIzol (Life Technologies) followed by an RNeasy MinElute kit vested cells was low relative to that of BM-differentiated DCs (6). (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. At least 500 ng at 50 ng/ml (evaluated by the absorbance 260/280 ratio Migration of BM-macrophages toward CSF-1 and CCL2 using a NanoDrop ND 1000 spectrophotometer; NanoDrop Technologies, Wilmington, DE) was sent to the Ramaciotti Centre for Genomics, Uni- Although CSF-1 is necessary for macrophage growth and differ- versity of New South Wales, Sydney, NSW, Australia, for analysis using entiation, it is also a potent chemokine that stimulates macrophage Affymetrix GeneChip HT MG-430 PM Array. The Affy package from migration (24). The chemotaxis of fully differentiated (day 9) http://www.jimmunol.org/ Bioconductor 3.2 was used to read the Affymetrix CEL files into R 3.3.1 macrophages was investigated. In three independent experiments, (https://www.r-project.org/) and to preprocess the arrays using robust multichip average methodology. Quality control of the arrays was per- macrophages from the BM of UV-irradiated mice migrated sig- formed using the arrayQualityMetrics package (29). After initial explo- nificantly less efficiently than those differentiated from the BM of ration of all of the array data by PCA (total n = 32), we found the data nonirradiated mice (Fig. 1A, 1B). This was not due to differences primarily separated by LPS exposure, and thus we conducted the differ- in expression of the CSF-1R (CD115), which was expressed at ential gene analysis of UV versus control in the LPS (n = 16) and non-LPS (n = 16) environments separately. The limma package (30) was used to similar levels by macrophages differentiated from the BM of conduct differential expression analysis, with gene targets with an unad- nonirradiated and UV-irradiated mice (Fig. 1C). In three experi-

justed p # 0.05 considered of significance for further analyses. The raw ments, aliquots of macrophages were activated with LPS (1 mg/ml) by guest on October 1, 2021 CEL files and processed data are available from NCBI Gene Expression for 24 h before assessing their migration. Expression of the CSF- Omnibus under accession number GSE98840 (https://www.ncbi.nlm.nih. 1R was reduced by LPS exposure (Fig. 1C) and correlated with a gov/geo/query/acc.cgi?acc=GSE98840). decrease in the number of migrated cells (Fig. 1A, 1B). Thus, the Detection of reticulon-1, paxillin, and motility-related proteins by inefficient migration by macrophages from the BM of UV- Western blotting irradiated mice remained even after LPS stimulation. Migration Macrophages were grown to subconfluence (8-d culture) before CSF-1 toward CCL2 was also reduced for macrophages differentiated starvation for 16 h to upregulate CSF-1R expression and then incubated from the BM of UV-irradiated mice (6 LPS activation) (Fig. 1D). with or without 120 ng/ml CSF-1 at 37˚C for 2.5 min (24, 25). After in- CCL2 was originally called MCP-1 because of its ability to recruit cubation, the cells were washed twice with PBS (4˚C) and lysed by monocytes to sites of inflammation associated with tissue injury or scraping in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet infection (32). CCL2 has been implicated in the pathogenesis of P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mMZnCl2,500mMNa3VO4, 1 mM benzamidine, 10 mg/ml leupeptin, and 10 mg/ml aprotinin [pH 7.2]) diseases such as rheumatoid arthritis and atherosclerosis, which for SDS-PAGE analysis. Polyvinylidene difluoride membranes were in- are characterized by monocytic infiltrates. There were no signifi- cubated with HRP substrates (EMS-Millipore), and the chemiluminescent cant differences in CCR2 (CD192) expression by macrophages signal was detected with an ImageQuant LAS 4000 biomolecular imager differentiated from the BM of control or UV-irradiated mice (GE Healthcare, Piscataway, NJ). The Abs used included anti–reticulon- 1A as described earlier, anti-phosphotyrosine (4G10; EMS-Millipore, (Fig. 1E). Upon activation by LPS, CCR2 expression was not Billerica, MA), anti–b-actin (Sigma-Aldrich), anti-pY397 FAK (BD changed (Fig. 1E), and the cells differentiated from the BM of Transduction Laboratories, Lexington, KY), anti-pY402 Pyk2 (BD UV-irradiated mice continued to have reduced mobility toward Transduction Laboratories), anti-p85 PI3K (Millipore, Billerica, MA), CCL2 (Fig. 1D). anti-pS473 Akt (Cell Signaling Technology, Danvers, MA), anti-p44/42 Erk (Cell Signaling Technology), anti-paxillin (BD Transduction Labora- Migration of BM-macrophages from chimeric mice toward tories), and anti-phosphoY118paxillin (Invitrogen, Camarillo, CA). CSF-1 and CCL2 CSF-1R Ab (clone C19) was a kind gift from Dr. E.R. Stanley. HRP- conjugated secondary Abs were obtained from Cell Signaling Technology. To further analyze the longevity of the reduced migratory capacity of macrophages differentiating from the BM of UV-irradiated mice, Detection of reticulon-1 subcellular localization by we used BM cells from UV-irradiated mice to establish UV- immunofluorescence microscopy chimeric mice. Control-chimeric mice were created with BM BM-differentiated macrophages (9 d in culture) were seeded onto cells from nonirradiated mice. After 12 wk and ∼90% engraftment fibronectin-coated coverslips (BD Biosciences) and grown for 48 h before of donor CD45.1 cells into the BM, spleen, and peripheral lymph CSF-1 starvation for a further 16 h, then incubated with or without 120 ng/ml CSF-1 at 37˚C for 2.5 min. After stimulation, coverslips were se- nodes of recipient mice (Table II), macrophages were differenti- quentially fixed in 4% paraformaldehyde and permeabilized in 0.25% ated as described earlier from the BM of the chimeric mice, and Triton X-100 before staining for F-actin (Alexa568 phalloidin, 1:100) as their migratory properties were analyzed. As shown in Fig. 2A, 4 BASIS OF POOR MIGRATION BY MACROPHAGES FROM UV MICE

Table I. Macrophage yields from the BM of wild-type mice (6 UV), and control- and UV-chimeric mice, and after culture with CSF-1 in 10-cm Petri dishes

Wild-Type Mice Chimeric Mice

Cell Yields (mean 6 SEM), n $ 3 2UV +UV 2UV +UV BM harvest 3 106 40.9 6 2.0 40.1 6 1.2 67.0 6 5.8 66.0 6 3.5 Day 2 (yield 3106 per dish, 107 14.7 6 1.0 16.1 6 1.0 13.4 6 0.7 12.1 6 0.7 seeded per dish) Day 4 (yield 3106 per dish, 2 3 106 3.7 6 0.3 4.0 6 0.4 3.3 6 0.5 2.2 6 0.9 seeded per dish on day 2) Day 9 (yield 3106 per dish, 106 4.4 6 0.2 4.6 6 0.5 3.6 6 0.5 3.7 6 0.4 seeded per dish on day 4)

2B, and 2D, macrophages differentiated from the BM of UV- ∼90% similar to BM-differentiated macrophages at the gene ex- chimeric mice migrated less efficiently toward CSF-1 and pression level (33). Fully engrafted chimeric mice (12 wk since CCL2. Activation by LPS exposure significantly reduced CSF-1 BM cell transfer) were injected with 1 ml of 0.9% saline, 1 ml of receptor expression (Fig. 2C) but did not override or negate the thioglycollate (3.8% Medium Brewer Modified), or 2 mg of alum migration differences measured for macrophages differentiated in a volume of 0.2 ml. After 3 d, cells that had collected in the from BM of control- and UV-chimeric mice. In contrast, there peritoneal cavity were measured (Fig. 3). In response to thio- were no changes in CCR2 (CD192) expression on the macro- glycollate injection, a significantly greater number of cells were Downloaded from phages with LPS exposure, and macrophages differentiated from harvested from the control-chimeric mice than from the UV- BM of control- and UV-chimeric mice expressed similar levels of chimeric mice (Fig. 3A). There was a trend for a similar finding CCR2 (data not shown). in the peritoneal cavity of UV-chimeric mice injected with alum. The phenotyping of peritoneal cells harvested after saline and Responses to inflammatory stimuli injected into the peritoneal thioglycollate injection is shown in Fig. 3B and 3C, respectively. cavity of chimeric mice The number of macrophages in the peritoneal cavity of control- http://www.jimmunol.org/ To investigate the migratory capacity of monocytes/macrophages chimeric mice was greater than that measured in UV-chimeric differentiated in vivo, but still with an origin from the donor mice after injection of thioglycollate and alum; these were princi- BM of UV- or nonirradiated mice, we injected inflammatory pally defined as elicited macrophages (Fig. 3D, 3E). The responses mediators into the peritoneal cavity of chimeric mice. i.p. injection to thioglycollate and alum differed; other than large numbers of of inflammatory stimuli such as thioglycollate and LPS induces the macrophages, the former stimulated an influx of eosinophils, and recruitment of monocytes to the peritoneal cavity that differentiate the latter neutrophils (Fig. 3F, 3G). In summary, monocytes/ into macrophages. After LPS exposure, peritoneal macrophages are macrophages developing in vivo from the BM of UV-chimeric by guest on October 1, 2021

FIGURE 1. Reduced migration toward CSF-1 and CCL2 by macrophages differentiated from the BM of UV-irradiated mice. (A and B) Response to CSF- 1 (120 ng/ml) by macrophages differentiated for 9 d from the BM of nonirradiated and UV-irradiated mice (n = 3 independent experiments). Aliquots of cells were also exposed to LPS for the final 24 h of culture. (A) Migration of cells differentiated from the BM of UV-irradiated mice expressed as a proportion of those from the BM of nonirradiated mice. (B) As for (A), but the migration of macrophages 6 LPS has been expressed relative to the migration of cells from nonirradiated mice that were not exposed to LPS. (C) Expression of CSF-1R (mean fluorescence intensity [MFI]) by differentiated macrophages, half of which were incubated with LPS for the final 24 h of 9 d in culture. (D)Asfor(A), but macrophages were responding to CCL2 (20 ng/ml). (E) Expression of CCR2 (MFI) by differentiated macrophages, half of which were incubated with LPS for the final 24 h of 9 d in culture. (A, B, and D) Each point represents macrophages differentiated from the BM of a single mouse (two filters, 10 images per filter; mean 6 SEM). (C and E) The intensity (MFI) on cells differentiated from the BM of three mice (mean + SEM). An asterisk indicates a significant difference in migration (*p , 0.05) by cells differentiated from the BM of control and UV-irradiated mice. The Journal of Immunology 5

mice were poor to accumulate in the peritoneal cavity in response to different inﬂammatory agents; this result supports the ﬁndings 2.8 2.2 1.6 0.3 of the in vitro migration assays. 6 6 6 6 — — 89.9 97.0 99.3 92.9 Cytokine production by macrophages differentiating from the BM of UV-chimeric mice The possibility was investigated that autocrine production of 2.21.0 36.8 1.0 27.2 0.2 30.0 0.8 cytokines or chemokines was controlling the differences in mi- 6 6 6 6 — —

99.4 94.0 95.8 97.9 gration of the BM-differentiated macrophages from the control- C-chim UV-chim

Lymph Nodes and UV-chimeric mice. Following 8 d in culture, BM cells from six control-chimeric and six UV-chimeric were incubated for 24 h, with or without LPS (1 mg/ml), and 23 different cytokines and

1.40.8 35.9 1.2 27.5 0.2 31.8 0.7 chemokines were analyzed in the culture supernatants (Fig. 4A 6 6 6 6 0.0 0.0 0.0 6.2 WT and 4B for two potential macrophage chemokines). No differences in cytokine or chemokine levels were detected in the absence of LPS exposure. Generally, cytokine and chemokine levels were increased upon LPS exposure, and statistically sig- niﬁcant differences were recorded for some cytokines in super- 7.5 — 22.5 — 2.51.4 39.5 2.2 28.5 0.3 22.9 0.9 6 6 6 6 6 6 natants from cells differentiated from the BM of control- and Downloaded from 99.3 89.6 90.0 99.1

UV-chim UV-chimeric mice (up to a 10% change for some cytokines). However, because signiﬁcant differences in macrophage migratory potential were detected for both LPS-stimulated and non-LPS-exposed macrophages, it was unlikely that autocrine 4.5 77.5 18.6 106.5 1.90.6 55.4 0.6 10.0 0.8 13.3 2.9 cytokine production controlled differences in macrophage mi- 6 6 6 6 6 6 gration capabilities. Spleen 99.2 92.0 93.7 98.2 http://www.jimmunol.org/ SEM for three individual mice. C-chim

6 Lactate production by macrophages differentiated from the BM of UV-irradiated and UV-chimeric mice Because the cytoskeletal rearrangements necessary for migration 6.5 101.5 4.0 83.8 5.00.8 59.2 1.2 7.7 0.3 10.1 3.2 are one of the most energy-consuming cellular processes (34–36), 6 6 6 6 6 6 0.0 0.1 0.0 9.6

WT metabolic responses were analyzed. Levels of lactate, an end- product of glycolysis, were assessed upon incubation of the differentiated macrophages for 24 h in medium containing 0.5% FCS and 120 ng/ml CSF-1. LPS significantly stimulated by guest on October 1, 2021 6.4 74.5 1.7 103.2 0.90.4 41.8 0.1 10.3 1.2 12.2 1.9 lactate production and consequently glycolysis in macrophages 6 6 6 6 6 6 over 24 h, but there were no differences in the levels produced by 96.4 81.1 76.7 99.7 macrophages from the BM of nonirradiated and UV-irradiated mice, and control- and UV-chimeric mice, respectively (Fig. 4C, 4D). Macrophages differentiated from the BM of chimeric mice were also investigated in a glycolytic stress test using real-time met- 19.5 66.2 2.4 49.3 2.30.1 29.7 0.1 3.2 0.8 1.0 10.8 abolic flux analysis under CSF-1–free conditions (Seahorse Flux 6 6 6 6 6 6 BM C-chim UV-chim 97.4 85.7 83.1 99.6 Analyzer). LPS exposure increased the glycolytic response, but there were no differences detected between macrophages from the BM on control- and UV-chimeric mice (6LPS), confirming the absence of metabolic adaptions (Fig. 4E). 11.8 71.7 1.9 55.3 0.20.1 3.1 1.4 1.2 9.3 3.6 29.7 Microarray of genes expressed by macrophages differentiated 6 6 6 6 6 6 WT 0.9 5.5 0.10 0.04 from the BM of UV-chimeric mice 2.9 1.0 8.9 62.0 55.8 25.0 To further probe the mechanism underlying differences in migration both in vitro and in vivo by macrophages differentiating from the BM of control- and UV-chimeric mice, we examined mRNA from fully differentiated BM-macrophages (after 9 d in

4 culture) by microarray. Because exposure to LPS does not cancel donor cells into the BM, spleen, and lymph nodes of control- and UV-chimeric mice 12 12 12 12 12

+ differences in migration ability, cells that had been exposed to LPS, or not, for 24 h (between days 8 and 9 of differentiation) were Weeks Reconstituted analyzed by microarray; this approach allowed identiﬁcation of genes responsible for variations in migration against a background ) + 6 of different activation states. Microarray of RNA from cells under + 10

2 + different activation states also provided an inbuilt control for 3 B220 CD11b replication of results. RNA was extracted from eight paired pop- + + CD4 CD4 + + ulations of BM-differentiated macrophages, each exposed for 24 h to LPS or control medium. The paired cell preparations had dif- % CD45.1 % CD45.1 % CD45.1 % CD45.1 % CD19 % CD3 % CD3 % F4/80 Total cells ( Comparisons of cell yields and cell types from wild-type (WT; nonchimeric) mice are shown at 4 and 12 wk after injection of cells. Mean C-chim, control-chimeric; UV-chim, UV-chimeric. ferentiated from the BM of eight pairs of control- and UV-chimeric

Table II. Engraftment of CD45.1 mice, each pair euthanized on separate days between 12 and 15 wk 6 BASIS OF POOR MIGRATION BY MACROPHAGES FROM UV MICE

FIGURE 2. Reduced migration toward CSF-1 and CCL2 by macrophages differentiated from the BM of UV-chimeric mice. (A and B) Response to CSF-1 (120 ng/ml) by macrophages differentiated for 9 d from the BM of control- and UV-chimeric mice (UVc) (n = 4 independent experiments). In three experiments, aliquots of cells were also exposed to LPS for the final 24 h of culture. (A) Migration of cells differentiated from the BM of UVc expressed as a proportion of those from the BM of control-chimeric mice. (B) As for (A), but the migration of macrophages 6 LPS has been expressed relative to the migration of cells from control chimeric mice that were not exposed to LPS. (C) Expression of CSF-1R (mean fluorescence intensity [MFI]) by differentiated macrophages, half of which were incubated with LPS for the final 24 h of 9 d in culture. (D) As for (A), but macrophages were responding to CCL2 (20 ng/ml). (A, B, and D) Each point represents macrophages from the BM of a single Downloaded from mouse (two filters, 10 images per filter; mean + SEM). (C) The intensity (MFI) on cells from the BM of three mice (mean + SEM). An asterisk indicates a significant difference in migration (*p , 0.05) by cells differentiated from the BM of control-chimeric mice and UVc. http://www.jimmunol.org/ after establishing the chimeric mice. When the array data were When the genes with an unadjusted p # 0.05 were further fil- examined for subsets of genes that have a nominal (unadjusted) tered for a log2 fold change $0.4 (considered a biologically sig- p # 0.05 and were similarly regulated in cells both exposed and not nificant change), we found one gene, for reticulon-1, that was exposed to LPS, there were 15 genes that were reduced in macro- differentially expressed in the same direction in both the LPS- phages differentiated from the BM of UV-chimeric mice compared exposed and not exposed macrophages from the BM of UV- with those from the BM of control-chimeric mice, and 25 genes that chimeric mice compared with those from control-chimeric mice. were increased in expression (Supplemental Table I). In macrophages differentiated from the BM of UV-chimeric mice, by guest on October 1, 2021

FIGURE 3. Reduced accumulation of macrophages in the peritoneal cavity of UV-chimeric mice (UVc) after injection of inflammatory reagents. Control-chimeric mice (Cc) and UVc were injected i.p. with 1 ml of saline (n = 3 and 4), 1 ml of thioglycollate (3.8%) (n = 8 and 6), or 0.2 ml of alum (n = 3 and 3). Cells were harvested from the peritoneal cavity after 3 d. (A) Total cells. (B) Gating strategy for identification of peritoneal cells after saline injection. (C) Gating strategy for identification of peritoneal cells after thioglycollate injection. (D) Total number of peritoneal macrophages (resident and elicited). (E) Number of elicited macrophages. (F) Number of neutrophils. (G) Number of eosinophils. Data are mean 6 SEM. An asterisk indicates a significant difference (*p , 0.05) in cells harvested from the peritoneal cavity of Cc and UVc. The Journal of Immunology 7 Downloaded from

FIGURE 4. Cytokine and lactate production by macrophages differentiating from the BM of UV-irradiated or UV-chimeric mice (UVc). (A) MIP-1a and (B) MIP-1b in the ﬁnal 24 h culture supernatant of macrophages differentiated for 9 d from the BM of six control-chimeric mice and six UVc (6 LPS; six independent experiments). Data are mean + SEM. Lactate production by fully differentiated macrophages from the BM of (C) UV-irradiated mice and (D) UVc. Macrophages were incubated as triplicate wells for 24 h, and lactate was measured in cell-free supernatants; a mean value for cells from individual mice and then a mean value for six mice in the group were calculated. Means + SEM (n = 6) are shown. (E) ECAR by macrophages differentiated from the

BM of control-chimeric mice and UVc. LPS exposure for 24 h stimulated a greater ECAR response (open symbols), but there were no differences in http://www.jimmunol.org/ response by macrophages differentiated from the BM of control-chimeric mice (triangles) and UVc (circles). reticulon-1 mRNA levels were reduced by 27 and 36% for (2LPS) (for enhanced migration) and focal adhesions (for greater adhesion and (+LPS) cells, respectively. and reduced chemotaxis) (24, 37, 38). When normalized to the expression of actin, there were no apparent changes in the levels of Expression of reticulon-1 protein in macrophages differentiated phosphorylated motility-related proteins and signaling molecules from the BM of control- and UV-chimeric mice in macrophages differentiated from the BM of UV-chimeric mice Reticulon-1 expression was first examined by flow cytometry using compared with those from control-chimeric mice. However, an Ab to reticulon-1A previously recorded for staining macro- macrophages differentiated from the BM of UV-chimeric mice by guest on October 1, 2021 phages (http://www.ProteinAtlas.org/ENSG00000139970-Rtn1/ expressed significantly higher levels of paxillin (with propor- tissue). As shown in Fig. 5A and 5B, .90% BM-differentiated tionally similar amounts of expression of phosphorylated paxillin) macrophages after 9 d in culture expressed reticulon-1 on their after CSF-1 stimulation (Fig. 5D, Supplemental Fig. 1) and sug- surface, as well as intracellularly. Differences in expression were gested that CSF-1R signaling in these macrophages may stimulate not detected between cells from the BM of control- and UV- enhanced adhesion and, in turn, reduce their capacity to migrate chimeric mice. For examination by Western blotting, macro- toward a chemoattractant. phages differentiated for 8 d from the BM of four control- and four UV-chimeric mice were starved of CSF-1 for 16 h and then pulsed Reticulon-1 translocates to the plasma membrane upon CSF-1 for 2.5 min with 120 ng/ml CSF-1, or control medium, before lysis stimulation and analysis. Again, the total levels of reticulon-1 expression were Reticulon-3 was recently identified as a critical regulator of significant, and there were no detected differences (as a measure nonclathrin-mediated endocytosis of the epidermal growth factor of actin) in lysates from macrophages differentiated from the BM (EGF) receptor (EGFR) (39). EGF stimulated contact between the of control- or UV-chimeric mice (Fig. 5C, Supplemental Fig. 1), endoplasmic reticulum and the plasma membrane in a reticulon- confirming the earlier flow cytometry data. Unexpectedly, 3–dependent manner. We determined whether CSF-1 induced reticulon-1 levels were significantly reduced in all lysates of plasma membrane reticulon-1 localization by starving macro- BM-macrophages after the 2.5-min CSF-1 stimulation (Fig. 5C, phages differentiated from the BM of control-chimeric mice of Supplemental Fig. 1). The reduction in the reticulon-1 band for CSF-1 for 16 h and then pulsing them for 2.5 min with 120 ng/ml lysates of BM-macrophages from UV-chimeric mice was signifi- CSF-1 or control medium. As shown in Fig. 6, reticulon-1 local- cantly greater (49.8%) than that measured for BM-macrophages from ized to the F-actin–rich cell cortex following CSF-1 stimulation, control-chimeric mice (30.8%). These results suggest that reticulon-1 suggesting that CSF-1 triggers contact between the endoplasmic is involved in responses to CSF-1 because it is either activated and reticulum and the plasma membrane. downregulated or becomes inaccessible to the Ab through protein complex formation following activation of CSF-1R signaling path- Reticulon-1 is involved in macrophage migration toward CSF-1 ways. Importantly, reticulon-1 levels are reduced to a significantly Because reticulon-1 is expressed on the surface by most BM- greater extent in BM-macrophages from UV-chimeric mice. differentiated macrophages in the presence of CSF-1, its role in Lysates were also examined for expression of adhesion- and macrophage migration was investigated. In three independent migration-associated proteins (Supplemental Table II), particularly experiments examining the migration of macrophages toward CSF-1, as macrophage spreading, adhesion, and migration through the incubation with an Ab to reticulon-1 significantly reduced the ability extracellular matrix requires dynamic remodeling of the actin of BM-differentiated macrophages to migrate toward CSF-1 cytoskeleton associated with integrin clustering in podosomes (Fig. 7A). Incubation with the Ab to reticulon-1 also significantly 8 BASIS OF POOR MIGRATION BY MACROPHAGES FROM UV MICE Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021 FIGURE 5. Expression of reticulon-1 (RTN1) in macrophages differentiated from the BM of UV-chimeric mice (UVc). (A) Surface and intracellular flow cytometry profiles for RTN1 expression by BM-differentiated macrophages. (B) RTN1 expression (mean fluorescence intensity [MFI]) by macrophages differentiated from the BM of each of three control-chimeric mice and three UVc. Means + SEM are shown. (C and D) Western blots. Macrophages were differentiated for 8 d from the BM of four control-chimeric mice and four UVc. They were starved of CSF-1 for 16 h and then pulsed for 2.5 min with CSF-1 or medium before lysis. Band density for (C) RTN1 and (D) paxillin normalized to the expression of actin and then as a percentage of the value for macrophages from the BM of control-chimeric mice not pulsed with CSF-1. Means + SEM are shown. An asterisk indicates a significant difference (*p , 0.05) between macrophages exposed to CSF-1 or not, or macrophages differentiated from the BM of control-chimeric mice and UVc. reduced the ability of BM-differentiated macrophages prepared response to CSF-1 have sustained reduced migration in response to from four independent control-chimeric mice to migrate toward multiple chemoattractants. We sought mRNA differences in these CCL2 (Fig. 7B). This result suggests that reticulon-1 is required for BM-differentiated macrophages by microarray; reticulon-1 tran- macrophage migration toward both CSF-1 and CCL2, and if scripts were significantly reduced in macrophages from UV-chimeric reticulon-1 levels are reduced, macrophage migration toward mice, both in a basal state and after activation by LPS for 24 h. chemoattractants is impaired. Expression of reticulon-1 on the surface of BM-differentiated macrophages was demonstrated, and by use of a blocking Ab, it was Discussion confirmed that reticulon-1 was functionally important in macrophage Macrophages fully differentiated from the BM of UV-irradiated migration toward both CSF-1 and CCL2. When reticulon-1 was ex- and UV-chimeric mice, compared with those from nonirradiated amined by Western blotting in BM-macrophages after a CSF-1 pulse, and control-chimeric mice, migrate less efficiently in vitro to a levels were reduced more significantly in cells from UV-chimeric gradient of chemoattractants. The different behaviors could not be mice than from control-chimeric mice. The same CSF-1 pulse also explained by altered expression of the receptors for those che- induced translocation of reticulon-1 to the plasma membrane. moattractants (CSF-1, CCL2). Reduced migration was also seen in The reticulons are a group of membrane-associated proteins with the UV-chimeric mice after injection of thioglycollate or alum; in much experimentation supporting their involvement in shaping the both scenarios, the cells accumulating would have differentiated tubular endoplasmic reticulum network (41). There are four from monocytes and, in turn, their precursors in the BM (40). The members in mammals with varied N-terminal domains that help UV-chimeric mice were generated with BM from UV-irradiated mice confer specific functions. They are expressed in the endoplasmic and allowed analysis of the longevity of the effects of UVR on he- reticulum, Golgi, and plasma membranes, and can be involved in matopoietic precursor cells in the BM. The hypothesis proposed was secretory pathways, membrane trafficking, apoptosis, and with that in response to UVR, macrophage progenitor cells in BM were relevance to our study, cell migration. Reticulon-1 is an interacting altered epigenetically such that their progeny that differentiate in partner of the GTPase activation protein, TBC1D20 (42). The The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 6. CSF-1 stimulates translocation of reticulon-1 to the cell periphery. Macrophages differentiated for 9 d from the BM of control-chimeric mice were plated on fibronectin-coated coverslips for 2 d, including overnight CSF-1 starvation followed by CSF-1 stimulation (120 ng/ml) for 0 or 2.5 min, and stained for reticulon-1 localization (n = 2 independent experiments). The degree of reticulon-1 (green in merge) colocalization with cortical F-actin (red) at by guest on October 1, 2021 the cell periphery is shown in the merged panels and more clearly demonstrated in the insets. Nuclei are DAPI-stained (blue), dashed squares signify insert areas, and arrows indicate cell outlines. Scale bar, 10 mm. small GTPase activated by TBC1D20 is Rab1, a molecule that reticulon-1 will also localize to the cell membrane after a short can regulate integrin b1 recycling to lipid rafts to promote cell exposure to CCL2. Migratory responses toward thioglycollate and migration (43). In our microarrays, a significant reduction in alum in vivo were reduced when monocytes/macrophages were TBC1D20 transcripts was measured in non-LPS-exposed macro- differentiated from the BM of UV-chimeric mice. Thus, we pro- phages differentiated from the BM of UV-chimeric mice pose that reticulon-1 may be involved more broadly in monocyte (p = 0.03). The involvement of another reticulon, reticulon-4, in macrophage migration has been previously reported; macrophages from mice unable to express reticulon-4 had reduced spreading and chemotaxis (44). In our analyses, reticulon-4 transcripts were not differentially expressed in BM-macrophages from control- and UV-chimeric mice. A new role for reticulon-3 located at endoplasmic reticulum–plasma membrane contact sites has recently been described as a critical regulator of EGFR nonclathrin endocytosis (39). At higher concentrations of EGF, ∼40% of EGFRs use nonclathrin endocytosis to traffic to the lysosome for degra- dation, with reticulon-3 acting as a tethering factor between the endoplasmic reticulum–plasma membrane (39). FIGURE 7. Anti-mouse reticulon-1 IgG inhibits macrophage migration This study has shown for the first time, to our knowledge, that toward CSF-1 and CCL2. Macrophages were differentiated for 9 d from reticulon-1 is expressed both on the surface and intracellularly the BM of three (A) or four (B) control-chimeric mice before incubation of BM-differentiated macrophages. The macrophages required with the anti–reticulon-1 IgG (5 mg/ml) (anti-RTN1) or, as a control, a reticulon-1 to migrate sufficiently toward the chemoattractants, mouse IgG (5 mg/ml). Macrophages were seeded into transwell inserts, and their migration over 5 h toward CSF-1 (A) or CCL2 (B) was assessed. The CSF-1 and CCL2, supporting reticulon-1 as an important mem- migration of macrophages incubated with anti-RTN1 has been expressed brane protein. The Western blotting and immunofluorescence as a proportion of that of macrophages incubated with the control IgG. experiments also confirmed involvement of reticulon-1 in re- Each point represents the migration of macrophages from an independent sponses to CSF-1. Just as reticulon-3 can regulate the type of experiment (two filters, 10 images per filter; mean 6 SEM). An asterisk endocytosis induced by EGFRs (39), reticulon-1 may deter- indicates a significant difference in migration (*p , 0.05) between mac- mine responses of macrophages to CSF-1. We hypothesize that rophages incubated with the anti-RTN1 or control Ab. 10 BASIS OF POOR MIGRATION BY MACROPHAGES FROM UV MICE and macrophage responses to chemoattractants and inflammatory In summary, macrophages differentiated from the BM of UV- agents, and that surface expression of reticulon-1 is reduced in irradiated and UV-chimeric mice, compared with those from non- macrophages differentiated in vivo from the BM of UV-chimeric irradiated or control-chimeric mice, have an inherent reduced ability mice. The mechanisms by which reticulon-1 and migratory re- to migrate toward chemoattractants and inflammatory agents. Fur- sponses may be linked are the subject of further experimentation. thermore, the effect of UV exposure is long lasting with a sustained The outcomes of Western blotting may represent proteolytic reduction in macrophage migration toward chemoattractants for cleavage or decreased expression of reticulon-1 after 2.5 min of 12–15 wk in the UV-chimeric mice. Microarray analysis of non- CSF-1 exposure. However, the immunofluorescence staining stimulated and LPS-activated macrophages and Western blotting suggested that reticulon-1 moves within the cell. The Western support involvement of reticulon-1, a confirmed surface membrane blotting experiments also detected increased levels of paxillin, protein, in determining the differences in macrophage migration to with proportionally similar amounts of expression of phosphory- CSF-1 and CCL2, and we propose thioglycollate and alum. The lated paxillin, in lysates of macrophages from the BM of UV- studies using immunofluorescence microscopy suggested that chimeric mice. This suggests paxillin involvement in responses reticulon-1 moves around the cell and can translocate to the cell to CSF-1 by the less migratory BM-macrophages from UV- membrane in response to chemoattractants. Thus, future experi- chimeric mice, possibly through increased formation of adhesion ments should concentrate on the subcellular localization of structures (45). Links between reticulon-1 and paxillin in macro- reticulon-1, rather than measure levels of reticulon-1 in lysates from phages from the BM of UV-chimeric mice after a short exposure macrophages differentiated from the BM of both nonirradiated and to CSF-1, or CCL2, warrant further examination. control-chimeric mice, in direct comparison with those from UV- Activation of the BM-differentiated macrophages by LPS did not irradiated or UV-chimeric mice, respectively. Our current data (from help dissect the basis of different migratory responses. In fact, gene array analysis, migration response of macrophages incubated Downloaded from differences in response to CSF-1 and CCL2 were similar regardless with the anti–reticulon-1 Ab, and immunofluorescence microscopy) of the biochemical pathways activated by LPS. The possibility of suggest that there is less reticulon-1 localized to the cell membrane other functional differences in macrophages differentiated from the of macrophages differentiated from the BM of UV-chimeric mice, BM of control- and UV-chimeric mice were examined; there were although confirmatory experiments are required. From this study, we relatively small differences in cytokine and chemokine production report that macrophages in UV-irradiated mice are indeed slow to (with or without LPS), which suggested that their autocrine activity collect at a site of inflammation, and thus pathways stimulated by http://www.jimmunol.org/ was not at play. Differences in flux through activation pathways UV irradiation of skin may help regulate macrophage-associated were accounted for in the completed microarrays that examined outcomes during infections and inflammatory challenges. Whether inherent differences in the macrophages when not stimulated and these outcomes contribute to UVR-induced immunosuppression in after LPS exposure. The accumulation of cells in the peritoneal humans is a matter for further study. More broadly, reticulon-1, and cavity was also examined, and confirmed that the outcomes we pathways involved in determining its function, may become a new were measuring were not an artifact of the in vitro chemotaxis assay target for biochemical manipulation to regulate monocyte and with a semipermeable membrane. Instead, monocytes, macro- macrophage migration capabilities. phages, and other cells were being attracted to migrate into, and by guest on October 1, 2021 accumulate in, the peritoneal cavity. Acknowledgments Like BM-macrophages differentiated in vitro, monocyte-derived We thank Deborah Strickland for assistance creating the chimeric mice and peritoneal macrophages in UV-chimeric mice inefficiently mi- gratefully acknowledge Bright Blue for donation of the Nikon C2 confocal grated toward the chemoattractants thioglycollate and alum. We microscope. had proposed that differences in metabolic programming may influence different migratory responses by macrophages. However, Disclosures there were no differences in lactate production or metabolic flux The authors have no financial conflicts of interest. through glycolytic or mitochondrial processes, the latter performed in the absence of CSF-1. Furthermore, LPS could stimulate significant glycolytic activity, stimulated lactate production, and control References metabolic pathways in macrophages, but was unable to influence 1. Hart, P. H., S. Gorman, and J. J. Finlay-Jones. 2011. 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Reduced Expression +LPS -LPS Gene Name Log2Fold Probe Mean P.Value Log2Fold Probe Mean P.Value change Expression change Expression RIKEN cDNA 3110099E03 gene -0.2652 1.6075 0.0260 -0.2209 1.4986 0.0084 reticulon 1 -0.6622 3.8441 0.0095 -0.4624 4.6989 0.0154 centromere protein B -0.2669 2.4130 0.0036 -0.2017 4.0960 0.0269 collagen, type XVIII, alpha 1 -0.3028 4.5397 0.0057 -0.2760 5.5336 0.0251 EGF-like repeats and discoidin I- -0.4375 2.4615 0.0007 -0.3653 3.3219 0.0073 like domains 3 glycoprotein galactosyl-transferase -0.2590 8.0591 0.0291 -0.2359 6.8286 0.0282 alpha 1, 3 kelch-like 36 -0.2735 3.8968 0.0326 -0.2818 4.1118 0.0456 NPC1-like 1 -0.2539 1.9919 0.0013 -0.2764 1.7859 0.0137 a disintegrin and metallopeptidase -0.2407 1.3791 0.0440 -0.3423 1.5897 0.0139 domain 6B discs, large (Drosophila) homolog- -0.2910 1.8991 0.0136 -0.2173 1.5141 0.0487 associated protein 2 2-4-dienoyl-Coenzyme A reductase -0.3534 5.9705 0.0337 -0.1527 6.8225 0.0134 2, peroxisomal thioredoxin reductase 1 -0.3438 5.9131 0.0293 -0.3469 5.8240 0.0324 cadherin-related family member 5 -0.2360 1.7370 0.0174 -0.3059 2.0282 0.0035 coactosin-like 1 (Dictyostelium) -0.1927 8.5868 0.0186 -0.1406 9.8611 0.0072 pseudouridylate synthase 7 homolog -0.3318 4.3148 0.0298 -0.2683 6.3299 0.0054 (S. cerevisiae)

Increased Expression RIKEN cDNA 1810010D01 gene 0.2117 3.5159 0.0370 0.3277 2.9983 0.0001 predicted gene 10941 0.33797 1.8304 0.0457 0.2836 2.9432 0.0245 predicted gene 35677 0.3232 1.9031 0.0451 0.3763 2.3358 0.0010 complement component 4 binding protein, pseudogene 1 0.3730 1.8115 0.0034 0.3318 1.7738 0.0066 cell division cycle 25A 0.1743 5.1265 0.0477 0.2344 5.7825 0.0052 cystin 1 0.2055 1.8080 0.0237 0.1643 1.7696 0.0487 NFk light polypeptide gene enhancer in B cells inhibitor 1 0.2749 4.9858 0.0213 0.2858 4.5799 0.0489 prepronociceptin 0.2350 1.8037 0.0424 0.3997 1.7500 0.0006 suppressor of cytokine signaling 7 0.1797 4.5181 0.0166 0.1400 4.8637 0.0315 predicted gene 4787 0.3188 1.4338 0.0414 0.2766 1.8809 0.0382 non-SMC condensin I complex, subunit H 0.2170 3.5045 0.0424 0.1550 6.8265 0.0254 T cell receptor gamma, variable 4 0.3393 1.3234 0.0037 0.2088 1.1654 0.0445 cytochrome P450, family 26, subfamily b, polypeptide 1 0.2793 2.2331 0.0439 0.2600 3.3314 0.0314 insulin-like 6 0.1535 8.1255 0.0432 0.1492 5.4351 0.0440 rhomboid domain containing 3 0.2549 4.4271 0.0068 0.1667 5.1889 0.0138 keratin 26 0.2043 1.3722 0.0489 0.2259 1.5326 0.0174 expressed sequence AA415398 0.6767 2.0144 0.0033 0.2399 2.1104 0.0179 RIKEN cDNA D930028M14 gene 0.2308 2.9724 0.0424 0.3599 1.9903 0.0008 proline-rich nuclear receptor coactivator 2 0.1593 9.1186 0.0346 0.1497 9.6116 0.0141 regulatory factor X, 5 (influences HLA class II) 0.1967 6.3315 0.0408 0.1680 7.0857 0.0151 ankyrin repeat domain 49 0.2340 5.1614 0.0371 0.1880 6.1490 0.0152 predicted gene 9866 0.3629 1.7717 0.0258 0.3917 2.1220 0.0215 makorin, ring finger protein 2, opposite strand 0.3376 1.7455 0.0334 0.3233 1.8538 0.0016 RIKEN cDNA 1700023E05 gene 0.2924 2.7695 0.0441 0.3674 3.0405 0.0004 tetratricopeptide repeat domain 25 0.4685 1.8064 0.0103 0.3987 1.9127 0.0078

Supplementary Table II. Western blots of lysates of macrophages differentiated for 9 days from the bone marrow of control- and UV-chimeric mice. Macrophages were starved for 16 h of CSF-1, before a 2.5 minute pulse at 37oC with, or without CSF-1 (120 ng/ml). Densities for binding of antibodies to the different signalling molecules were quantified and standardized to expression of actin. Lysates were prepared from 4 pairs of macrophage cultures, with each pair prepared on separate days from a control- and matched UV-chimeric mouse. Mean + SEM. -CSF-1 +CSF-1 (2.5 min) C-chim UV-chim C-chim UV-chim CSF-1R 47.0 ± 24.3 38.5 ± 14.4 173.1 ± 33.5 182.6 ± 25.3 pErk 33.5 ± 16.2 24.9 ± 10.9 184.8 ± 24.6 184.8 ± 28.4 PI3K 75.3 ± 10.1 78.1 ± 13.2 103.6 ± 10.0 118.9 ± 10.6 pY FAK 58.1 ± 13.0 49.9 ± 8.9 149.7 ± 11.0 158.1 ± 23.6 P85 143.6 ± 37.2 127.5 ± 27.1 43.7 ± 12.9 67.8 ± 12.0 pY Pyk2 84.6 ± 24.6 96.9 ± 17.2 129.4 ± 21.2 154.3 ± 26.9 P110d 79.1 ± 11.2 73.6 ± 9.9 105.0 ± 8.7 120.2 ± 9.9 paxillin 105.4 ± 33.2 144.5 ± 30.7 120.7 ± 25.9 178.9 ± 22.6* pS Akt 69.5 ± 16.2 66.2 ± 18.4 149.0 ± 48.3 146.2 ± 26.9 * p<0.01, paired t-test

1 Supplementary Figure 1. Western blot of reticulon-1, paxillin, phosphoY118paxillin and

2 actin expression. Macrophages were differentiated from the BM of 4 control- and 4 UV-

3 chimeric mouse, and exposed or not to CSF-1 for 2.5 min. The density of the bands for

4 reticulon-1, paxillin and actin were quantified and are shown in Fig 5C and D.