In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-R cells as a model of peritoneal macrophages should be carefully validated. All MS data are deposited in ProteomeXchange with the identifier PXD022133.

5 N Orekhovich Research Institute of Biomedical Chemistry Pogodinskaja Str 10 119121 Moscow Russia

BioCeV Institute of Microbiology of the CAS Prumyslova 595 252 50 Vestec Czech Republic

Faculty of Science University of South Bohemia Branišovská 1760 370 05 České Budějovice Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Branišovská 31 370 05 České Budějovice Czech Republic

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Pamies D., Bal-Price A., Chesné C., Coecke S., Dinnyes A., Eskes C., Grillari R., Gstraunthaler G., Hartung T., Jennings P., et al. Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems. ALTEX. 2018;35:353–378. doi: 10.14573/altex.1710081. PubMed DOI

Lee C.Z.W., Kozaki T., Ginhoux F. Studying tissue macrophages in vitro: Are iPSC-derived cells the answer? Nat. Rev. Immunol. 2018;18:716–725. doi: 10.1038/s41577-018-0054-y. PubMed DOI

Fitzgerald M.L., Moore K.J., Freeman M.W., Reed G.L. Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: A divergent response relative to human THP-1 monocyte/macrophages. J. Immunol. 2000;164:2692–2700. doi: 10.4049/jimmunol.164.5.2692. PubMed DOI

Chaudhry M.Z., Kasmapour B., Plaza-Sirvent C., Bajagic M., Casalegno Garduño R., Borkner L., Lenac Roviš T., Scrima A., Jonjic S., Schmitz I., et al. UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene. Front. Cell. Infect. Microbiol. 2017;7:312. doi: 10.3389/fcimb.2017.00312. PubMed DOI PMC

Guo M., Hartlova A., Dill B.D., Prescott A.R., Gierlinski M., Trost M. High-resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages. Proteomics. 2015;15:3169–3174. doi: 10.1002/pmic.201400431. PubMed DOI PMC

Campbell-Valois F.X., Trost M., Chemali M., Dill B.D., Laplante A., Duclos S., Sadeghi S., Rondeau C., Morrow I.C., Bell C., et al. Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome. Mol. Cell. Proteomics. 2012;11:M111-016378. doi: 10.1074/mcp.M111.016378. PubMed DOI PMC

Trost M., English L., Lemieux S., Courcelles M., Desjardins M., Thibault P. The phagosomal proteome in interferon-gamma-activated macrophages. Immunity. 2009;30:143–154. doi: 10.1016/j.immuni.2008.11.006. PubMed DOI

Marcantonio M., Trost M., Courcelles M., Desjardins M., Thibault P. Combined enzymatic and data mining approaches for comprehensive phosphoproteome analyses: Application to cell signaling events of interferon-gamma-stimulated macrophages. Mol. Cell. Proteomics. 2008;7:645–660. doi: 10.1074/mcp.M700383-MCP200. PubMed DOI

Bell C., English L., Boulais J., Chemali M., Caron-Lizotte O., Desjardins M., Thibault P. Quantitative proteomics reveals the induction of mitophagy in tumor necrosis factor-alpha-activated (TNFalpha) macrophages. Mol. Cell. Proteomics. 2013;12:2394–2407. doi: 10.1074/mcp.M112.025775. PubMed DOI PMC

Adami C., Brunda M.J., Palleroni A.V. In vivo immortalization of murine peritoneal macrophages: A new rapid and efficient method for obtaining macrophage cell lines. J. Leukoc. Biol. 1993;53:475–478. doi: 10.1002/jlb.53.4.475. PubMed DOI

Rusanov A.L., Stepanov A.A., Zgoda V.G., Kaysheva A.L., Selinger M., Maskova H., Loginov D., Sterba J., Grubhoffer L., Luzgina N.G. Proteome dataset of mouse macrophage cell line infected with tick-borne encephalitis virus. Data Brief. 2019;28:105029. doi: 10.1016/j.dib.2019.105029. PubMed DOI PMC

Hume D.A. The Many Alternative Faces of Macrophage Activation. Front. Immunol. 2015;6:370. doi: 10.3389/fimmu.2015.00370. PubMed DOI PMC

Shkurupiy V.A., Tkachev V.O., Potapova O.V., Luzgina N.G., Bugrimova J.S., Obedinskaya K.S., Zaiceva N.S., Chechushkov A.V. Morphofunctional characteristics of the immune system in CBA and C57BL/6 mice. Bull. Exp. Biol. Med. 2011;150:725–728. doi: 10.1007/s10517-011-1234-y. PubMed DOI

Heinz S., Romanoski C.E., Benner C., Allison K.A., Kaikkonen M.U., Orozco L.D., Glass C.K. Effect of natural genetic variation on enhancer selection and function. Nature. 2013;503:487–492. doi: 10.1038/nature12615. PubMed DOI PMC

Raza S., Barnett M.W., Barnett-Itzhaki Z., Amit I., Hume D.A., Freeman T.C. Analysis of the transcriptional networks underpinning the activation of murine macrophages by inflammatory mediators. J. Leukoc. Biol. 2014;96:167–183. doi: 10.1189/jlb.6HI0313-169R. PubMed DOI PMC

Wells C.A., Ravasi T., Faulkner G.J., Carninci P., Okazaki Y., Hayashizaki Y., Sweet M., Wainwright B.J., Hume D.A. Genetic control of the innate immune response. BMC Immunol. 2003;4:5. doi: 10.1186/1471-2172-4-5. PubMed DOI PMC

Aderem A., Underhill D.M. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 1999;17:593–623. doi: 10.1146/annurev.immunol.17.1.593. PubMed DOI

Pustylnikov S., Sagar D., Jain P., Khan Z.K. Targeting the C-type lectins-mediated host-pathogen interactions with dextran. J. Pharm. Pharm. Sci. 2014;17:371–392. doi: 10.18433/J3N590. PubMed DOI PMC

Shapouri-Moghaddam A., Mohammadian S., Vazini H., Taghadosi M., Esmaeili S.A., Mardani F., Seifi B., Mohammadi A., Afshari J.T., Sahebkar A. Macrophage plasticity, polarization, and function in health and disease. J. Cell Physiol. 2018;233:6425–6440. doi: 10.1002/jcp.26429. PubMed DOI

Wynn T.A., Vannella K.M. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity. 2016;44:450–462. doi: 10.1016/j.immuni.2016.02.015. PubMed DOI PMC

Mogilenko D.A., Kudriavtsev I.V., Trulioff A.S., Shavva V.S., Dizhe E.B., Missyul B.V., Zhakhov A.V., Ischenko A.M., Perevozchikov A.P., Orlov S.V. Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Toll-like receptor 4 activation in human macrophages. J. Biol. Chem. 2012;287:5954–5968. doi: 10.1074/jbc.M111.289322. PubMed DOI PMC

Ruan C.-C., Ge Q., Li Y., Li X.-D., Chen D.-R., Ji K.-D., Wu Y.-J., Sheng L.-J., Yan C., Zhu D.-L., et al. Complement-mediated macrophage polarization in perivascular adipose tissue contributes to vascular injury in deoxycorticosterone acetate-salt mice. Arter. Thromb. Vasc. Biol. 2015;35:598–606. doi: 10.1161/ATVBAHA.114.304927. PubMed DOI

Afzal Khan M., Assiri A.M., Broering D.C. Complement and macrophage crosstalk during process of angiogenesis in tumor progression. J. Biomed. Sci. 2015;22:58. doi: 10.1186/s12929-015-0151-1. PubMed DOI PMC

Serrander L., Skarman P., Rasmussen B., Witke W., Lew D.P., Krause K.H., Stendahl O., Nüsse O. Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils. J. Immunol. 2000;165:2451–2457. doi: 10.4049/jimmunol.165.5.2451. PubMed DOI

Perry D.G., Daugherty G.L., Martin W.J. 2nd. Clathrin-coated pit-associated proteins are required for alveolar macrophage phagocytosis. J. Immunol. 1999;162:380–386. PubMed

Cheng Y.-L., Kuo C.-F., Lu S.-L., Hiroko O., Wu Y.-N., Hsieh C.-L., Noda T., Wu S.-R., Anderson R., Lin C.-F., et al. Group A Streptococcus Induces LAPosomes via SLO/β1 Integrin/NOX2/ROS Pathway in Endothelial Cells That Are Ineffective in Bacterial Killing and Suppress Xenophagy. mBio. 2019;10:e02148-19. doi: 10.1128/mBio.02148-19. PubMed DOI PMC

Hawk C.S., Coelho C., Lima de Oliveira D.S., Paredes V., Albuquerque P., Bocca A.L., Dos Santos A.C., Rusakova V., Holemon H., Silva-Pereira I., et al. Integrin β1 Promotes the Interaction of Murine IgG3 with Effector Cells. J. Immunol. 2019;202:2782–2794. doi: 10.4049/jimmunol.1701795. PubMed DOI PMC

Guo M., Härtlova A., Gierliński M., Prescott A., Castellvi J., Losa J.H., Petersen S.K., Wenzel U.A., Dill B.D., Emmerich C.H., et al. Triggering MSR1 promotes JNK-mediated inflammation in IL-4-activated macrophages. EMBO J. 2019;38:e100299. doi: 10.15252/embj.2018100299. PubMed DOI PMC

Willingham S.B., Volkmer J.P., Gentles A.J., Sahoo D., Dalerba P., Mitra S.S., Wang J., Contreras-Trujillo H., Martin R., Cohen J.D., et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl. Acad. Sci. USA. 2012;109:6662–6667. doi: 10.1073/pnas.1121623109. PubMed DOI PMC

Okazawa H., Motegi S., Ohyama N., Ohnishi H., Tomizawa T., Kaneko Y., Oldenborg P.-A., Ishikawa O., Matozaki T. Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system. J. Immunol. 2005;174:2004–2011. doi: 10.4049/jimmunol.174.4.2004. PubMed DOI

Gauss K.A., Bunger P.L., Crawford M.A., McDermott B.E., Swearingen R., Nelson-Overton L.K., Siemsen D.W., Kobayashi S.D., Deleo F.R., Quinn M.T. Variants of the 5′-untranslated region of human NCF2: Expression and translational efficiency. Gene. 2006;366:169–179. doi: 10.1016/j.gene.2005.09.012. PubMed DOI

Thomas D.C. The phagocyte respiratory burst: Historical perspectives and recent advances. Immunol. Lett. 2017;192:88–96. doi: 10.1016/j.imlet.2017.08.016. PubMed DOI

Pradip D., Peng X., Durden D.L. Rac2 specificity in macrophage integrin signaling: Potential role for Syk kinase. J. Biol. Chem. 2003;278:41661–41669. doi: 10.1074/jbc.M306491200. PubMed DOI

Chiriaco M., Salfa I., Di Matteo G., Rossi P., Finocchi A. Chronic granulomatous disease: Clinical, molecular, and therapeutic aspects. Pediatr. Allergy Immunol. 2016;27:242–253. doi: 10.1111/pai.12527. PubMed DOI

Marikovsky M., Ziv V., Nevo N., Harris-Cerruti C., Mahler O. Cu/Zn superoxide dismutase plays important role in immune response. J. Immunol. 2003;170:2993–3001. doi: 10.4049/jimmunol.170.6.2993. PubMed DOI

Sarkar A., Tindle C., Pranadinata R.F., Reed S., Eckmann L., Stappenbeck T.S., Ernst P.B., Das S. ELMO1 Regulates Autophagy Induction and Bacterial Clearance During Enteric Infection. J. Infect. Dis. 2017;216:1655–1666. doi: 10.1093/infdis/jix528. PubMed DOI PMC

Gong P., Chen S., Zhang L., Hu Y., Gu A., Zhang J., Wang Y. RhoG-ELMO1-RAC1 is involved in phagocytosis suppressed by mono-butyl phthalate in TM4 cells. Environ. Sci. Pollut. Res. Int. 2018;25:35440–35450. doi: 10.1007/s11356-018-3503-z. PubMed DOI

Katoh H., Hiramoto K., Negishi M. Activation of Rac1 by RhoG regulates cell migration. J. Cell Sci. 2006;119:56–65. doi: 10.1242/jcs.02720. PubMed DOI

Csépányi-Kömi R., Sirokmány G., Geiszt M., Ligeti E. ARHGAP25, a novel Rac GTPase-activating protein, regulates phagocytosis in human neutrophilic granulocytes. Blood. 2012;119:573–582. doi: 10.1182/blood-2010-12-324053. PubMed DOI

Csépányi-Kömi R., Lévay M., Ligeti E. Rho/RacGAPs Embarras de richesse? Small GTPases. 2012;3:178–182. doi: 10.4161/sgtp.20040. PubMed DOI PMC

Wheeler A.P., Wells C.M., Smith S.D., Vega F.M., Henderson R.B., Tybulewicz V.L., Ridley A.J. Rac1 and Rac2 regulate macrophage morphology but are not essential for migration. Cell Sci. 2006;119 (Pt 13):2749–2757. doi: 10.1242/jcs.03024. PubMed DOI

Hoppe A.D., Swanson J.A. Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol. Biol. Cell. 2004;15:3509–3519. doi: 10.1091/mbc.e03-11-0847. PubMed DOI PMC

Park H., Chan M.M., Iritani B.M. Hem-1: Putting the “WAVE” into actin polymerization during an immune response. FEBS Lett. 2010;584:4923–4932. doi: 10.1016/j.febslet.2010.10.018. PubMed DOI PMC

Park H., Cox D. Cdc42 regulates Fc gamma receptor-mediated phagocytosis through the activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP) and neural-WASP. Mol. Biol. Cell. 2009;20:4500–4508. doi: 10.1091/mbc.e09-03-0230. PubMed DOI PMC

Park H., Staehling-Hampton K., Appleby M.W., Brunkow M.E., Habib T., Zhang Y., Ramsdell F., Liggitt H.D., Freie B., Tsang M., et al. A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic protein 1 in lymphopoiesis and innate immunity. J. Exp. Med. 2008;205:2899–2913. doi: 10.1084/jem.20080340. PubMed DOI PMC

Chan M.M., Wooden J.M., Tsang M., Gilligan D.M., Hirenallur-S D.K., Finney G.L., Rynes E., Maccoss M., Ramirez J.A., Park H., et al. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes. PLoS ONE. 2013;8:e54902. doi: 10.1371/journal.pone.0054902. PubMed DOI PMC

Roskoski R., Jr. Src protein-tyrosine kinase structure and regulation. Biochem. Biophys. Res. Commun. 2004;324:1155–1164. doi: 10.1016/j.bbrc.2004.09.171. PubMed DOI

Roskoski R., Jr. Src kinase regulation by phosphorylation and dephosphorylation. Biochem. Biophys. Res. Commun. 2005;331:1–14. doi: 10.1016/j.bbrc.2005.03.012. PubMed DOI

Tardif M., Savard M., Flamand L., Gosselin J. Impaired protein kinase C activation/translocation in Epstein-Barr virus-infected monocytes. J. Biol. Chem. 2002;277:24148–24154. doi: 10.1074/jbc.M109036200. PubMed DOI

Thorslund S.E., Edgren T., Pettersson J., Nordfelth R., Sellin M.E., Ivanova E., Francis M.S., Isaksson E.L., Wolf-Watz H., Fällman M. The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function. PLoS ONE. 2011;6:e16784. doi: 10.1371/journal.pone.0016784. PubMed DOI PMC

McCahill A., Warwicker J., Bolger G.B., Houslay M.D., Yarwod S.J. The RACK1 scaffold protein: A dynamic cog in cell response mechanisms. Mol. Pharmacol. 2002;62:1261–1273. doi: 10.1124/mol.62.6.1261. PubMed DOI

Wang F., Yamauchi M., Muramatsu M., Osawa T., Tsuchida R., Shibuya M. RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway. J. Biol. Chem. 2011;286:9097–9106. doi: 10.1074/jbc.M110.165605. PubMed DOI PMC

Tait J.F., Frankenberry D.A., Miao C.H., Killary A.M., Adler D.A., Disteche C.M. Chromosomal localization of the human annexin III (ANX3) gene. Genomics. 1991;10:441–448. doi: 10.1016/0888-7543(91)90330-H. PubMed DOI

Diakonova M., Gerke V., Ernst J., Liautard J.P., van der Vusse G., Griffiths G. Localization of five annexins in J774 macrophages and on isolated phagosomes. J. Cell Sci. 1997;110:1199–1213. doi: 10.1242/jcs.110.10.1199. PubMed DOI

Ye W., Li Y., Fan L., Zhao Q., Yuan H., Tan B., Zhang Z. Effect of annexin A7 suppression on the apoptosis of gastric cancer cells. Mol. Cell. Biochem. 2017;429:33–43. doi: 10.1007/s11010-016-2934-4. PubMed DOI

Wang L., Li X., Ren Y., Geng H., Zhang Q., Cao L., Meng Z., Wu X., Xu M., Xu K. Cancer-associated fibroblasts contribute to cisplatin resistance by modulating ANXA3 in lung cancer cells. Cancer Sci. 2019;110:1609–1620. doi: 10.1111/cas.13998. PubMed DOI PMC

Rosenbaum S., Kreft S., Etich J., Frie C., Stermann J., Grskovic I., Frey B., Mielenz D., Pöschl E., Gaipl U., et al. Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif. J. Biol. Chem. 2011;286:5708–5716. doi: 10.1074/jbc.M110.193086. PubMed DOI PMC

Shirakabe K., Hattori S., Seiki M., Koyasu S., Okada Y. VIP36 protein is a target of ectodomain shedding and regulates phagocytosis in macrophage Raw 264.7 cells. J. Biol. Chem. 2011;286:43154–43163. doi: 10.1074/jbc.M111.275586. PubMed DOI PMC

Otani Y., Yamaguchi Y., Sato Y., Furuichi T., Ikenaka K., Kitani H., Baba H. PLD4 is involved in phagocytosis of microglia: Expression and localization changes of PLD4 are correlated with activation state of microglia. PLoS ONE. 2011;6:e27544. doi: 10.1371/journal.pone.0027544. PubMed DOI PMC

Gao L., Zhou Y., Zhou S.-X., Yu X.-J., Xu J.-M., Zuo L., Luo Y.-H., Li X.-A. PLD4 promotes M1 macrophages to perform antitumor effects in colon cancer cells. Oncol. Rep. 2017;37:408–416. doi: 10.3892/or.2016.5216. PubMed DOI

Click R.E. Review: 2-mercaptoethanol alteration of in vitro immune functions of species other than murine. J. Immunol. Methods. 2014;402:1–8. doi: 10.1016/j.jim.2013.11.007. PubMed DOI PMC

Walker J.M., editor. Basic Protein and Peptide Protocols. 1st ed. Humana Press; Totowa, NJ, USA: 1994. The bicinchoninic acid (BCA) assay for protein quantitation; pp. 5–8. PubMed

Tyanova S., Temu T., Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 2016;11:2301–2319. doi: 10.1038/nprot.2016.136. PubMed DOI

Barsnes H., Vaudel M. A Highly Adaptable Common Interface for Proteomics Search and de Novo Engines. Proteome Res. 2018;17:2552–2555. doi: 10.1021/acs.jproteome.8b00175. PubMed DOI

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D.J., Inuganti A., Griss J., Mayer G., Eisenacher M., et al. The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Nucleic Acids Res. 2019;47:D442–D450. doi: 10.1093/nar/gky1106. PubMed DOI PMC

Conway J.R., Lex A., Gehlenborg N. UpSetR: An R package for the visualization of intersecting sets and their properties. Bioinformatics. 2017;33:2938–2940. doi: 10.1093/bioinformatics/btx364. PubMed DOI PMC

YIshihama Y., Oda T., Tabata T., Sato T., Nagasu J., Rappsilber M. Mann, Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell. Proteom. 2005;4:1265–1272. doi: 10.1074/mcp.M500061-MCP200. PubMed DOI

Doncheva N.T., Morris J.H., Gorodkin J., Jensen L.J. Cytoscape StringApp: Network Analysis and Visualization of Proteomics Data. J. Proteome Res. 2019;18:623–632. doi: 10.1021/acs.jproteome.8b00702. PubMed DOI PMC

Morris J.H., Apeltsin L., Newman A.M., Baumbach J., Wittkop T., Su G., Bader G.D., Ferrin T.E. clusterMaker: A multi-algorithm clustering plugin for Cytoscape. BMC Bioinformatics. 2011;12:436. doi: 10.1186/1471-2105-12-436. PubMed DOI PMC

Johnston D.G.W., Kearney J., Zasłona Z., Williams M.A., O’Neill L.A.J., Corr S.C. MicroRNA-21 Limits Uptake of Listeria monocytogenes by Macrophages to Reduce the Intracellular Niche and Control Infection. Front. Cell. Infect. Microbiol. 2017;7:201. doi: 10.3389/fcimb.2017.00201. PubMed DOI PMC

Carpenter A.E., Jones T.R., Lamprecht M.R., Clarke C., Kang I.H., Friman O., Guertin D.A., Chang J.H., Lindquist R.A., Moffat J., et al. CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100. doi: 10.1186/gb-2006-7-10-r100. PubMed DOI PMC

Genotype-driven sensitivity of mice to tick-borne encephalitis virus correlates with differential host responses in peripheral macrophages and brain