Top

Published in:

Open Access 01-12-2020 | Stroke | Research

Potential caveats of putative microglia-specific markers for assessment of age-related cerebrovascular neuroinflammation

Authors: Pedram Honarpisheh, Juneyoung Lee, Anik Banerjee, Maria P. Blasco-Conesa, Parisa Honarpisheh, John d’Aigle, Abdullah A. Mamun, Rodney M. Ritzel, Anjali Chauhan, Bhanu P. Ganesh, Louise D. McCullough

Published in: Journal of Neuroinflammation | Issue 1/2020

Abstract

Background

The ability to distinguish resident microglia from infiltrating myeloid cells by flow cytometry-based surface phenotyping is an important technique for examining age-related neuroinflammation. The most commonly used surface markers for the identification of microglia include CD45 (low-intermediate expression), CD11b, Tmem119, and P2RY12.

Methods

In this study, we examined changes in expression levels of these putative microglia markers in in vivo animal models of stroke, cerebral amyloid angiopathy (CAA), and aging as well as in an ex vivo LPS-induced inflammation model.

Results

We demonstrate that Tmem119 and P2RY12 expression is evident within both CD45^int and CD45^high myeloid populations in models of stroke, CAA, and aging. Interestingly, LPS stimulation of FACS-sorted adult microglia suggested that these brain-resident myeloid cells can upregulate CD45 and downregulate Tmem119 and P2RY12, making them indistinguishable from peripherally derived myeloid populations. Importantly, our findings show that these changes in the molecular signatures of microglia can occur without a contribution from the other brain-resident or peripherally sourced immune cells.

Conclusion

We recommend future studies approach microglia identification by flow cytometry with caution, particularly in the absence of the use of a combination of markers validated for the specific neuroinflammation model of interest. The subpopulation of resident microglia residing within the “infiltrating myeloid” population, albeit small, may be functionally important in maintaining immune vigilance in the brain thus should not be overlooked in neuroimmunological studies.

Available only for authorised users

Greenhalgh AD, Passos Dos Santos R, Zarruk JG, Salmon CK, Kroner A, David S. Arginase-1 is expressed exclusively by infiltrating myeloid cells in CNS injury and disease. Brain Behav Immun. 2016;56:61–7 Available from: http://www.ncbi.nlm.nih.gov/pubmed/27126514.PubMedCrossRef

Ford AL, Goodsall AL, Hickey WF, Sedgwick JD. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol. 1995;154:4309–21 Available from: http://www.ncbi.nlm.nih.gov/pubmed/7722289.PubMed

Lassmann H, Schmied M, Vass K, Hickey WF. Bone marrow derived elements and resident microglia in brain inflammation. Glia. 1993;7:19–24 Available from: http://www.ncbi.nlm.nih.gov/pubmed/7678581.PubMedCrossRef

Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FMV. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci. 2007;10:1538–43 Available from: http://www.ncbi.nlm.nih.gov/pubmed/18026097.PubMedCrossRef

Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, et al. Functional differences between microglia and monocytes after ischemic stroke. J Neuroinflammation. 2015;12:106 Available from: http://www.ncbi.nlm.nih.gov/pubmed/26022493.PubMedPubMedCentralCrossRef

Rheinländer A, Schraven B, Bommhardt U. CD45 in human physiology and clinical medicine. Immunol Lett. 2018;196:22–32 Available from: http://www.ncbi.nlm.nih.gov/pubmed/29366662.PubMedCrossRef

Hermiston ML, Xu Z, Weiss A. CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol. 2003;21:107–37 Available from: http://www.ncbi.nlm.nih.gov/pubmed/12414720.PubMedCrossRef

Jordão MJC, Sankowski R, Brendecke SM, Sagar LG, Tai Y-H, et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science. 2019;363:eaat7554 Available from: http://www.ncbi.nlm.nih.gov/pubmed/30679343.PubMedCrossRef

Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, et al. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat Neurosci. 2014;17:131–43 Available from: http://www.ncbi.nlm.nih.gov/pubmed/24316888.PubMedCrossRef

10.

Chauhan A, Al Mamun A, Spiegel G, Harris N, Zhu L, McCullough LD. Splenectomy protects aged mice from injury after experimental stroke. Neurobiol Aging. 2018;61:102–11 Available from: http://www.ncbi.nlm.nih.gov/pubmed/29059593.PubMedCrossRef

11.

Kolter J, Kierdorf K, Henneke P. Origin and differentiation of nerve-associated macrophages. J Immunol. 2020;204:271–9 Available from: http://www.ncbi.nlm.nih.gov/pubmed/31907269.PubMedCrossRef

12.

Felger JC, Abe T, Kaunzner UW, Gottfried-Blackmore A, Gal-Toth J, McEwen BS, et al. Brain dendritic cells in ischemic stroke: time course, activation state, and origin. Brain Behav Immun. 2010;24:724–37 Available from: http://www.ncbi.nlm.nih.gov/pubmed/19914372.PubMedCrossRef

13.

Fumagalli S, Perego C, Ortolano F, De Simoni M-G. CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice. Glia. 2013;61:827–42 Available from: http://www.ncbi.nlm.nih.gov/pubmed/23440897.PubMedCrossRef

14.

Ritzel RM, Lai Y-J, Crapser JD, Patel AR, Schrecengost A, Grenier JM, et al. Aging alters the immunological response to ischemic stroke. Acta Neuropathol. 2018;136:89–110 Available from: http://www.ncbi.nlm.nih.gov/pubmed/29752550.PubMedPubMedCentralCrossRef

15.

Martin E, El-Behi M, Fontaine B, Delarasse C. Analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry. J Vis Exp. 2017; Available from: http://www.ncbi.nlm.nih.gov/pubmed/28671658.

16.

Weitbrecht L, Berchtold D, Zhang T, Jagdmann S, Dames C, Winek K, et al. CD4+ T cells promote delayed B cell responses in the ischemic brain after experimental stroke. Brain Behav Immun. 2020; Available from: http://www.ncbi.nlm.nih.gov/pubmed/33002634.

17.

Finneran DJ, Morgan D, Gordon MN, Nash KR. CNS-wide over expression of fractalkine improves cognitive functioning in a tauopathy model. J Neuroimmune Pharmacol. 2019;14:312–25 Available from: http://link.springer.com/10.1007/s11481-018-9822-5.PubMedCrossRef

18.

Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, et al. New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci U S A. 2016;113:E1738–46 Available from: http://www.ncbi.nlm.nih.gov/pubmed/26884166.PubMedPubMedCentralCrossRef

19.

Stein VM, Baumgärtner W, Schröder S, Zurbriggen A, Vandevelde M, Tipold A. Differential expression of CD45 on canine microglial cells. J Vet Med A Physiol Pathol Clin Med. 2007;54:314–20 Available from: http://www.ncbi.nlm.nih.gov/pubmed/17650152.PubMedCrossRef

20.

Grabert K, Michoel T, Karavolos MH, Clohisey S, Baillie JK, Stevens MP, et al. Microglial brain region-dependent diversity and selective regional sensitivities to aging. Nat Neurosci. 2016;19:504–16 Available from: http://www.ncbi.nlm.nih.gov/pubmed/26780511.PubMedPubMedCentralCrossRef

21.

Hickman SE, Kingery ND, Ohsumi TK, Borowsky ML, Wang L, Means TK, et al. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci. 2013;16:1896–905 Available from: http://www.ncbi.nlm.nih.gov/pubmed/24162652.PubMedPubMedCentralCrossRef

22.

Wes PD, Holtman IR, Boddeke EWGM, Möller T, Eggen BJL. Next generation transcriptomics and genomics elucidate biological complexity of microglia in health and disease. Glia. 2016;64:197–213 Available from: http://www.ncbi.nlm.nih.gov/pubmed/26040959.PubMedCrossRef

23.

Guerreiro R, Wojtas A, Bras J, Carrasquillo M, Rogaeva E, Majounie E, et al. TREM2 variants in Alzheimer’s disease. N Engl J Med. 2013;368:117–27 Available from: http://www.ncbi.nlm.nih.gov/pubmed/23150934.PubMedCrossRef

24.

Masuda T, Sankowski R, Staszewski O, Prinz M. Microglia heterogeneity in the single-cell era. Cell Rep. 2020;30:1271–81 Available from: http://www.ncbi.nlm.nih.gov/pubmed/32023447.PubMedCrossRef

25.

Prinz M, Jung S, Priller J. Microglia biology: one century of evolving concepts. Cell. 2019;179:292–311 Available from: http://www.ncbi.nlm.nih.gov/pubmed/31585077.PubMedCrossRef

26.

Zhu C, Kros JM, van der Weiden M, Zheng P, Cheng C, Mustafa DAM. Expression site of P2RY12 in residential microglial cells in astrocytomas correlates with M1 and M2 marker expression and tumor grade. Acta Neuropathol Commun. 2017;5:4 Available from: http://www.ncbi.nlm.nih.gov/pubmed/28073370.PubMedPubMedCentralCrossRef

27.

Cattaneo M. P2Y12 receptors: structure and function. J Thromb Haemost. 2015;13(Suppl 1):S10–6 Available from: http://www.ncbi.nlm.nih.gov/pubmed/26149010.PubMedCrossRef

28.

Davis J, Xu F, Deane R, Romanov G, Previti ML, Zeigler K, et al. Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor. J Biol Chem. 2004;279:20296–306 Available from: http://www.ncbi.nlm.nih.gov/pubmed/14985348.PubMedCrossRef

29.

Jäkel L, Van Nostrand WE, Nicoll JAR, Werring DJ, Verbeek MM. Animal models of cerebral amyloid angiopathy. Clin Sci (Lond). 2017;131:2469–88 Available from: http://www.ncbi.nlm.nih.gov/pubmed/28963121.CrossRef

30.

Ronaldson PT, Davis TP. Regulation of blood–brain barrier integrity by microglia in health and disease: a therapeutic opportunity. J Cereb Blood Flow Metab. 2020. https://doi.org/10.1177/0271678X20951995.

31.

Korin B, Ben-Shaanan TL, Schiller M, Dubovik T, Azulay-Debby H, Boshnak NT, et al. High-dimensional, single-cell characterization of the brain’s immune compartment. Nat Neurosci. 2017;20:1300–9 Available from: http://www.nature.com/articles/nn.4610.PubMedCrossRef

32.

Popa-Wagner A, Petcu EB, Capitanescu B, Hermann DM, Radu E, Gresita A. Ageing as a risk factor for cerebral ischemia: underlying mechanisms and therapy in animal models and in the clinic. Mech Ageing Dev. 2020;190:111312 Available from: https://linkinghub.elsevier.com/retrieve/pii/S0047637420301081.PubMedCrossRef

33.

Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, et al. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 1996;274:99–102 Available from: http://www.ncbi.nlm.nih.gov/pubmed/8810256.PubMedCrossRef

34.

Saito S, Yamamoto Y, Maki T, Hattori Y, Ito H, Mizuno K, et al. Taxifolin inhibits amyloid-β oligomer formation and fully restores vascular integrity and memory in cerebral amyloid angiopathy. Acta Neuropathol Commun. 2017;5:26 Available from: http://www.ncbi.nlm.nih.gov/pubmed/28376923.PubMedPubMedCentralCrossRef

35.

Kokjohn TA, Roher AE. Amyloid precursor protein transgenic mouse models and Alzheimer’s disease: understanding the paradigms, limitations, and contributions. Alzheimers Dement. 2009;5:340–7 Available from: http://www.ncbi.nlm.nih.gov/pubmed/19560104.PubMedPubMedCentralCrossRef

36.

Spychala MS, Venna VR, Jandzinski M, Doran SJ, Durgan DJ, Ganesh BP, et al. Age-related changes in the gut microbiota influence systemic inflammation and stroke outcome. Ann Neurol. 2018;84:23–36 Available from: http://www.ncbi.nlm.nih.gov/pubmed/29733457.PubMedPubMedCentralCrossRef

37.

Miao J, Xu F, Davis J, Otte-Höller I, Verbeek MM, Van Nostrand WE. Cerebral microvascular amyloid beta protein deposition induces vascular degeneration and neuroinflammation in transgenic mice expressing human vasculotropic mutant amyloid beta precursor protein. Am J Pathol. 2005;167:505–15 Available from: http://www.ncbi.nlm.nih.gov/pubmed/16049335.PubMedPubMedCentralCrossRef

38.

Chauhan A, Moser H, McCullough LD. Sex differences in ischaemic stroke: potential cellular mechanisms. Clin Sci (Lond). 2017;131:533–52 Available from: http://www.ncbi.nlm.nih.gov/pubmed/28302915.CrossRef

39.

Verma R, Ritzel RM, Harris NM, Lee J, Kim T, Pandi G, et al. Inhibition of miR-141-3p ameliorates the negative effects of poststroke social isolation in aged mice. Stroke. 2018;49:1701–7 Available from: http://www.ncbi.nlm.nih.gov/pubmed/29866755.PubMedPubMedCentralCrossRef

40.

Lee J, D’Aigle J, Atadja L, Quaicoe V, Honarpisheh P, Ganesh BP, et al. Gut microbiota-derived short-chain fatty acids promote post-stroke recovery in aged mice. Circ Res. 2020; Available from: http://www.ncbi.nlm.nih.gov/pubmed/32354259.

41.

Lee J, d’Aigle J, Atadja L, Quaicoe V, Honarpisheh P, Ganesh BP, et al. Gut microbiota-derived short-chain fatty acids promote post-stroke recovery in aged mice. Circ Res. 2020. https://doi.org/10.1161/CIRCRESAHA.119.316448.

42.

Chen H-R, Sun Y-Y, Chen C-W, Kuo Y-M, Kuan IS, Tiger Li Z-R, et al. Fate mapping via CCR2-CreER mice reveals monocyte-to-microglia transition in development and neonatal stroke. Sci Adv. 2020;6:eabb2119 Available from: http://www.ncbi.nlm.nih.gov/pubmed/32923636.PubMedPubMedCentralCrossRef

43.

Garner KM, Amin R, Johnson RW, Scarlett EJ, Burton MD. Microglia priming by interleukin-6 signaling is enhanced in aged mice. J Neuroimmunol. 2018;324:90–9 Available from: http://www.ncbi.nlm.nih.gov/pubmed/30261355.PubMedPubMedCentralCrossRef

44.

Woollard KJ, Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nat Rev Cardiol. 2010;7:77–86 Available from: http://www.ncbi.nlm.nih.gov/pubmed/20065951.PubMedPubMedCentralCrossRef

45.

Tay TL, Sagar, Dautzenberg J, Grün D, Prinz M. Unique microglia recovery population revealed by single-cell RNAseq following neurodegeneration. Acta Neuropathol Commun. 2018;6:87 Available from: http://www.ncbi.nlm.nih.gov/pubmed/30185219.PubMedPubMedCentralCrossRef

46.

Koellhoffer EC, McCullough LD, Ritzel RM. Old maids: aging and its impact on microglia function. Int J Mol Sci. 2017;18 Available from: http://www.ncbi.nlm.nih.gov/pubmed/28379162.

47.

Yang H, Graham LC, Reagan AM, Grabowska WA, Schott WH, Howell GR. Transcriptome profiling of brain myeloid cells revealed activation of Itgal, Trem1, and Spp1 in western diet-induced obesity. J Neuroinflammation. 2019;16:169 Available from: http://www.ncbi.nlm.nih.gov/pubmed/31426806.PubMedPubMedCentralCrossRef

48.

Bennett FC, Bennett ML, Yaqoob F, Mulinyawe SB, Grant GA, Hayden Gephart M, et al. A combination of ontogeny and CNS environment establishes microglial identity. Neuron. 2018;98:1170–1183.e8 Available from: http://www.ncbi.nlm.nih.gov/pubmed/29861285.PubMedPubMedCentralCrossRef

49.

Grassivaro F, Menon R, Acquaviva M, Ottoboni L, Ruffini F, Bergamaschi A, et al. Convergence between microglia and peripheral macrophages phenotype during development and neuroinflammation. J Neurosci. 2020;40:784–95 Available from: http://www.ncbi.nlm.nih.gov/pubmed/31818979.PubMedPubMedCentralCrossRef

50.

Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, et al. A unique microglia type associated with restricting development of Alzheimer’s disease. Cell. 2017;169:1276–1290.e17 Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867417305780.PubMedCrossRef

51.

Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity. 2017;47:566–581.e9 Available from: http://www.ncbi.nlm.nih.gov/pubmed/28930663.PubMedPubMedCentralCrossRef

52.

Walker DG, Tang TM, Mendsaikhan A, Tooyama I, Serrano GE, Sue LI, et al. Patterns of expression of purinergic receptor P2RY12, a putative marker for non-activated microglia, in aged and Alzheimer’s disease brains. Int J Mol Sci. 2020;21 Available from: http://www.ncbi.nlm.nih.gov/pubmed/31968618.

53.

Getts DR, Terry RL, Getts MT, Müller M, Rana S, Shrestha B, et al. Ly6c + “inflammatory monocytes” are microglial precursors recruited in a pathogenic manner in West Nile virus encephalitis. J Exp Med. 2008;205:2319–37 Available from: http://www.ncbi.nlm.nih.gov/pubmed/18779347.PubMedPubMedCentralCrossRef

54.

Rangaraju S, Raza SA, Li NX, Betarbet R, Dammer EB, Duong D, et al. Differential phagocytic properties of CD45^low microglia and CD45^high brain mononuclear phagocytes-activation and age-related effects. Front Immunol. 2018;9:405 Available from: http://www.ncbi.nlm.nih.gov/pubmed/29552013.PubMedPubMedCentralCrossRef

55.

Sousa C, Golebiewska A, Poovathingal SK, Kaoma T, Pires-Afonso Y, Martina S, et al. Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures. EMBO Rep. 2018;19 Available from: http://www.ncbi.nlm.nih.gov/pubmed/30206190.

56.

Bedi SS, Smith P, Hetz RA, Xue H, Cox CS. Immunomagnetic enrichment and flow cytometric characterization of mouse microglia. J Neurosci Methods. 2013;219:176–82 Available from: http://www.ncbi.nlm.nih.gov/pubmed/23928152.PubMedCrossRef

57.

Stratoulias V, Venero JL, Tremblay M-È, Joseph B. Microglial subtypes: diversity within the microglial community. EMBO J. 2019;38:e101997 Available from: http://www.ncbi.nlm.nih.gov/pubmed/31373067.PubMedPubMedCentralCrossRef

Title: Potential caveats of putative microglia-specific markers for assessment of age-related cerebrovascular neuroinflammation
Authors: Pedram Honarpisheh
Juneyoung Lee
Anik Banerjee
Maria P. Blasco-Conesa
Parisa Honarpisheh
John d’Aigle
Abdullah A. Mamun
Rodney M. Ritzel
Anjali Chauhan
Bhanu P. Ganesh
Louise D. McCullough
Publication date: 01-12-2020
Publisher: BioMed Central
Keyword: Stroke
Published in: Journal of Neuroinflammation / Issue 1/2020
Electronic ISSN: 1742-2094
DOI: https://doi.org/10.1186/s12974-020-02019-5

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Potential caveats of putative microglia-specific markers for assessment of age-related cerebrovascular neuroinflammation

Abstract

Background

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 1/2020

Novel alterations in corneal neuroimmune phenotypes in mice with central nervous system tauopathy

Astragaloside IV inhibits astrocyte senescence: implication in Parkinson’s disease

Pinocembrin ameliorates intermittent hypoxia-induced neuroinflammation through BNIP3-dependent mitophagy in a murine model of sleep apnea

Autonomic nervous system and inflammation interaction in endometriosis-associated pain

Neuronal aldosterone elicits a distinct genomic response in pain signaling molecules contributing to inflammatory pain

Macrophage MSR1 promotes the formation of foamy macrophage and neuronal apoptosis after spinal cord injury