Alveolar macrophages (AM) are critical to the homeostasis of the inflammatory environment in the lung. Differential expression of surface markers classifies macrophages to either classically (M1) or alternatively activated (M2). We investigated the phenotype of human alveolar macrophages (AM) in adults living in two different geographical locations: UK and Malawi. We show that the majority of AM express high levels of M1 and M2 markers simultaneously, with the M1/M2 phenotype being stable in individuals from different geographical locations. The combined M1/M2 features confer to AM a hybrid phenotype, which does not fit the classic macrophage classification. This hybrid phenotype may confer to alveolar macrophages an ability to quickly switch between M1 or M2 associated functions allowing for appropriate responses to stimuli and tissue environment

Ferreira, Daniela

Jambo, Kondwani

Kamng'ona, Raphael

Mitsi, Elena

Mwandumba, Henry

Reiné, Jesús

Rylance, Jamie

SolorzanoGonzalez, Carla

LSTM Online Archive

LETTER TO THE EDITOR Open AccessHuman alveolar macrophagespredominately express combined classicalM1 and M2 surface markers in steady stateElena Mitsi1, Raphael Kamng’ona2, Jamie Rylance1, Carla Solórzano1, J. Jesus Reiné1, Henry C. Mwandumba1,2,Daniela M. Ferreira1† and Kondwani C. Jambo1,2*†AbstractAlveolar macrophages (AM) are critical to the homeostasis of the inflammatory environment in the lung. Differentialexpression of surface markers classifies macrophages to either classically (M1) or alternatively activated (M2). Weinvestigated the phenotype of human alveolar macrophages (AM) in adults living in two different geographicallocations: UK and Malawi. We show that the majority of AM express high levels of M1 and M2 markers simultaneously,with the M1/M2 phenotype being stable in individuals from different geographical locations. The combined M1/M2features confer to AM a hybrid phenotype, which does not fit the classic macrophage classification. This hybridphenotype may confer to alveolar macrophages an ability to quickly switch between M1 or M2 associated functionsallowing for appropriate responses to stimuli and tissue environment.IntroductionThe healthy human alveoli are dominated by alveolarmacrophages (AM) [1]. These cells play a critical role inregulating immune responses [2] and maintaininghomeostasis in the lung [1, 3]. Their plasticity to adaptto changes in their microenvironment is fundamental inretaining the lung health [4]. Macrophages have beencategorised as “polarised” towards either M1 (proinflam-matory, mediating resistance to pathogens), or M2 mode(anti-inflammatory, promoting tissue remodelling) [5].This categorisation is derived from murine models, andfrom in vitro polarisation of human macrophages usinga combination of cytokines. However, whether AMpolarisation in steady state fits the traditional M1/M2 di-chotomy is still not well understood. We therefore inves-tigated human AM polarisation in steady state, andascertained the stability of the phenotype in two geo-graphical locations and following in vivo exposure tobacterial and viral stimuli.MethodsBriefly, in the UK, healthy, non-smoking participantsaged 18–50 years were recruited from an ongoingstudy of the Experimental human pneumococcal chal-lenge (EHPC) model [6]. A subset of 25 participants,16 individuals who had become colonized (carriage +)with Streptococcus pneumoniae, and 9 who had not(carriage –) underwent research bronchoscopy at 4 to7 weeks after the bacterial challenge. Alveolar macro-phages were obtained from bronchoalveolar lavage(BAL), as described previously [7]. In Queen ElizabethCentral Hospital (QECH), Malawi, BAL samples [8]were obtained from selected asymptomatic adults(≥18 yrs), comprising ten HIV-1-uninfected healthycontrols and ten age-matched ART-naïve HIV-1-infected individuals. We excluded participants withclinical evidence of active disease or recent history ofsevere respiratory illness.Flow cytometry-based immunophenotyping wasused to characterise AM phenotype obtained fromBAL fluid. The expressional levels of key surfacemarkers were measured immediately after samplecollection.* Correspondence: Kondwani.Jambo@lstmed.ac.uk†Equal contributors1Department of Clinical Sciences, Liverpool School of Tropical Medicine,Liverpool, UK2Malawi Liverpool Wellcome Trust Clinical Research Programme, College ofMedicine, P.O Box 30096, Chichiri, Blantyre 3, Malawi© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Mitsi et al. Respiratory Research  (2018) 19:66 https://doi.org/10.1186/s12931-018-0777-0ResultsHuman alveolar macrophages simultaneously express M1and M2 markers in steady stateData from the two human cohorts from distinct geograph-ical locations, UK (Additional file 1 Table S1) and Malawi(Additional file 1 Table S2), were collected independentlyand their comparison revealed a similar pattern of surfacemarker expression by AM. In both cohorts, the majorityof AM were characterised as CD206hiCD86hi. M1-likephenotype (CD206loCD86hi) and M2-like phenotype(CD206hiCD86lo) subsets represented a small proportion(less than 1%) of the total AM population (Fig. 1a and c).In addition, the CD206hiCD86hi subset expressed greaterlevels of the M2 marker, CD163, compared to the M1 andM2 subpopulations (Fig. 1b and d). UK samples werefurther analysed for additional M1 markers (CD80, CD64)and an activation marker (HLADR). In these, theCD206hiCD86hi subset expressed the highest levels ofCD80, CD64 and HLADR expression in comparison tothe other AM subsets (Fig. 1e-g).Colonization with pneumococcus does not alterthe AM phenotype, whereas chronic HIV infectionis associated with a lower expression of CD163 onCD206loCD86hi AMWe have previously shown that live intranasal pneumo-coccal challenge alters the alveolar environment by in-creasing the levels of pneumococcal-specific memoryCD4+ Th17 cells in the human lung [9], and that HIVinfection disrupts the alveolar cytokine microenvironment[10]. We therefore investigated whether in vivo exposureto intranasal pneumococcal challenge or HIVinfection polarise the AM phenotype. We comparedexperimentally pneumococcal-colonized individualswith non-colonized individuals, as well as, asymptom-atic ART-naïve HIV-infected adults with age-matchedHIV-uninfected healthy individuals. We found nodifference in M1 or M2-like phenotype between indi-viduals who became colonized following inoculationwith S. pneumoniae compared with those who clearedthe bacteria (Fig. 1a). Specifically, the expression ofCD163 (Fig. 1b), CD80, CD64 and HLADR measuredin the four AM subsets remain at the same levelsbetween these groups (Fig. 1e-g).Interestingly, in the Malawian population, AM sub-sets from HIV-infected individuals followed the samepattern of subdivision described in healthy volunteers.No difference was found in the proportion of the in-vestigated subsets between the ART-naïve HIV-infected adults and HIV-uninfected healthy individuals(Fig. 1c). However, the expression of CD163 was sig-nificantly reduced in the CD206loCD86hi subset inHIV-infected individuals (p = 0.0147) (Fig. 1d).DiscussionIn our study, we compared the polarisation of AM iso-lated from human volunteers from distinct geographicallocations, UK and Malawi. We report that in healthy hu-man pulmonary mucosa, AM adopt a hybrid phenotype,which shares features of both M1 (CD80, CD86, CD64)and M2 (CD206 and CD163) polarized macrophages.The same phenotype has been previously reported onthe decidual macrophages (resident macrophages of theuterine lining), which exhibit characteristics of both pro-inflammatory and tolerogenic macrophages [11].We also found that nasal pneumococcal colonization,even though it alters CD4+ T cell responses in thealveoli [9], does not alter the expression of key M1 andM2 polarization markers on the surface of AM. Thisleads to the speculation that a nasal pneumococcalexposure episode is either incompetent to modulate AMpolarization or its effect is transient and quicklyirreversible due to macrophages high plasticity [4]. Bycontrast, chronic HIV infection did affect the expressionof CD163 on the M1-like AM subset (CD206loCD86hi),with a lower expression of CD163 observed in HIV-infected individuals than HIV-uninfected controls. Thisfinding is intriguing and may be due to active HIV infec-tion of this alveolar macrophage subset [12] which hasbeen shown to strongly repress CD163 expression oninfected-macrophages [13]. Furthermore, CD163 is shedduring activation as soluble CD163 (sCD163) [14] andthis might in part explain the reduction of CD163 onthe surface M1-like AM in HIV-infected individuals.However, even with in vivo exposure to pneumococcior HIV, the CD206hiCD86hi AM subset remained themajor population, and this ascertains the stability ofthis phenotype.Our data provides strong evidence suggesting that notall macrophages fall into M1 and M2 subsets. The majorAM population in steady state expresses a duo M1/M2phenotype. This phenotype is present in individuals fromtwo distinct geographical location and is stable evenafter exposure to stimuli known to alter the alveolar en-vironment. This is consistent with recent call for a re-think of the M1/M2 macrophage paradigm [5]. It ispossible that the lack of stark polarisation is helpful inmaintaining a healthy balance between immune toler-ance and protective immunity in the alveolar space.However, whether other tissue macrophages, beyond de-cidual and alveolar macrophages, exhibit similar pheno-types in vivo warrants further investigation.ConclusionThe clear majority of alveolar macrophages combine M1and M2 features in steady state, a phenotype that mayallow brisk and adaptive responsiveness to multipleelements in the local milieu.Mitsi et al. Respiratory Research  (2018) 19:66 Page 2 of 4a bc defgFig. 1 Comparison of human alveolar macrophage phenotype between UK and Malawi healthy adults (a) Percentages of AM subsets gated forCD206 and CD86 (UK cohort, n = 9 for grey bars and n = 16 for black bars) and the (b) the degree of CD163 expression in each of the subsets.The data shows that CD206hiCD86hi subset occupies the highest proportion and expresses the highest levels of CD163 when compared with theother three. c-d The same pattern is observed on the AM derived from the healthy Malawian individuals (grey bars, n = 10). The effect ofpneumococcal colonization and HIV infection on AM polarisation. a-b AM subsets are compared between non-colonized (carriage -) and colonizedgroup (carriage +) post experimental nasal pneumococcal inoculation. There is no alteration of AM phenotype associated with nasal pneumococcalcolonization. CD206hiCD86hi is the dominant subset with the highest CD163 expression amongst the rest (p < 0.0001 when comparedwith CD206loCD86hi or CD206hiCD86lo. c AM subsets are compared between HIV-uninfected healthy adults (n = 10) and HIV-infected individuals(n = 10). AM collected from HIV-infected individuals follows the same pattern with healthy HIV-uninfected individuals when gated for CD206 andCD86. d CD206hiCD86hi is the dominant subset with the highest CD163 expression amongst the rest (p < 0.002 when compared with CD206loCD86hior CD206hiCD86lo in both HIV-uninfected and HIV-infected individuals. However, the HIV infection regresses the expression of CD163 in the CD206loCD86hisubset. e-g Levels of CD80, CD64 and HLADR expression respectively between carriage negative and carriage positive individuals. There is no significantdifference on levels of CD80, CD64 and HLADR expression mediated by nasopharynx pneumococcal colonization. Within-group comparisons usedWilcoxon tests, and between-cohort comparisons used Mann-Whitney U testMitsi et al. Respiratory Research  (2018) 19:66 Page 3 of 4Additional fileAdditional file 1: Table S1. Demographics of the EHPC studyparticipants- UK. Table S2. Demographics of study participants – Malawi.Table S3. Summary of the panel composition at the UK site (no shading) andMalawi site (grey shading). Fig. S1. Gating strategy used to identify humanalveolar macrophages in the UK Cohort. Fig. S2. Gating strategy used toidentify human alveolar macrophages in the Malawi Cohort. (DOCX 681 kb)AbbreviationsAM: Alveolar macrophage; BAL: Bronchoalveolar lavageAcknowledgmentsThe authors gratefully would like to thank all participants who participated inthis study, all staff of the Clinical Research Unit at the Royal Liverpool Hospitaland Queen Elizabeth Central Hospital, as well as, the clinical staff of theRespiratory Infection Group at the Liverpool School of Tropical Medicine andImmunology Research Group at the Malawi-Liverpool-Wellcome Trust Clinicalresearch programme (MLW).FundingThis work was funded by Wellcome (UK) through an Intermediate Fellowshipnumber 105831/Z/14/Z awarded to KCJ, the Bill and Melinda GatesFoundation (OPP1117728) and Medical Research Council (Grant number:MR/M011569/) grant awarded to D.M.F. The MLW is supported by astrategic award from the Wellcome.Availability of data and materialsThe datasets used and analysed during the current study will be availablefrom the Liverpool school of Tropical Medicine online repository. This can beaccessed via: http://archive.lstmed.ac.uk/.Authors’ contributionsConception and design: EM, HCM, JR, DMF, KCJ. Analysis and interpretation:EM, RK, JR, CDS, HCM, DMF, KCJ. Drafting the manuscript for importantintellectual content: EM, HCM, JR DMF, KCJ.Ethics approval and consent to participateAt the UK site, the study was approved by the NHS Research Ethics Committee(REC Reference 14/NW/1460). At the Malawi site, approved the study wasapproved by research ethics committee of Malawi College of Medicine, underapproval number P.03/16/1907. All participants provided written informedconsent.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.Received: 9 March 2018 Accepted: 12 April 2018References1. Wissinger E, Goulding J, Hussell T. Immune homeostasis in the respiratorytract and its impact on heterologous infection. Semin Immunol. 2009;21(3):147–55.2. Gordon SB, Read RC. Macrophage defences against respiratory tractinfections. Br Med Bull. 2002;61:45–61.3. Snelgrove RJ, Godlee A, Hussell T. Airway immune homeostasis andimplications for influenza-induced inflammation. Trends Immunol. 2011;32(7):328–34.4. Stout RD, Suttles J. Functional plasticity of macrophages: reversible adaptationto changing microenvironments. J Leukoc Biol. 2004;76(3):509–13.5. Hussell T, Bell TJ. Alveolar macrophages: plasticity in a tissue-specificcontext. Nat Rev Immunol. 2014;14(2):81–93.6. Gritzfeld JF, Wright AD, Collins AM, Pennington SH, Wright AK, Kadioglu A,Ferreira DM, Gordon SB. Experimental human pneumococcal carriage. J VisExp. 2013;(72):50115.7. Zaidi SR, Collins AM, Mitsi E, Reine J, Davies K, Wright AD, Owugha J,Fitzgerald R, Ganguli A, Gordon SB, et al. Single use and conventionalbronchoscopes for Broncho alveolar lavage (BAL) in research: a comparativestudy (NCT 02515591). BMC Pulm Med. 2017;17(1):83.8. Mtunthama N, Malamba R, French N, Molyneux ME, Zijlstra EE, Gordon SB.Malawians permit research bronchoscopy due to perceived need forhealthcare. J Med Ethics. 2008;34(4):303–7.9. Wright AK, Bangert M, Gritzfeld JF, Ferreira DM, Jambo KC, Wright AD,Collins AM, Gordon SB. Experimental human pneumococcal carriageaugments IL-17A-dependent T-cell defence of the lung. PLoS Pathog. 2013;9(3):e1003274.10. Jambo KC, Tembo DL, Kamng'ona AW, Musicha P, Banda DH, KankwatiraAM, Malamba RD, Allain TJ, Heyderman RS, Russell DG, et al. HIV-associateddisruption of lung cytokine networks is incompletely restored inasymptomatic HIV-infected Malawian adults on antiretroviral therapy. ERJOpen Res. 2017;3(4):00097–2017.11. Quillay H, El Costa H, Marlin R, Duriez M, Cannou C, Chretien F, FernandezH, Lebreton A, Ighil J, Schwartz O, et al. Distinct characteristics of endometrialand decidual macrophages and regulation of their permissivity to HIV-1infection by SAMHD1. J Virol. 2015;89(2):1329–39.12. Jambo KC, Banda DH, Kankwatira AM, Sukumar N, Allain TJ, Heyderman RS,Russell DG, Mwandumba HC. Small alveolar macrophages are infectedpreferentially by HIV and exhibit impaired phagocytic function. MucosalImmunol. 2014;7(5):1116–26.13. Porcheray F, Samah B, Leone C, Dereuddre-Bosquet N, Gras G. Macrophageactivation and human immunodeficiency virus infection: HIV replicationdirects macrophages towards a pro-inflammatory phenotype while previousactivation modulates macrophage susceptibility to infection and viralproduction. Virology. 2006;349(1):112–20.14. Burdo TH, Lentz MR, Autissier P, Krishnan A, Halpern E, Letendre S,Rosenberg ES, Ellis RJ, Williams KC. Soluble CD163 made by monocyte/macrophages is a novel marker of HIV activity in early and chronic infectionprior to and after anti-retroviral therapy. J Infect Dis. 2011;204(1):154–63.Mitsi et al. Respiratory Research  (2018) 19:66 Page 4 of 4

Human alveolar macrophages predominately express combined classical M1 and M2 surface markers in steady state.

Abstract Alveolar macrophages (AM) are critical to the homeostasis of the inflammatory environment in the lung. Differential expression of surface markers classifies macrophages to either classically (M1) or alternatively activated (M2). We investigated the phenotype of human alveolar macrophages (AM) in adults living in two different geographical locations: UK and Malawi. We show that the majority of AM express high levels of M1 and M2 markers simultaneously, with the M1/M2 phenotype being stable in individuals from different geographical locations. The combined M1/M2 features confer to AM a hybrid phenotype, which does not fit the classic macrophage classification. This hybrid phenotype may confer to alveolar macrophages an ability to quickly switch between M1 or M2 associated functions allowing for appropriate responses to stimuli and tissue environment

Elena Mitsi

Raphael Kamng’ona

Jamie Rylance

Carla Solórzano

J. Jesus Reiné

Henry C. Mwandumba

Daniela M. Ferreira

Kondwani C. Jambo

Directory of Open Access Journals

Human alveolar macrophages predominately express combined classical M1 and M2 surface markers in steady state

Crossref

http://archive.lstmed.ac.uk/8560/1/respiratory_research_Human%20alveolar%20macrophages.pdf

Human alveolar macrophages predominately express combined classical M1 and M2 surface markers in steady state.

Abstract

Similar works

Full text

Available Versions

LSTM Online Archive

Directory of Open Access Journals

Crossref