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Flow cytometry evaluation of CD14/CD16 monocyte subpopulations in systemic sclerosis patients: a cross sectional controlled study

Flow cytometry evaluation of CD14/CD16 monocyte subpopulations in systemic sclerosis patients: a... Background: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by vasculopathy and fibrosis, which can be subclassified into diffuse cutaneous (dSSc) and limited cutaneous (lSSc) subtypes. Previous studies suggest that an increase in monocytes can be a hallmark of various inflammatory diseases, including SSc. Our aim was to evaluate circulating blood monocyte subpopulations (classical, intermediate and non-classical) of SSc patients and their possible association with disease manifestations. Methods: Fifty consecutive patients fulfilling the 2013 ACR/EULAR classification criteria for SSc were included in a cross-sectional study. Monocyte subpopulations were identified based on their expression of CD64, CD14 and CD16, evaluated by flow cytometry, and were correlated with the clinical characteristics of the patients; furthermore, the expression of HLA-DR, CD163, CD169 and CD206 in the monocytes was studied. Thirty-eight age- and sex-matched healthy individuals were recruited as a control group. Results: SSc patients had an increased number of circulating peripheral blood monocytes with an activated phenotypic profile compared to healthy subjects. Absolute counts of CD16+ (intermediary and non-classical) monocyte subpopulations were higher in SSc patients. There was no association between monocyte subpopulations and the clinical manifestations evaluated. Conclusion: We identified higher counts of all monocyte subpopulations in SSc patients compared to the control group. There was no association between monocyte subpopulations and major fibrotic manifestations. CD169 was shown to be more representative in dSSc, being a promising marker for differentiating disease subtypes. Keywords: Systemic sclerosis, Monocytes, Monocyte subpopulations, Flow cytometry, Pathogenesis * Correspondence: laia_schneider@hotmail.com Serviço de Reumatologia, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, 2350 Ramiro Barcelos St, Room 645, Porto Alegre, RS 90035-903, Brazil Programa de Pós Graduação em Medicina: Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 2 of 11 Background Peripheral blood monocytes heterogeneity still lacks a Systemic sclerosis (SSc) is a chronic inflammatory sys- clear comprehension of its role in SSc pathophysiology temic disease, mainly characterized by expansion of the and clinical manifestations. The aim of our study was to secondary extracellular matrix due to an exaggerated pro- characterize the three monocytes subpopulations of SSc tein production, especially collagen. Collagen is deposited patients, analyze the expression of CD163, CD169, as a result of the abnormal interaction between endothe- CD206 and HLA-DR (function and activation monocytes lial cells, mononuclear cells (lymphocytes and monocytes) receptors), and their association with clinical findings. and fibroblasts. Monocytes express several receptors, which monitor and detect environmental changes. Those Methods cells present great plasticity and heterogeneity and have Patients the ability to modify their functional phenotype in re- Fifty consecutive patients fulfilling the 2013 ACR/ sponse to stimulation [1]. There seems to be a relationship EULAR classification criteria for SSc [16] were recruited between the monocytes/macrophages activation and mi- from the Outpatient Rheumatology Clinic of the Hos- gration [2, 3], and SSc pathogenesis [4]. pital de Clínicas de Porto Alegre, Brazil. Clinical form of Evidences from human and murine studies suggest that disease was classified as diffuse SSc (dSSc) or limited an increase in monocytes can be an indicator of various SSc (lSSc) according to the revised classification of inflammatory diseases and that these cells could differenti- LeROY [17, 18]. Medications under use were obtained ate into inflammatory or anti-inflammatory subgroups [5]. from medical records and were confirmed during med- For almost two decades studies have shown the presence ical appointments. Patients receiving immunosuppres- of two subsets of cells: classical and non-classical mono- sive drugs for SSc at the time of inclusion were carefully cytes. Classical monocytes, characterized by expression of evaluated since those medications can influence mono- CD14++CD16-, represent about 80% of all monocytes and cytes counts and function [9]. Only patients that re- have phagocytic function, with production of reactive oxy- ceived cyclophosphamide treatment at least 6 months gen species and secretion of pro- and anti-inflammatory prior to the interview were included. Disease duration cytokines in response to agonist stimulation of toll-like was defined as the time interval since the onset of Ray- receptor-4 (TLR-4) [6]. Whereas non-classical monocytes, naud’s phenomenon or skin symptoms (whichever came which express CD16+, have a higher activity in response first). Severity of skin involvement was assessed using to viruses and produce proinflammatory cytokines the modified Rodnan skin score [19]. Laboratory test re- through TLR-8 and TLR-9 [7, 8]. sults including serology, presence of autoantibodies and In recent years, a third group of monocytes has been pulmonary function tests such as assessment of lung dif- proposed, dividing the CD16+ population into non- fusion capacity for carbon monoxide (DLCO) and forced classical and intermediate monocytes (respectively, vital capacity (FVC) performed in the period of 3 CD14+/−CD16++ and CD14 + CD16+) [8–10]. These months before or after the peripheral blood sample col- CD16+ monocytes appear to be related to alternative ac- lection were obtained in medical records. Patients with tivated M2 macrophages, and their activation pathway scleroderma in overlap syndrome with other connective showed that they are associated with T helper (Th) 2 re- tissue disease and/or infectious diseases (HIV, hepatitis sponse [5, 11]. The differences between these monocyte C or B) were excluded. subsets have been made clear, with distinct phenotypes and different functions, such as defense against patho- gens, homeostasis and tissue repair [9, 10, 12]. Controls Evaluation of mannose receptor-1 (MRC1 or CD206) Thirty-eight subjects age- and sex-matched to the SSc pa- and CD163 (macrophage scavenger receptors) that are tients, with no previous history of autoimmune or typical of M2 macrophage activation pathway, showed hematological diseases and a normal complete blood cell an association of these markers with tissue repair and fi- (CBC) count (hemoglobin > 11.0 g/dl; white blood cells > brosis [13], and in SSc patients those markers are associ- 3 3 3 3 4×10 /mm and < 12 × 10 /mm ; and platelets > 150 × ated with high pulmonary artery pressure and increased 3 3 10 /mm ) were recruited from a local blood donation cen- mortality [14]. Other marker that may play a key role in ter and at a Primary Health Care Service unit. The criteria the monocyte activation present in SSc is CD169 (sialic for inclusion were the same as those used in blood acid-binding Ig-like lectine I or Siglec-1), also known as donation. sialoadhesin, which has been shown to have a higher ex- pression in a subset of SSc patients monocytes [15]. Monocytes of SSc patients have a greater expression of Study design these molecules, which is even higher in patients with This was a monocentric cross-sectional study carried tissue disease and pulmonary artery hypertension. out from October, 2016, to May, 2017. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 3 of 11 Flow cytometry protocol Biosciences) for 10 min. Samples were washed and resus- Peripheral blood samples were collected in ethylenedi- pended in phosphate buffered saline (PBS). At least 30, aminetetraacetic acid (EDTA) coated tubes. CBC was 000 gated monocytes were acquired in an 8-color BD performed in a Sysmex XE-2100™ (Sysmex, Kobe, JPN) FACSCanto™ II flow cytometer (BD Biosciences) using hematological counter and the total amount of periph- FACSDiva™ software (BD Biosciences). Flow cytometer eral blood monocytes was determined. technical parameters were automatically set with BD Flow cytometry immunophenotyping (FCI) to identify Cytometer Setup and Tracking Beads (BD Biosciences). and characterize monocyte subpopulations was per- Daily internal quality control (QC) was performed with formed less than 4 h after blood sampling. About 1,000, IMMUNO-TROL Cells (BD Biosciences). 000 cells were placed into polystyrene tubes and were subjected to surface staining by incubation with titrated Phenotypic analysis of monocyte subpopulations amounts of fluorochrome-conjugated monoclonal anti- Figure 1 shows the gating strategy used. Initially, dou- bodies (MoAbs) against CD14 APC-H7 (clone MφP9), blets were excluded and total nucleated cells were se- CD64 BV421 (clone 10.1), CD16 BV510 (clone 3G8), lected based on FSC (forward scatter) versus SSC (side HLA-DR PerCP-Cy5.5 (clone G46–6), CD206 APC scatter) distribution. Monocytes were gated based on cell (clone 19.2), CD169 BB515 (clone 7–239) and CD163 size and complexity, as well as CD64+ expression [20], PE (clone GHI/61) for 30 min at room temperature and and were plotted in a CD14 versus CD16 graph in order protected from light. All MoAbs were purchased from to characterize all three monocyte subpopulations: clas- BD Biosciences (San Diego, USA). Red blood cells were sical monocytes (CD14++CD16-), intermediate mono- lysed by incubation with 2 ml of lysing buffer (BD cytes (CD14++CD16+) and non-classical monocytes Fig. 1 Identification of monocyte subpopulations. a Cells were gated based on FSC (forward scatter) versus SSC (side scatter) distribution and doublets were excluded. b Monocytes were gated based on cell size and complexity, as well as CD64 expression. c Monocyte subpopulations were plotted in a CD14 versus CD16 expression in a representative healthy donor Schneider et al. Advances in Rheumatology (2021) 61:27 Page 4 of 11 (CD14+/−CD16++). Fluorescence-minus-one (FMO) monocyte subpopulation were recorded. All FCI data methodology was used to differentiate autoflourescent were analyzed using Kaluza Analysis Software (Beckman events from events with low concentrations of cell sur- Coulter Life Sciences, Indianapolis, USA) by two experi- face markers [21]. All values obtained in this analysis enced operators concomitantly. were considered FMO limit and all values of fluores- cence above the limit were considered as true positive. Compliance with ethical standards Percentage of expression and mean fluorescence inten- This study was approved by the Research Ethics Com- sity (MFI) of any given marker within the defined mittee of the institution (CAAE number Table 1 Demographic, clinical, and laboratory features of systemic sclerosis patients and controls Systemic sclerosis patients Healthy controls (n = 50) (n = 38) a a Female gender (%) 47 (94.0) 36 (94.7) b b Age (years) 57.2 ± 12.8 55.2 ± 11.4 European-derived (%) 40 (80) 24 (68.6) Smoking status (%) Never 25 (50) 23 (65.7) Previous 21 (42) 30 (29.7) Current 4 (8) 2 (5.7) Disease duration (years) 15 (7–21) Diffuse cutaneous involvement (%) 14 (28.0) Modified Rodnan skin score 6 (2.8–10.3) Renal crisis 4 (8.0) Gastrointestinal symptoms (%) Heartburn 25 (50.0) Dysphagia 26 (52.0) Diarrhea / constipation 21 (42.0) DLCO (% predicted) 70.1 ± 16.3 Reduced DLCO (< 80% predicted) 52.9 (15.9) FVC (% predicted) 79.2 ± 18.9 Reduced FVC (< 80% of predicted) 22 (44) PASP 31.7 ± 14.4 Pulmonary hypertension (PASP ≥40 mmHg) (%) 5 (10) Autoantibodies (%) ANA 39/50 (78.0) Anti-Ro/SSA 1/45 (2.2) Anti-La/SSB 0/45 (0) Anti-Scl-70 4/44 (9.1) RF 35/50 (75.0) Medications (%) Prednisolone < 20 mg/day 6 (12) Mycophenolate mofetil 4 (8) Previous methotrexate administration 10 (20) Previous rituximab administration 2 (4) Previous cyclophosphamide administration 12 (24) Data are shown as mean (± standard deviation), median (interquartile ranges) or number (n) ANA anti-nuclear antibodies, Anti-dsDNA anti-double-stranded DNA, Anti-Scl70 antitopoisomeraseI, DLCO diffusion capacity of carbon monoxide, FVC forced vital capacity, PASP arterial systolic pressure on Doppler echocardiography, RF rheumatoid factor, SSc systemic sclerosis Mann-Whitney test, p = 1.000 Student’s T test, p = 0.448 Schneider et al. Advances in Rheumatology (2021) 61:27 Page 5 of 11 3 3 49299515.0.0000.5327) and was conducted in accordance mm , p < 0.001; and 387.4 (310.4–551.1)/mm versus with the Declaration of Helsinki and current laws in 273.9 (179.3–352.2)/mm , p = 0.002, respectively) (data Brazil. Participants were included after written informed not shown). Among monocyte subpopulations, SSc pa- consent. tients had higher absolute counts of all subpopulations (classical, intermediate and non-classical) when com- Statistical analysis pared to HC (classical monocytes 346.2 (260.9–450.8)/ 3 3 Variables distribution was analyzed by Shapiro-Wilk’s mm versus 209.8 (146.1–287.1)/mm , p < 0.001; inter- test and continuous variables were compared by Stu- mediate monocytes 38.2 (24.6–47.1)/mm versus 25.4 dent’s T or Mann–Whitney’s U test, whereas propor- (12.6–41.2)/mm , p = 0.005; non-classical monocytes 3 3 tions were compared by Pearson’s chi-square or Fisher’s 41.2 (20.8–58.3)/mm versus 28.3 (11.5–46.1)/mm , p = exact test. ANOVA or Kruskall-Wallis, with a Tukey or 0.006) (data not shown). Regarding disease subtype, lSSc Dunn correction test was used for multiple comparisons. patients presented increased absolute values of all mono- Pearson or Spearman correlation test was used to evalu- cyte subpopulations when compared to HC (classical ate association between variables. The statistical signifi- monocytes 363.2 (262.9–451.8)/mm versus 209.8 cance level adopted was 5% (p < 0.05). Data analysis was (146.1–287.1)/mm , p < 0.001 (A); intermediate mono- carried out with SPSS version 21.0® (IBM, Chicago, cytes 38.5 (24.7–47.5)/mm versus 25.4 (12.6–41.2)/ USA) and Prisma 8 software (Graphpad Inc. San Diego, mm , p = 0.009 (B); non-classical monocytes 42.5 (20.8– 3 3 USA). 69.0)/mm versus 28.3 (11.5–46.1)/mm , p = 0.005) (C). While in dSSc patients, only classical monocytes had in- Results creased absolute counts compared to HC (209.8 (146.1– 3 3 Patients and controls characteristics 287.1)/mm versus 326.5 (231.2–461.9)/mm , p = 0.003) Demographic data and clinical characteristics of SSc and (A). There was no difference in monocytes absolute or healthy control (HC) subjects are shown in Table 1. SSc relative values between lSSc and dSSc subgroups (p = patients mean age was 57.2 years and 94% were female. 0.450 and p = 0.914, respectively). Limited form of disease was present in 72% of SSc pa- tients. Median Rodnan skin score was 5 (2.0–8.0) in lSSc Circulating CD16+ monocyte subpopulations patients and 11 (3.8–16.8) in the dSSc patients (p = When analyzing intermediate and non-classical mono- 0.007). Pulmonary function also differed among disease cytes as a single group of CD16+ monocytes, we identi- subgroups when comparing forced vital capacity fied a higher absolute count of this subpopulation in (FVC%predicted) results: lSSc subjects had higher mean lSSc patients compared to HC (79.9 (53.4–103.5)/mm values when compared to dSSc patients (83.8 ± 17.4% versus 55.9 (26.8–85.8)/mm , p = 0.003) (Fig. 2d). versus 67.5 ± 18.2%, p = 0.005). Circulating monocyte subpopulations Monocyte subpopulations and clinical manifestations Main results of monocytes evaluation are show in Fig. 2. Absolute counts of monocyte subpopulations did not SSc patients had higher absolute and relative number of correlate with severity of cutaneous fibrosis assessed by circulating monocytes when compared to HC (459.6 the modified Rodnan skin score, pulmonary involvement 3 3 (322.9–550.3)/mm versus 273.9 (179.3–352.2)/mm , assessed by DLCOc/VA, FVC and pulmonary artery sys- p < 0.001), this difference remained significant when tolic pressure, or disease duration when evaluated comparing lSSc or dSSc patients with HC independently among the total of patients or in lSSc and dSSc subtypes (478.2 (342.9–555.5)/mm versus 273.9 (179.3–352.2)/ (Fig. 3) (Additional files 1 and 2). Fig. 2 Monocyte subpopulations according to their membrane expression of CD14 and CD16 in systemic sclerosis patients with limited (lSSc) (n = 36) and diffuse (dSSc) (n = 14) subtypes, and in healthy controls (HC) (n = 38). The absolute count of all monocyte subpopulations - classical a, intermediate b and non-classical c - is increased in lSSc compared to HC. d When analyzing intermediate and non-classical monocytes as a single group of CD16+ monocytes, their absolute value is increased in lSSc compared to HC. *p < 0.05; **p < 0.01; ***p < 0.001 Schneider et al. Advances in Rheumatology (2021) 61:27 Page 6 of 11 Expression of surface receptors in monocyte expression was lower in both lSSc and dSSc classical subpopulations monocytes when compared to HC. CD163, macrophage Results of surface expression of HLA-DR, CD206, scavenger receptor, was more expressed in classical (per- CD169 and CD163 receptors are shown in Fig. 4 and centage and MFI) and intermediate (percentage) mono- Additional file 3. There were different expression pat- cytes of SSc patients as compared to HC. In subgroup terns among the evaluated monocyte subpopulations. analysis, a difference in CD163 expression was demon- HLA-DR, an activation marker, was consistently strated in classical monocytes of lSSc subjects compared expressed in all monocytes from SSc and HC, and its in- to the HC group. CD169 receptor had a higher percent- tensity of expression was higher in all monocyte subsets age of expression in SSc versus HC. When evaluating from lSSc and dSSc patients when compared to control. SSc subtypes, classical and intermediate monocytes from CD206 expression varied among the monocyte subsets, dSSc patients had higher MFI and percentage of CD169 the percentage of expression was higher in classical and expression than lSSc and HC, whereas non-classical intermediate monocytes of SSc patients when compared monocytes of dSSc patients had higher percentage of this to HC and its MFI expression was lower in SSc as com- marker expression (Fig. 4). pared to HC. When analyzing SSc subgroups, percentage Further analyses were performed for comparison of of CD206, the mannose receptor-1, expression was the markers MFI of expression among the different higher only in classical monocytes of lSSc patients com- monocyte subpopulations. Except for CD163 expres- pared to HC and in intermediate monocytes of lSSc and sion, the monocyte subpopulations exhibited distinct dSSc compared to HC; whereas MFI of CD206 expression patterns when compared to one another, Fig. 3 Correlations regarding the CD16+ monocyte subpopulation. The CD16+ monocyte subpopulation of SSc patients (n = 50) and in the subgroup of patients presenting lSSc (n = 36) and dSSc (n = 14) was tested for association with cutaneous fibrosis assessed by the modified Rodnan skin score (mRSS), pulmonary involvement assessed by DLCOc/VA and FVC. Disease duration was not associated with CD16 + values. DLCO: diffusion capacity for carbon monoxide; FVC: forced vital capacity Schneider et al. Advances in Rheumatology (2021) 61:27 Page 7 of 11 Fig. 4 Expression of surface receptors under study on monocyte subpopulations of systemic sclerosis patients divided by disease subtype in lSSc (n = 36) and dSSc (n = 14) compared with healthy controls (HC) (n = 38). ANOVA with Tukey correction for symmetric distribution and Kruskal- Wallis with Dunn correction for asymmetric distribution. MFI: mean fluorescence intensity. SSc: systemic sclerosis; HC: healthy controls; lSSc: limited systemic sclerosis; dSSc: diffuse systemic sclerosis. *p < 0.05; **p < 0.01; ***p < 0.001 Schneider et al. Advances in Rheumatology (2021) 61:27 Page 8 of 11 which was similar among patients and HC subgroups increased amount of circulating monocytes with CD16+ (Additional file 3). monocytes as the main representative monocyte subset in SSc patients [5, 32]. Nevertheless, our results differ re- Discussion garding correlations with clinical manifestations. The In our study, monocytes percentage and absolute count fact that we did not find a correlation of monocyte sub- were significantly increased in SSc patients as compared sets with clinical manifestations reinforces the hypoth- to healthy subjects, regardless of disease subtype. Monocy- esis of cellular modulation and activation in loco, since tosis in SSc patients has been reported previously [5, 22]. the different tissues are able to direct the inflammatory Assessment of CD14 and CD16 receptors expression response in order to exacerbate inflammation with pro- allowed the identification of all three monocyte subpopu- inflammatory mediators or into resolution and repair lations, as already described for healthy individuals and [33]. Hence, findings of monocyte/macrophage activa- some disease states [9, 23–25]. However, our study is the tion in the bloodstream may not be observed at the tis- first to describe these subpopulations in SSc patients, sue level, specifically, in internal organs affected by SSc. using specific markers CD14 and CD16 for such distinc- HLA-DR is a cell activation marker, so it is consistent tion according to positivity and MFI of expression. to find a greater expression intensity of this marker in We identified an increase in all monocyte subpopula- patients with autoimmune diseases when compared to tions in SSc patients, which was even more significant in normal subjects. We found an increase in MFI expres- patients with lSSc. Interestingly, lSSc patients showed an sion of HLA-DR in all monocyte subpopulations of SSc increase of all three monocyte subpopulations compared patients compared to HC. MFI of HLA-DR expression to controls, whereas in dSSc patients that increase was in CD16+ monocyte subpopulations (intermediate and at expense of classical monocytes, indicating that in this non-classical) was significantly higher than that of form of disease the CD16+ monocyte subpopulations CD16- classical monocytes, indicating a greater activa- does not increase. Recent studies in SSc patients have tion of CD16+ antigen-presenting cells, which is ex- shown an increase in CD16+ monocytes in the periph- pected in an autoimmune state. eral blood of these patients, however, these studies did Other molecules, such as capture receptor CD163 and not distinguish between intermediate and non-classical CD206 were also associated with M2 macrophages and subpopulations [5, 22]. could be involved into further differentiation processes. A Previous studies have shown a significant correlation previous study comparing the expression of monocyte and of the absolute CD16+ monocyte subpopulation count macrophage markers in peripheral blood and skin biopsy and severity of skin fibrosis and pulmonary fibrosis [5, specimens showed higher percentages of circulating cells 26]. In our study, we did not find correlations between expressing CD204, CD163 and CD14, furthermore these monocyte subpopulations and the clinical manifestations findings were correlated with skin involvement [22]. evaluated. This difference may be related with inter- Additionally, circulating CD14+ monocytes/macro- mediate and non-classical monocyte subpopulations phages with CD206 coexpression have been shown to be grouped into CD16+ monocytes. Although differences associated with pulmonary artery hypertension [13]. It has among intermediate and non-classical monocyte subsets also been shown that macrophages derived from circulat- have been stablished [9, 10, 12], there is a strong prox- ing monocytes have coexpression of CD206, CD163 and imity between them [9], suggesting a direct developmen- CD169, and that the expression of these markers is signifi- tal relationship, where gain of CD16 expression and cantly higher in patients with pulmonary interstitial dis- decrease in CD14 expression may be indicative of mat- ease [3]. In our study, a higher percentage of classical and uration [27] and inflammatory activation for these cells intermediate monocytes with CD206 expression were to be recruited to the periphery, where they begin to act identified in SSc patients compared to HC, which demon- in localized inflammatory processes [5]. In addition, strates a greater amount of M2-like cells in those mono- there are resident tissue macrophages originated from cyte subgroups of SSc patients. the yolk sac or from fetal liver progenitors [28], that Using a flow cytometry approach for assessing surface have long tissue life and self-renewing ability by in situ markers from circulating monocytes/macrophages, Sol- proliferation, even in the absence of recruitment from dano et al. (2018) demonstrated that SSc patients have circulating monocytes [29–31]. The role of these macro- higher expression of the M2 related markers CD14, phages in tissues during inflammation is still unclear CD206, CD163 and CD204; or that there is a coexpres- and further research is needed on the role of circulating sion of M1 and M2 surface markers in those cells [34]; monocytes for their activation and proliferation, espe- providing evidence of plasticity in the expression of this cially in inflammation models. markers in circulating monocyte/macrophage and that Our data corroborate the results obtained by Lescoat monocytes can evolve to exhibit features that are shared et al. (2017) and Trombetta et al. (2018) concerning the by more than one macrophage population. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 9 of 11 Previous studies have suggested that CD169 may be a subpopulations between lSSc and dSSc disease subtypes. phagocyte activity marker in inflammatory disorders. Monocytes from SSc patients presented a more activated CD169 has been identified as a marker of highly patho- phenotypic profile which was suggestive of M2 cells, genic phagocytes in multiple sclerosis patients. Also, an with higher expression of CD206. Opposing to previous increased expression of Siglec-1 on peripheral blood reports, we did not find an association between mono- monocytes as well as its role in cell reactivity to autoan- cyte subpopulations and major fibrotic manifestations. tigen has been shown in rheumatoid arthritis [35, 36]. The intermediate and non-classical monocyte subpopu- CD169 expression in renal macrophages has been corre- lations of SSc patients have increase expression of HLA- lated with proteinuria and tissue damage in glomerulo- DR. CD169 appears to be more representative marker nephritis [37], however its expression in peripheral for dSSc subtype, however, more studies are necessary to blood cells was not assessed. CD169 expression has also confirm that this is a good marker for differentiation of been associated with involvement in the immune system disease subtype. regulation [38]. Biological functions of CD169 in macro- Abbreviations phages and monocytes have yet to be elucidated. In our CBC: Complete blood cell; DLCO: Lung diffusion capacity for carbon study, there was an increased expression of CD169 in monoxide; dSSc: Diffuse cutaneous systemic sclerosis; EDTA: Ethylenediaminetetraacetic acid; FCI: Flow cytometry classical monocytes from SSc patients compared to con- immunophenotyping; FMO: Fluorescence-minus-one; FSC: Forward scatter; trols, and CD169 expression was higher in all monocyte FVC: Forced vital capacity; HC: Healthy control; lSSc: Limited cutaneous subpopulations of dSSc patients compared to lSSc and systemic sclerosis; MFI: Mean fluorescence intensity; MoAbs: Monoclonal antibodies; MRC1: Mannose receptor-1; PBS: Phosphate buffered saline; controls. This finding could represent a greater disease QC: Quality control; Siglec-1: Sialic acid-binding Ig-like lectine I; SSc: Systemic activity in this subgroup of patients. sclerosis; SSC: Side scatter; Th: T helper; TLR: Toll-like receptor As to the use of medications, no association of these with subpopulations of monocytes was found. There Supplementary Information were no differences between patients monocyte subpop- The online version contains supplementary material available at https://doi. ulations regarding corticosteroids use (n = 6). Patients org/10.1186/s42358-021-00182-8. included in the study used low doses (less than 20 mg Additional file 1: Table S1. Spearman correlation coefficient values per day), similar to previous reports [5]. between monocyte subpopulations and clinical data. Our study has some limitations. First of all, the patient Additional file 2: Table S2. Analysis of the absolute of CD16 + in population appears to be relatively small. However, there relation to the time of disease separated by tercil. were many restrictions for patient inclusion in order to Additional file 3: Table S3. Comparison of the markers MFI of minimize treatment interference and disease overlap. expression among the different monocyte subpopulations. Evaluation of exclusively circulating cells is also a limita- tion. There is a need for simultaneous assessment of cir- Acknowledgements culating cells and tissue biopsies to better understand the We would like to thank Fabiane Spagnol Pedrazzani, at the Department of Specialized Diagnostics of the Hospital de Clínicas de Porto Alegre, for her participation of macrophages derived from circulating assistance in the design of the technical parameters for the flow cytometry monocytes in tissue fibrosis. We have evidence that protocol. monocytes and macrophages actually participate in SSc pathogenesis and that there are slight differences between Authors’ contributions LS, VH, RX and RC designed the project. LS, NAM, VH, IFSM, CYU and RRP tissues [1, 39], nevertheless, we need better strategies to collected the data and were responsible for data analysis. LS standardized analyze these cells and studies assessing different tissues and performed the flow cytometry experiments. LS and NAM wrote the and peripheral blood to determine whether these macro- manuscript. VH, RX and RC reviewed the manuscript. All authors read and approved the final manuscript. phages are derived from circulating monocytes or other cells. In addition, our findings should be interpreted in Funding light of our sample characteristics. Considering the We are grateful to the Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) and the Conselho Nacional de pathophysiological and clinical changes observed in SSc Desenvolvimento Científico e Tecnológico (CNPq) for the financial support. over time, different monocyte subpopulations could be observed in early phases of the disease. Also, monocytes Availability of data and materials activation may be influenced by different levels of im- The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. munosuppression and distinct mechanisms of action of immunosuppressive drug treatment. Declarations Conclusions Ethics approval and consent to participate This study was approved by the Research Ethics Committee of the institution In conclusion, our results showed that SSc patients have (CAAE number 49299515.0.0000.5327) and was conducted in accordance increased absolute counts of all three monocyte subpop- with the Declaration of Helsinki and current laws in Brazil. Participants were ulations and that there is no difference among monocyte included after written informed consent. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 10 of 11 Consent for publication macrophages are phenotypically and functionally distinct. Blood. 2014; Not applicable. 123(20):e110–22. https://doi.org/10.1182/blood-2013-08-520619. 15. York MR, Nagai T, Mangini AJ, Lemaire R, van Seventer JM, Lafyatis R. A macrophage marker, Siglec-1, is increased on circulating monocytes in Competing interests patients with systemic sclerosis and induced by type I interferons and toll- The authors declare that they have no competing interests. like receptor agonists. Arthritis Rheum. 2007;56(3):1010–20. https://doi.org/1 0.1002/art.22382. Author details 16. van den Hoogen F, Khanna D, Fransen J, Johnson SR, Baron M, Tyndall Serviço de Reumatologia, Hospital de Clínicas de Porto Alegre, Universidade A, et al. 2013 classification criteria for systemic sclerosis: an American Federal do Rio Grande do Sul, 2350 Ramiro Barcelos St, Room 645, Porto College of Rheumatology/European league against rheumatism Alegre, RS 90035-903, Brazil. Programa de Pós Graduação em Medicina: collaborative initiative. Arthritis Rheum. 2013;65(11):2737–47. https://doi. Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, org/10.1002/art.38098. 3 4 Brazil. Laboratório Zanol, Porto Alegre, Brazil. 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Flow cytometry evaluation of CD14/CD16 monocyte subpopulations in systemic sclerosis patients: a cross sectional controlled study

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Abstract

Background: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by vasculopathy and fibrosis, which can be subclassified into diffuse cutaneous (dSSc) and limited cutaneous (lSSc) subtypes. Previous studies suggest that an increase in monocytes can be a hallmark of various inflammatory diseases, including SSc. Our aim was to evaluate circulating blood monocyte subpopulations (classical, intermediate and non-classical) of SSc patients and their possible association with disease manifestations. Methods: Fifty consecutive patients fulfilling the 2013 ACR/EULAR classification criteria for SSc were included in a cross-sectional study. Monocyte subpopulations were identified based on their expression of CD64, CD14 and CD16, evaluated by flow cytometry, and were correlated with the clinical characteristics of the patients; furthermore, the expression of HLA-DR, CD163, CD169 and CD206 in the monocytes was studied. Thirty-eight age- and sex-matched healthy individuals were recruited as a control group. Results: SSc patients had an increased number of circulating peripheral blood monocytes with an activated phenotypic profile compared to healthy subjects. Absolute counts of CD16+ (intermediary and non-classical) monocyte subpopulations were higher in SSc patients. There was no association between monocyte subpopulations and the clinical manifestations evaluated. Conclusion: We identified higher counts of all monocyte subpopulations in SSc patients compared to the control group. There was no association between monocyte subpopulations and major fibrotic manifestations. CD169 was shown to be more representative in dSSc, being a promising marker for differentiating disease subtypes. Keywords: Systemic sclerosis, Monocytes, Monocyte subpopulations, Flow cytometry, Pathogenesis * Correspondence: laia_schneider@hotmail.com Serviço de Reumatologia, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, 2350 Ramiro Barcelos St, Room 645, Porto Alegre, RS 90035-903, Brazil Programa de Pós Graduação em Medicina: Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 2 of 11 Background Peripheral blood monocytes heterogeneity still lacks a Systemic sclerosis (SSc) is a chronic inflammatory sys- clear comprehension of its role in SSc pathophysiology temic disease, mainly characterized by expansion of the and clinical manifestations. The aim of our study was to secondary extracellular matrix due to an exaggerated pro- characterize the three monocytes subpopulations of SSc tein production, especially collagen. Collagen is deposited patients, analyze the expression of CD163, CD169, as a result of the abnormal interaction between endothe- CD206 and HLA-DR (function and activation monocytes lial cells, mononuclear cells (lymphocytes and monocytes) receptors), and their association with clinical findings. and fibroblasts. Monocytes express several receptors, which monitor and detect environmental changes. Those Methods cells present great plasticity and heterogeneity and have Patients the ability to modify their functional phenotype in re- Fifty consecutive patients fulfilling the 2013 ACR/ sponse to stimulation [1]. There seems to be a relationship EULAR classification criteria for SSc [16] were recruited between the monocytes/macrophages activation and mi- from the Outpatient Rheumatology Clinic of the Hos- gration [2, 3], and SSc pathogenesis [4]. pital de Clínicas de Porto Alegre, Brazil. Clinical form of Evidences from human and murine studies suggest that disease was classified as diffuse SSc (dSSc) or limited an increase in monocytes can be an indicator of various SSc (lSSc) according to the revised classification of inflammatory diseases and that these cells could differenti- LeROY [17, 18]. Medications under use were obtained ate into inflammatory or anti-inflammatory subgroups [5]. from medical records and were confirmed during med- For almost two decades studies have shown the presence ical appointments. Patients receiving immunosuppres- of two subsets of cells: classical and non-classical mono- sive drugs for SSc at the time of inclusion were carefully cytes. Classical monocytes, characterized by expression of evaluated since those medications can influence mono- CD14++CD16-, represent about 80% of all monocytes and cytes counts and function [9]. Only patients that re- have phagocytic function, with production of reactive oxy- ceived cyclophosphamide treatment at least 6 months gen species and secretion of pro- and anti-inflammatory prior to the interview were included. Disease duration cytokines in response to agonist stimulation of toll-like was defined as the time interval since the onset of Ray- receptor-4 (TLR-4) [6]. Whereas non-classical monocytes, naud’s phenomenon or skin symptoms (whichever came which express CD16+, have a higher activity in response first). Severity of skin involvement was assessed using to viruses and produce proinflammatory cytokines the modified Rodnan skin score [19]. Laboratory test re- through TLR-8 and TLR-9 [7, 8]. sults including serology, presence of autoantibodies and In recent years, a third group of monocytes has been pulmonary function tests such as assessment of lung dif- proposed, dividing the CD16+ population into non- fusion capacity for carbon monoxide (DLCO) and forced classical and intermediate monocytes (respectively, vital capacity (FVC) performed in the period of 3 CD14+/−CD16++ and CD14 + CD16+) [8–10]. These months before or after the peripheral blood sample col- CD16+ monocytes appear to be related to alternative ac- lection were obtained in medical records. Patients with tivated M2 macrophages, and their activation pathway scleroderma in overlap syndrome with other connective showed that they are associated with T helper (Th) 2 re- tissue disease and/or infectious diseases (HIV, hepatitis sponse [5, 11]. The differences between these monocyte C or B) were excluded. subsets have been made clear, with distinct phenotypes and different functions, such as defense against patho- gens, homeostasis and tissue repair [9, 10, 12]. Controls Evaluation of mannose receptor-1 (MRC1 or CD206) Thirty-eight subjects age- and sex-matched to the SSc pa- and CD163 (macrophage scavenger receptors) that are tients, with no previous history of autoimmune or typical of M2 macrophage activation pathway, showed hematological diseases and a normal complete blood cell an association of these markers with tissue repair and fi- (CBC) count (hemoglobin > 11.0 g/dl; white blood cells > brosis [13], and in SSc patients those markers are associ- 3 3 3 3 4×10 /mm and < 12 × 10 /mm ; and platelets > 150 × ated with high pulmonary artery pressure and increased 3 3 10 /mm ) were recruited from a local blood donation cen- mortality [14]. Other marker that may play a key role in ter and at a Primary Health Care Service unit. The criteria the monocyte activation present in SSc is CD169 (sialic for inclusion were the same as those used in blood acid-binding Ig-like lectine I or Siglec-1), also known as donation. sialoadhesin, which has been shown to have a higher ex- pression in a subset of SSc patients monocytes [15]. Monocytes of SSc patients have a greater expression of Study design these molecules, which is even higher in patients with This was a monocentric cross-sectional study carried tissue disease and pulmonary artery hypertension. out from October, 2016, to May, 2017. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 3 of 11 Flow cytometry protocol Biosciences) for 10 min. Samples were washed and resus- Peripheral blood samples were collected in ethylenedi- pended in phosphate buffered saline (PBS). At least 30, aminetetraacetic acid (EDTA) coated tubes. CBC was 000 gated monocytes were acquired in an 8-color BD performed in a Sysmex XE-2100™ (Sysmex, Kobe, JPN) FACSCanto™ II flow cytometer (BD Biosciences) using hematological counter and the total amount of periph- FACSDiva™ software (BD Biosciences). Flow cytometer eral blood monocytes was determined. technical parameters were automatically set with BD Flow cytometry immunophenotyping (FCI) to identify Cytometer Setup and Tracking Beads (BD Biosciences). and characterize monocyte subpopulations was per- Daily internal quality control (QC) was performed with formed less than 4 h after blood sampling. About 1,000, IMMUNO-TROL Cells (BD Biosciences). 000 cells were placed into polystyrene tubes and were subjected to surface staining by incubation with titrated Phenotypic analysis of monocyte subpopulations amounts of fluorochrome-conjugated monoclonal anti- Figure 1 shows the gating strategy used. Initially, dou- bodies (MoAbs) against CD14 APC-H7 (clone MφP9), blets were excluded and total nucleated cells were se- CD64 BV421 (clone 10.1), CD16 BV510 (clone 3G8), lected based on FSC (forward scatter) versus SSC (side HLA-DR PerCP-Cy5.5 (clone G46–6), CD206 APC scatter) distribution. Monocytes were gated based on cell (clone 19.2), CD169 BB515 (clone 7–239) and CD163 size and complexity, as well as CD64+ expression [20], PE (clone GHI/61) for 30 min at room temperature and and were plotted in a CD14 versus CD16 graph in order protected from light. All MoAbs were purchased from to characterize all three monocyte subpopulations: clas- BD Biosciences (San Diego, USA). Red blood cells were sical monocytes (CD14++CD16-), intermediate mono- lysed by incubation with 2 ml of lysing buffer (BD cytes (CD14++CD16+) and non-classical monocytes Fig. 1 Identification of monocyte subpopulations. a Cells were gated based on FSC (forward scatter) versus SSC (side scatter) distribution and doublets were excluded. b Monocytes were gated based on cell size and complexity, as well as CD64 expression. c Monocyte subpopulations were plotted in a CD14 versus CD16 expression in a representative healthy donor Schneider et al. Advances in Rheumatology (2021) 61:27 Page 4 of 11 (CD14+/−CD16++). Fluorescence-minus-one (FMO) monocyte subpopulation were recorded. All FCI data methodology was used to differentiate autoflourescent were analyzed using Kaluza Analysis Software (Beckman events from events with low concentrations of cell sur- Coulter Life Sciences, Indianapolis, USA) by two experi- face markers [21]. All values obtained in this analysis enced operators concomitantly. were considered FMO limit and all values of fluores- cence above the limit were considered as true positive. Compliance with ethical standards Percentage of expression and mean fluorescence inten- This study was approved by the Research Ethics Com- sity (MFI) of any given marker within the defined mittee of the institution (CAAE number Table 1 Demographic, clinical, and laboratory features of systemic sclerosis patients and controls Systemic sclerosis patients Healthy controls (n = 50) (n = 38) a a Female gender (%) 47 (94.0) 36 (94.7) b b Age (years) 57.2 ± 12.8 55.2 ± 11.4 European-derived (%) 40 (80) 24 (68.6) Smoking status (%) Never 25 (50) 23 (65.7) Previous 21 (42) 30 (29.7) Current 4 (8) 2 (5.7) Disease duration (years) 15 (7–21) Diffuse cutaneous involvement (%) 14 (28.0) Modified Rodnan skin score 6 (2.8–10.3) Renal crisis 4 (8.0) Gastrointestinal symptoms (%) Heartburn 25 (50.0) Dysphagia 26 (52.0) Diarrhea / constipation 21 (42.0) DLCO (% predicted) 70.1 ± 16.3 Reduced DLCO (< 80% predicted) 52.9 (15.9) FVC (% predicted) 79.2 ± 18.9 Reduced FVC (< 80% of predicted) 22 (44) PASP 31.7 ± 14.4 Pulmonary hypertension (PASP ≥40 mmHg) (%) 5 (10) Autoantibodies (%) ANA 39/50 (78.0) Anti-Ro/SSA 1/45 (2.2) Anti-La/SSB 0/45 (0) Anti-Scl-70 4/44 (9.1) RF 35/50 (75.0) Medications (%) Prednisolone < 20 mg/day 6 (12) Mycophenolate mofetil 4 (8) Previous methotrexate administration 10 (20) Previous rituximab administration 2 (4) Previous cyclophosphamide administration 12 (24) Data are shown as mean (± standard deviation), median (interquartile ranges) or number (n) ANA anti-nuclear antibodies, Anti-dsDNA anti-double-stranded DNA, Anti-Scl70 antitopoisomeraseI, DLCO diffusion capacity of carbon monoxide, FVC forced vital capacity, PASP arterial systolic pressure on Doppler echocardiography, RF rheumatoid factor, SSc systemic sclerosis Mann-Whitney test, p = 1.000 Student’s T test, p = 0.448 Schneider et al. Advances in Rheumatology (2021) 61:27 Page 5 of 11 3 3 49299515.0.0000.5327) and was conducted in accordance mm , p < 0.001; and 387.4 (310.4–551.1)/mm versus with the Declaration of Helsinki and current laws in 273.9 (179.3–352.2)/mm , p = 0.002, respectively) (data Brazil. Participants were included after written informed not shown). Among monocyte subpopulations, SSc pa- consent. tients had higher absolute counts of all subpopulations (classical, intermediate and non-classical) when com- Statistical analysis pared to HC (classical monocytes 346.2 (260.9–450.8)/ 3 3 Variables distribution was analyzed by Shapiro-Wilk’s mm versus 209.8 (146.1–287.1)/mm , p < 0.001; inter- test and continuous variables were compared by Stu- mediate monocytes 38.2 (24.6–47.1)/mm versus 25.4 dent’s T or Mann–Whitney’s U test, whereas propor- (12.6–41.2)/mm , p = 0.005; non-classical monocytes 3 3 tions were compared by Pearson’s chi-square or Fisher’s 41.2 (20.8–58.3)/mm versus 28.3 (11.5–46.1)/mm , p = exact test. ANOVA or Kruskall-Wallis, with a Tukey or 0.006) (data not shown). Regarding disease subtype, lSSc Dunn correction test was used for multiple comparisons. patients presented increased absolute values of all mono- Pearson or Spearman correlation test was used to evalu- cyte subpopulations when compared to HC (classical ate association between variables. The statistical signifi- monocytes 363.2 (262.9–451.8)/mm versus 209.8 cance level adopted was 5% (p < 0.05). Data analysis was (146.1–287.1)/mm , p < 0.001 (A); intermediate mono- carried out with SPSS version 21.0® (IBM, Chicago, cytes 38.5 (24.7–47.5)/mm versus 25.4 (12.6–41.2)/ USA) and Prisma 8 software (Graphpad Inc. San Diego, mm , p = 0.009 (B); non-classical monocytes 42.5 (20.8– 3 3 USA). 69.0)/mm versus 28.3 (11.5–46.1)/mm , p = 0.005) (C). While in dSSc patients, only classical monocytes had in- Results creased absolute counts compared to HC (209.8 (146.1– 3 3 Patients and controls characteristics 287.1)/mm versus 326.5 (231.2–461.9)/mm , p = 0.003) Demographic data and clinical characteristics of SSc and (A). There was no difference in monocytes absolute or healthy control (HC) subjects are shown in Table 1. SSc relative values between lSSc and dSSc subgroups (p = patients mean age was 57.2 years and 94% were female. 0.450 and p = 0.914, respectively). Limited form of disease was present in 72% of SSc pa- tients. Median Rodnan skin score was 5 (2.0–8.0) in lSSc Circulating CD16+ monocyte subpopulations patients and 11 (3.8–16.8) in the dSSc patients (p = When analyzing intermediate and non-classical mono- 0.007). Pulmonary function also differed among disease cytes as a single group of CD16+ monocytes, we identi- subgroups when comparing forced vital capacity fied a higher absolute count of this subpopulation in (FVC%predicted) results: lSSc subjects had higher mean lSSc patients compared to HC (79.9 (53.4–103.5)/mm values when compared to dSSc patients (83.8 ± 17.4% versus 55.9 (26.8–85.8)/mm , p = 0.003) (Fig. 2d). versus 67.5 ± 18.2%, p = 0.005). Circulating monocyte subpopulations Monocyte subpopulations and clinical manifestations Main results of monocytes evaluation are show in Fig. 2. Absolute counts of monocyte subpopulations did not SSc patients had higher absolute and relative number of correlate with severity of cutaneous fibrosis assessed by circulating monocytes when compared to HC (459.6 the modified Rodnan skin score, pulmonary involvement 3 3 (322.9–550.3)/mm versus 273.9 (179.3–352.2)/mm , assessed by DLCOc/VA, FVC and pulmonary artery sys- p < 0.001), this difference remained significant when tolic pressure, or disease duration when evaluated comparing lSSc or dSSc patients with HC independently among the total of patients or in lSSc and dSSc subtypes (478.2 (342.9–555.5)/mm versus 273.9 (179.3–352.2)/ (Fig. 3) (Additional files 1 and 2). Fig. 2 Monocyte subpopulations according to their membrane expression of CD14 and CD16 in systemic sclerosis patients with limited (lSSc) (n = 36) and diffuse (dSSc) (n = 14) subtypes, and in healthy controls (HC) (n = 38). The absolute count of all monocyte subpopulations - classical a, intermediate b and non-classical c - is increased in lSSc compared to HC. d When analyzing intermediate and non-classical monocytes as a single group of CD16+ monocytes, their absolute value is increased in lSSc compared to HC. *p < 0.05; **p < 0.01; ***p < 0.001 Schneider et al. Advances in Rheumatology (2021) 61:27 Page 6 of 11 Expression of surface receptors in monocyte expression was lower in both lSSc and dSSc classical subpopulations monocytes when compared to HC. CD163, macrophage Results of surface expression of HLA-DR, CD206, scavenger receptor, was more expressed in classical (per- CD169 and CD163 receptors are shown in Fig. 4 and centage and MFI) and intermediate (percentage) mono- Additional file 3. There were different expression pat- cytes of SSc patients as compared to HC. In subgroup terns among the evaluated monocyte subpopulations. analysis, a difference in CD163 expression was demon- HLA-DR, an activation marker, was consistently strated in classical monocytes of lSSc subjects compared expressed in all monocytes from SSc and HC, and its in- to the HC group. CD169 receptor had a higher percent- tensity of expression was higher in all monocyte subsets age of expression in SSc versus HC. When evaluating from lSSc and dSSc patients when compared to control. SSc subtypes, classical and intermediate monocytes from CD206 expression varied among the monocyte subsets, dSSc patients had higher MFI and percentage of CD169 the percentage of expression was higher in classical and expression than lSSc and HC, whereas non-classical intermediate monocytes of SSc patients when compared monocytes of dSSc patients had higher percentage of this to HC and its MFI expression was lower in SSc as com- marker expression (Fig. 4). pared to HC. When analyzing SSc subgroups, percentage Further analyses were performed for comparison of of CD206, the mannose receptor-1, expression was the markers MFI of expression among the different higher only in classical monocytes of lSSc patients com- monocyte subpopulations. Except for CD163 expres- pared to HC and in intermediate monocytes of lSSc and sion, the monocyte subpopulations exhibited distinct dSSc compared to HC; whereas MFI of CD206 expression patterns when compared to one another, Fig. 3 Correlations regarding the CD16+ monocyte subpopulation. The CD16+ monocyte subpopulation of SSc patients (n = 50) and in the subgroup of patients presenting lSSc (n = 36) and dSSc (n = 14) was tested for association with cutaneous fibrosis assessed by the modified Rodnan skin score (mRSS), pulmonary involvement assessed by DLCOc/VA and FVC. Disease duration was not associated with CD16 + values. DLCO: diffusion capacity for carbon monoxide; FVC: forced vital capacity Schneider et al. Advances in Rheumatology (2021) 61:27 Page 7 of 11 Fig. 4 Expression of surface receptors under study on monocyte subpopulations of systemic sclerosis patients divided by disease subtype in lSSc (n = 36) and dSSc (n = 14) compared with healthy controls (HC) (n = 38). ANOVA with Tukey correction for symmetric distribution and Kruskal- Wallis with Dunn correction for asymmetric distribution. MFI: mean fluorescence intensity. SSc: systemic sclerosis; HC: healthy controls; lSSc: limited systemic sclerosis; dSSc: diffuse systemic sclerosis. *p < 0.05; **p < 0.01; ***p < 0.001 Schneider et al. Advances in Rheumatology (2021) 61:27 Page 8 of 11 which was similar among patients and HC subgroups increased amount of circulating monocytes with CD16+ (Additional file 3). monocytes as the main representative monocyte subset in SSc patients [5, 32]. Nevertheless, our results differ re- Discussion garding correlations with clinical manifestations. The In our study, monocytes percentage and absolute count fact that we did not find a correlation of monocyte sub- were significantly increased in SSc patients as compared sets with clinical manifestations reinforces the hypoth- to healthy subjects, regardless of disease subtype. Monocy- esis of cellular modulation and activation in loco, since tosis in SSc patients has been reported previously [5, 22]. the different tissues are able to direct the inflammatory Assessment of CD14 and CD16 receptors expression response in order to exacerbate inflammation with pro- allowed the identification of all three monocyte subpopu- inflammatory mediators or into resolution and repair lations, as already described for healthy individuals and [33]. Hence, findings of monocyte/macrophage activa- some disease states [9, 23–25]. However, our study is the tion in the bloodstream may not be observed at the tis- first to describe these subpopulations in SSc patients, sue level, specifically, in internal organs affected by SSc. using specific markers CD14 and CD16 for such distinc- HLA-DR is a cell activation marker, so it is consistent tion according to positivity and MFI of expression. to find a greater expression intensity of this marker in We identified an increase in all monocyte subpopula- patients with autoimmune diseases when compared to tions in SSc patients, which was even more significant in normal subjects. We found an increase in MFI expres- patients with lSSc. Interestingly, lSSc patients showed an sion of HLA-DR in all monocyte subpopulations of SSc increase of all three monocyte subpopulations compared patients compared to HC. MFI of HLA-DR expression to controls, whereas in dSSc patients that increase was in CD16+ monocyte subpopulations (intermediate and at expense of classical monocytes, indicating that in this non-classical) was significantly higher than that of form of disease the CD16+ monocyte subpopulations CD16- classical monocytes, indicating a greater activa- does not increase. Recent studies in SSc patients have tion of CD16+ antigen-presenting cells, which is ex- shown an increase in CD16+ monocytes in the periph- pected in an autoimmune state. eral blood of these patients, however, these studies did Other molecules, such as capture receptor CD163 and not distinguish between intermediate and non-classical CD206 were also associated with M2 macrophages and subpopulations [5, 22]. could be involved into further differentiation processes. A Previous studies have shown a significant correlation previous study comparing the expression of monocyte and of the absolute CD16+ monocyte subpopulation count macrophage markers in peripheral blood and skin biopsy and severity of skin fibrosis and pulmonary fibrosis [5, specimens showed higher percentages of circulating cells 26]. In our study, we did not find correlations between expressing CD204, CD163 and CD14, furthermore these monocyte subpopulations and the clinical manifestations findings were correlated with skin involvement [22]. evaluated. This difference may be related with inter- Additionally, circulating CD14+ monocytes/macro- mediate and non-classical monocyte subpopulations phages with CD206 coexpression have been shown to be grouped into CD16+ monocytes. Although differences associated with pulmonary artery hypertension [13]. It has among intermediate and non-classical monocyte subsets also been shown that macrophages derived from circulat- have been stablished [9, 10, 12], there is a strong prox- ing monocytes have coexpression of CD206, CD163 and imity between them [9], suggesting a direct developmen- CD169, and that the expression of these markers is signifi- tal relationship, where gain of CD16 expression and cantly higher in patients with pulmonary interstitial dis- decrease in CD14 expression may be indicative of mat- ease [3]. In our study, a higher percentage of classical and uration [27] and inflammatory activation for these cells intermediate monocytes with CD206 expression were to be recruited to the periphery, where they begin to act identified in SSc patients compared to HC, which demon- in localized inflammatory processes [5]. In addition, strates a greater amount of M2-like cells in those mono- there are resident tissue macrophages originated from cyte subgroups of SSc patients. the yolk sac or from fetal liver progenitors [28], that Using a flow cytometry approach for assessing surface have long tissue life and self-renewing ability by in situ markers from circulating monocytes/macrophages, Sol- proliferation, even in the absence of recruitment from dano et al. (2018) demonstrated that SSc patients have circulating monocytes [29–31]. The role of these macro- higher expression of the M2 related markers CD14, phages in tissues during inflammation is still unclear CD206, CD163 and CD204; or that there is a coexpres- and further research is needed on the role of circulating sion of M1 and M2 surface markers in those cells [34]; monocytes for their activation and proliferation, espe- providing evidence of plasticity in the expression of this cially in inflammation models. markers in circulating monocyte/macrophage and that Our data corroborate the results obtained by Lescoat monocytes can evolve to exhibit features that are shared et al. (2017) and Trombetta et al. (2018) concerning the by more than one macrophage population. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 9 of 11 Previous studies have suggested that CD169 may be a subpopulations between lSSc and dSSc disease subtypes. phagocyte activity marker in inflammatory disorders. Monocytes from SSc patients presented a more activated CD169 has been identified as a marker of highly patho- phenotypic profile which was suggestive of M2 cells, genic phagocytes in multiple sclerosis patients. Also, an with higher expression of CD206. Opposing to previous increased expression of Siglec-1 on peripheral blood reports, we did not find an association between mono- monocytes as well as its role in cell reactivity to autoan- cyte subpopulations and major fibrotic manifestations. tigen has been shown in rheumatoid arthritis [35, 36]. The intermediate and non-classical monocyte subpopu- CD169 expression in renal macrophages has been corre- lations of SSc patients have increase expression of HLA- lated with proteinuria and tissue damage in glomerulo- DR. CD169 appears to be more representative marker nephritis [37], however its expression in peripheral for dSSc subtype, however, more studies are necessary to blood cells was not assessed. CD169 expression has also confirm that this is a good marker for differentiation of been associated with involvement in the immune system disease subtype. regulation [38]. Biological functions of CD169 in macro- Abbreviations phages and monocytes have yet to be elucidated. In our CBC: Complete blood cell; DLCO: Lung diffusion capacity for carbon study, there was an increased expression of CD169 in monoxide; dSSc: Diffuse cutaneous systemic sclerosis; EDTA: Ethylenediaminetetraacetic acid; FCI: Flow cytometry classical monocytes from SSc patients compared to con- immunophenotyping; FMO: Fluorescence-minus-one; FSC: Forward scatter; trols, and CD169 expression was higher in all monocyte FVC: Forced vital capacity; HC: Healthy control; lSSc: Limited cutaneous subpopulations of dSSc patients compared to lSSc and systemic sclerosis; MFI: Mean fluorescence intensity; MoAbs: Monoclonal antibodies; MRC1: Mannose receptor-1; PBS: Phosphate buffered saline; controls. This finding could represent a greater disease QC: Quality control; Siglec-1: Sialic acid-binding Ig-like lectine I; SSc: Systemic activity in this subgroup of patients. sclerosis; SSC: Side scatter; Th: T helper; TLR: Toll-like receptor As to the use of medications, no association of these with subpopulations of monocytes was found. There Supplementary Information were no differences between patients monocyte subpop- The online version contains supplementary material available at https://doi. ulations regarding corticosteroids use (n = 6). Patients org/10.1186/s42358-021-00182-8. included in the study used low doses (less than 20 mg Additional file 1: Table S1. Spearman correlation coefficient values per day), similar to previous reports [5]. between monocyte subpopulations and clinical data. Our study has some limitations. First of all, the patient Additional file 2: Table S2. Analysis of the absolute of CD16 + in population appears to be relatively small. However, there relation to the time of disease separated by tercil. were many restrictions for patient inclusion in order to Additional file 3: Table S3. Comparison of the markers MFI of minimize treatment interference and disease overlap. expression among the different monocyte subpopulations. Evaluation of exclusively circulating cells is also a limita- tion. There is a need for simultaneous assessment of cir- Acknowledgements culating cells and tissue biopsies to better understand the We would like to thank Fabiane Spagnol Pedrazzani, at the Department of Specialized Diagnostics of the Hospital de Clínicas de Porto Alegre, for her participation of macrophages derived from circulating assistance in the design of the technical parameters for the flow cytometry monocytes in tissue fibrosis. We have evidence that protocol. monocytes and macrophages actually participate in SSc pathogenesis and that there are slight differences between Authors’ contributions LS, VH, RX and RC designed the project. LS, NAM, VH, IFSM, CYU and RRP tissues [1, 39], nevertheless, we need better strategies to collected the data and were responsible for data analysis. LS standardized analyze these cells and studies assessing different tissues and performed the flow cytometry experiments. LS and NAM wrote the and peripheral blood to determine whether these macro- manuscript. VH, RX and RC reviewed the manuscript. All authors read and approved the final manuscript. phages are derived from circulating monocytes or other cells. In addition, our findings should be interpreted in Funding light of our sample characteristics. Considering the We are grateful to the Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) and the Conselho Nacional de pathophysiological and clinical changes observed in SSc Desenvolvimento Científico e Tecnológico (CNPq) for the financial support. over time, different monocyte subpopulations could be observed in early phases of the disease. Also, monocytes Availability of data and materials activation may be influenced by different levels of im- The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. munosuppression and distinct mechanisms of action of immunosuppressive drug treatment. Declarations Conclusions Ethics approval and consent to participate This study was approved by the Research Ethics Committee of the institution In conclusion, our results showed that SSc patients have (CAAE number 49299515.0.0000.5327) and was conducted in accordance increased absolute counts of all three monocyte subpop- with the Declaration of Helsinki and current laws in Brazil. Participants were ulations and that there is no difference among monocyte included after written informed consent. Schneider et al. Advances in Rheumatology (2021) 61:27 Page 10 of 11 Consent for publication macrophages are phenotypically and functionally distinct. Blood. 2014; Not applicable. 123(20):e110–22. https://doi.org/10.1182/blood-2013-08-520619. 15. York MR, Nagai T, Mangini AJ, Lemaire R, van Seventer JM, Lafyatis R. 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