Bone Marrow Monocytes and Derived Dendritic Cells From

Bone Marrow Monocytes and Derived Dendritic Cells from Myelodysplastic Patients Have Functional Abnormalities Associated with Defective Response to This information is current as Bacterial Infection of October 2, 2021. Laiz C. Bento, Nydia S. Bacal, Fernanda A. Rocha, Patricia Severino and Luciana C. Marti J Immunol published online 16 March 2020

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 16, 2020, doi:10.4049/jimmunol.1900328 The Journal of Immunology

Bone Marrow Monocytes and Derived Dendritic Cells from Myelodysplastic Patients Have Functional Abnormalities Associated with Defective Response to Bacterial Infection

Laiz C. Bento,* Nydia S. Bacal,* Fernanda A. Rocha,† Patricia Severino,† and Luciana C. Marti†

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell diseases characterized by dysplasia of one or more hematologic lineages and a high risk of developing into acute myeloid leukemia. MDS patients have recurrent bacterial infections and abnormal expression of CD56 by monocytes. We investigated MDS patients’ bone marrow CD56+/CD562 monocytes and their in vitro–derived dendritic cell populations in comparison with cells obtained from disease-free subjects. We found that monocytes from MDS patients, irrespective of CD56 expression, have reduced phagocytosis activity and low expression of genes involved in triggering immune responses, regulation of immune and inﬂammatory response signaling pathways, and in Downloaded from the response to LPS. Dendritic cells derived in vitro from MDS monocytes failed to develop dendritic projections and had reduced expression of HLA-DR and CD86, suggesting that Ag processing and T cell activation capabilities are impaired. In conclusion, we identiﬁed, in both CD56+ and CD562 monocytes from MDS patients, several abnormalities that may be related to the increased susceptibility to infections observed in these patients. The Journal of Immunology, 2020, 204: 000–000.

yelodysplastic syndromes (MDS) include a hetero- Monocytes are cells with high plasticity. They were originally http://www.jimmunol.org/ geneous group of hematopoietic stem cell diseases classified according to morphological characteristics, but, more M characterized by dysplasia of one or more hemato- recently, the expression of CD14 and CD16 have become important logic lineages. The clinical course of MDS is variable, and patients monocyte markers. Classical monocytes, expressing high levels of are at high risk of developing acute myeloid leukemia (1, 2). In CD14 and absent CD16 (CD14hi/CD162), are ∼80% of all mono- addition, MDS patients frequently suffer from severe bacterial cytes and considered to be better at secreting proinflammatory cy- infections, but the mechanisms underlying their high suscepti- tokines and in generating reactive oxygen species. Intermediate bility to infection are unknown (3, 4). monocytes (CD14hi/CD16+) are more efficient in phagocytosis and In MDS, besides already described granulocyte defects, express higher levels of MHC class II and accessory molecules (7). by guest on October 2, 2021 monocytic lineage cells also exhibit several phenotypic alterations When monocytes are cultivated in presence of GM-CSF and IL-4, (2). The expression of CD56 in monocytes is often abnormal in they can exhibit features of immature dendritic cells (iDCs), and in chronic myelomonocytic leukemia, which is a relatively frequent response to inflammatory signals, they can differentiate into mature MDS. To be considered as aberrant, the percentage of CD56- dendritic cells (mDCs) with Ag presentation ability (8). expressing monocytes should be equal or higher than 20% (2, 5). The mechanisms underlying the immune response against patho- The main function of the CD56 molecule is to promote intercel- gens comprise both innate and adaptive immunity. The first response lular adhesion; it can also act as a receptor involved in cellular against microorganisms is that of innate immunity. It starts by host migration, proliferation, and differentiation (6). Nevertheless, until cells recognition of pathogen-associated molecular patterns (PAMPs). now, there has been no information on the consequences of altered These PAMPs are recognizable by evolutionary conserved receptor CD56 expression observed in malignant neoplasms. molecules present in a variety of host cells such as TLRs, RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin-like *Clinical Pathology Laboratory, Hospital Israelita Albert Einstein, Saõ Paulo 05652 receptors (CLRs), and DNA sensors (9–12). Among TLRs, TLR1, 000, Brazil; and †Experimental Research Laboratory, Hospital Israelita Albert TLR2, TLR4, and TLR6 are present in the cellular plasma mem- Einstein, Saõ Paulo 05652 000, Brazil brane and mainly recognize hydrophobic sugar complexed PAMPs ORCIDs: 0000-0002-6682-9343 (P.S.); 0000-0002-3890-0827 (L.C.M.). (such as LPS, whereas TLR3, TLR7, TLR8, and TLR9 are localized Received for publication March 19, 2019. Accepted for publication February 6, 2020. in endosomes and sense hydrophilic sugar complexed PAMPs such This work was supported by National Council of Technological and Scientific as 59-ppp-ssRNA) (11–13). Monocytes, macrophages, and dendritic Development process number 441665/2014-4. cells (DCs) express most PAMP receptors. These cells establish the The microarray data presented in this article have been submitted to the Gene Expression connection between the innate and adaptive immune responses. Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE142450. They act by engulfing and eliminating microorganisms, processing Address correspondence and reprint requests to Dr. Luciana C. Marti, Hospital Israelita Ag molecules, and presenting the derived peptides in association Albert Einstein, Experimental Research Department, Avenida Albert Einstein, 627, Bloco A, 2oSS, Morumbi, Saõ Paulo SP 05652 000, Brazil. E-mail address: with MHC molecules to lymphocyte recognition as the initial step [email protected] of adaptive immune responses. 2 The online version of this article contains supplemental material. The aim of the current study was to study CD56+ and CD56 Abbreviations used in this article: BM, bone marrow; DC, dendritic cell; iDC, bone marrow (BM) monocytes and thereof derived DCs obtained immature dendritic cell; mDC, mature dendritic cell; MDS, myelodysplastic syndrome; from MDS patients and from disease-free individuals. We analyzed PAMP, pathogen-associated molecular pattern. the following aspects: cell surface markers by flow cytometry, Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 morphology and phagocytic activity, and gene expression patterns.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900328 2 MOLECULAR ALTERATIONS IN MDS PATIENTS’ MONOCYTES

We found that cells from MDS patients had reduced phagocytic ac- The CD56-negative cells originating from the first-step separation were further separated (second-column separation) based on the CD14 tivity, low expression of MHC class II and of costimulatory mole- + 2 cules and TLRs, and reduced expression of cytokine and chemokine expression to yield a 100% of CD14 C56 population. Disease-free samples from BM donors contained ,3% CD56+ monocytes, so the genes. Taken together, results suggest functional abnormalities within separation was only based on CD14 expression. the monocyte population that may contribute to deficient response to bacterial infection, a common feature in MDS patients. Generation of DCs from BM monocytes CD14+CD562 and CD14+CD56+ monocytes were dispensed into six-well plates Materials and Methods containing X-VIVO 15 Medium (Cambrex) supplemented with anti- biotic–antimycotic solution (Life Technologies) containing 100 U/ml BM samples penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. To gen- BM samples from five MDS-diagnosed patients and from five individuals erate iDCs, the cells were cultured in the presence of human rIL-4 (20 ng/ml) without myelodysplasia or hematological disease (disease-free individuals) and GM-CSF (50 ng/ml) (both from R&D Systems, Minneapolis, MN) for were used in this study. The samples were remainders from diagnostic 6dat37˚Cina5%CO2 incubator. mDCs were obtained after iDCs stimu- routine laboratory exams. Disease-free individuals were BM donors with no lation with LPS 100 ng/ml for 24 h (Sigma-Aldrich). Because of low numbers MDS-associated abnormalities. The five MDS patients enrolled in this study ofmonocytesexpressingCD56indisease-freeBM(,3%, as mentioned in the were diagnosed based on current World Health Organization criteria (1, 14). previous section), we were only able to perform the differentiation procedures + 2 All had cells with myelodysplastic characteristics, and one patient presented in CD14 CD56 cells, whereas, in the MDS patients, we performed the + + + 2 cytogenetic abnormality (Fig. 1, Table I). MDS patients were also immu- experiments using CD14 CD56 and CD14 CD56 cell populations. nophenotyped by flow cytometry and had Ogata scores (15) equal or superior to 3 (Table I). All participants in the current study signed an informed Characterization of monocyte-derived DCs consent form, and the study and form were approved by the Hospital Israelita For the characterization of differentiation and maturation processes of Albert Einstein Research Ethics Committee (Certificate of Presentation for monocyte-derived DCs, monocytes and monocyte-derived DCs were Downloaded from Ethical Appreciation number: 33739214.8.0000.0071). analyzed by flow cytometry using the following Abs to cell surface markers labeled with the indicated fluorochrome: CD209-FITC, HLA- Flow cytometry characterization of BM mononuclear cells DR–PerCP.Cy5.5, CD3-allophycocyanin-H7, and CD33-allophycocyanin, from MDS patients CD16-PB (all from BioLegend); CD80-FITC, CD56-PE, and CD86-PE bought from Becton and Dickinson; and CD14-ECD and CD45-PC7 (from BM samples presenting MDS-associated morphological and/or cyto- Beckman Coulter). Fluorescence minus one was used as a fluorescence genetic abnormalities were immunophenotyped using the following background control. All monocytes and derived DCs that were analyzed labeled Abs (bought from Beckman Coulter) to cell surface markers: http://www.jimmunol.org/ expressed the myeloid marker CD33 as shown in the gating strategy (Fig. 2). CD4/k-FITC, CD8/L-PE, CD3/CD4-ECD, CD33-PC5.5, CD20/CD56-PC7, For acquisition, we used FACS LSR II Fortessa (Becton and Dickinson), and CD34-allophycocyanin, CD19-allophycocyanin-700, CD10-allophycocyanin- datasets were analyzed using the FlowJo software (Tree Star). 750, CD5-PB, and CD45–Krome Orange. Patient samples with confirmed immunophenotypic characteristics of MDS by Ogata scores $3 and having Morphological analysis of monocytes and abnormal CD56 expression were included in this study. monocyte-derived DCs + Selection of BM CD14 cells from disease-free donors or Morphological analysis of monocytes and monocyte-derived DCs from pa- MDS patients tients and disease-free individuals was performed using the cytospin tech- nique, followed by Rosenfeld staining. Briefly, 50 mlofcellularsuspension Unstained BM cell suspensions were diluted 1:3 with 0.01 M phosphate-

were used to prepare the slide, followed by a centrifugation of 1000 rpm/5 by guest on October 2, 2021 buffered 0.15 M NaCl (PBS), transferred to 15 ml conical tubes containing min. Cells were stained with 500 ml of Rosenfeld staining solution (meth- 5 ml of Ficoll-Paque PLUS (GE Althusser, Cardiff, U.K.) and centrifuged ylene blue eosin Maygrunwald combined with azurine methylene diluted in (30 min/500 3 g without brake) at 22˚C. The cells from the interface were ¨ methanol) for 2 min, followed by the addition of 1 ml PBS for 6 min. The removed, resuspended, and centrifuged again (5 min/500 3 g). Cell selection stained samples were analyzed by Eclipse 80i microscope (Nikon) using and separation according to CD56 and CD14 expression was done by magnetic 1003 magniﬁcation objective. selection columns (Miltenyi Biotec, Bergisch Gladbach, Germany). MDS patients BM cells were selected in two steps. The ﬁrst step was based on CD56 TLR4 expression by monocytes expression (CD56+ cells), which selected for monocytes (CD14+CD56+)and 2 NK cells (CD14 CD56+). We proceeded to eliminate NK cells by transferring BM samples from MDS patients and disease-free individuals were used to these selected population to a plastic culture dish in culture medium for 1 h at 37˚C verify TLR4 expression on the surface of monocytes using labeled Abs in a 5% CO2 incubator. The nonadherent cells (NK) were removed; the adherent to the following markers: CD14-FITC, TLR4-PE, and CD56-PC7 (all cell population was recovered and had around 98% of CD14+CD56+ cells. from Becton and Dickinson) and CD16-PB, CD45—Krome Orange, and

FIGURE 1. Myelodysplastic characteristics detected by morphologic features. (A) Regular erythroblast from a control patient. (B) Binucleated erythroblast from MDS patient. (C) Regular neutrophil from a control patient. (D) Hypogranular neutrophil with hypolobed nuclei from MDS patient (original magniﬁcation 3100). The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Monocytes (A) and DCs (B and C) gate strategy. All samples after CD14 selection (monocytes, iDCs, and mDCs) were gated as described: ﬁrst gated on side scatter–area (SSC-A) versus CD45-positive cells, followed by a gating on CD33-positive cells and CD14-positive/negative cells. Next, these cells were analyzed for the expression of CD209, HLA-DR, CD80, and CD86. Figure representative of ﬁve experiments (MDS patients = 5, and controls = 5). 4 MOLECULAR ALTERATIONS IN MDS PATIENTS’ MONOCYTES Downloaded from

FIGURE 3. CD56 expression in MDS and disease-free BM monocytes. All samples from the whole BM were gated as described: ﬁrst gated on side http://www.jimmunol.org/ scatter–area (SSC-A) versus CD45–Krome Orange (KO)-positive cells, followed by a gating on CD14-ECD with CD3-ECD; next, the lymphocytes T and B were excluded by gating the cells on CD33-PC5.5. Subsequent, only myeloid cells (CD33-positive cells) were gated for CD56-PC7. Figure representative of ﬁve independent experiments (MDS patients = 5, and controls = 5).

CD33-PC5 (from Beckman Coulter). Fluorescence minus one was used Statistical analysis as a ﬂuorescence background control. Acquisition and datasets were analyzed as described above for monocyte-derived DCs. The statistical analysis was performed using the GraphPad Prism program (version 6.02). Student t test was used for comparison between

Phagocytosis assay two groups and ANOVA with Bonferroni correction was used for by guest on October 2, 2021 , + + + multiple comparisons. Significance level was set at p 0.05 for all CD14 BM monocytes from disease-free individuals and CD14 /CD56 experiments. and CD14+/CD562 BM monocytes from MDS patients were incubated in 24-well dish in culture medium with green fluorescent Escherichia coli BioParticles (Life Technologies) for 5 h at 37˚C in a 5% CO2 incubator. Results The suspension was transferred to a tube and washed three times in PBS. Monocytes from MDS patients exhibit abnormal expression of Next, the monocytes were stained with anti–CD14-PE. Acquisition was CD56, decreased percentage of intermediate monocytes, and done in an FACS LSR II Fortessa, and datasets were analyzed using the reduced phagocytic capacity FACSDiva (Becton and Dickinson) or FlowJo software. Additionally, cells were also transferred to glass slides using a cytocentrifuge. The slides were BM monocytes from MDS patients and from disease-free BM samples incubated with anti–CD14-PE–labeled Ab and with DAPI. Analyses were were analyzed for their morphology, immunophenotype, and by performed using a Zeiss LSM 710 confocal microscope and the Zeiss ZEN 2.3 lite software for image generation. Gene expression CD14+ monocytes (regardless of CD56 coexpression) from two disease- free individuals and from two MDS patients (biological replicates) were processed for RNA extraction using the RNeasy Kit (QIAGEN). Clariom S Arrays (Thermo Fisher Scientific) were used for gene expression analyses according to the manufacturer’s protocols (GeneChip Whole-Transcript Expression Arrays; Thermo Fisher Scientific). Hybridization signals were detected using the GeneChip Scanner 3000 7G (Thermo Fisher Scientific) and the Affymetrix GeneChip Command Console Software. Array data quality analyses, normalization (robust multiarray average with detection above background), and statistical tests for differential expression (eBayes) were carried out with the Transcriptome Analysis Console Software (4.0.1) (cutoff for significance, p value , 0.05). Gene expression levels are presented in fold changes. Hierarchical clustering of differential gene expression is presented in the form of heat maps using Euclidean distance and the Ward method or complete linkage. Gene Ontology term and KEGG pathways enrichment analysis were carried out using Database for Annotation, FIGURE 4. Monocytes expressing CD56 observed in patients and control Visualization and Integrated Discovery Bioinformatics Resources 6.8, and 6 results were considered statistically significant when p value , 0.05 after samples. MDS patients expressed CD56 in about of 58.08 17.01% of Benjamini–Hochberg false discovery rate correction. Raw and processed monocytes, whereas only 3.22 6 1.30% monocytes of control samples data are available at Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/ expressed CD56 (p , 0.0001). Figure representative of five independent geo/) under accession number GSE142450. experiments (MDS patients = 5, and controls = 5). The Journal of Immunology 5

ﬂow cytometry for CD56 expression, as shown in Figs. 1, 2, and 3. When we looked at the phagocytic capacity of both (CD56+ and The frequencies of CD56-expressing monocytes were much CD562) MDS-BM monocytes, we found that both populations higher (58.08 6 17.01%) in BM samples from MDS patients had reduced uptake of green ﬂuorescent Gram-negative bacteria in comparison with those found in disease-free BM samples (E. coli) in comparison with similar BM monocyte populations (3.22 6 1.30%) (Fig. 4). from controls (Fig. 5). Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Differences in phagocytosis index between monocytes from patients and controls. Monocytes were cultivated for 5 h in presence of green fluorescent E. coli.(A) Flow cytometry gate strategy showing side scatter–area (SSC-A) 3 CD14. (B) Dot plot showing the percentage of monocytes that internalized E. coli. (C) Monocytes mean of fluorescence intensity (MFI) of E. coli fluorescent channel (FITC) obtained from a control sample and from CD56+ and CD562 MDS patient. (D–F) Confocal microscopy showing the E. coli internalization (green) by monocytes (red). Original magnification 3100. (G) Graph showing significant differences between the MFI from controls compared with the patients samples. Figure representative of five independent experiments (MDS patients = 5, and controls = 5). 6 MOLECULAR ALTERATIONS IN MDS PATIENTS’ MONOCYTES

Classical and intermediate circulating monocytes differ as to the Of interest is that monocytes from MDS patients clearly down- phagocytic function with intermediate monocytes being more effi- regulated genes involved in triggering immune responses (GO: cient in this aspect (7). Hence, we examined the frequencies of classical 0045087), in regulating immune and inflammatory response sig- (CD14hiCD16low) and intermediate monocytes (CD14hiCD16hi)in naling pathways (GO:0050776; GO:0006954), and in the response MDS-BM samples and in control BM samples. We observed significant to LPS (GO:0071222) (Table II). Among them, we highlight reduction in the percentage of intermediate monocytes in MDS-BM the downregulation of FCGR1A, FCGR3A, and FCGR3B, which when compared with controls (Fig. 6). We found higher numbers of encode the Fc receptors FcgRI (CD64), FcgRIIIa (CD16a), total monocytes in MDS-BMs (on average, 16,058 monocytes/mm3) and FcgRIIIb (CD16b), respectively; the TLRs TLR1, TLR7, and than in disease-free individuals (on average, 2966 monocytes/mm3), TLR8; cytokines and chemokines IL-32, CCL5, CXCL12, and which is a described feature of some myelodysplastic diseases CXCL16; MHC class II–related genes HLA-DPB1, HLA-DPA1, such as refractory anemia with excess of blasts II and chronic HLA-DQA2, HLA-DQA1, HLA-DQB1,andHLA-DRB1; CIITA, myelomonocytic leukemia (14). However, in contrast, the number of and LPS pathway genes, such as CD14, MyD88,andMAPK14. intermediate monocytes was lower in four MDS patients (on average, Among the tree most upregulated genes found in MDS-BM 195 intermediate monocytes/mm3) compared with controls (on av- monocytes in comparison with normal BM monocytes (Table III), erage, 373 intermediate monocytes/mm3). One MDS patient was we highlight the upregulation of CD56 (also known as NCAM1), as an outlier, having BM leukocytosis (367,900 leukocytes/mm3)anda was expected. The other two mainly upregulated genes in MDS-BM corresponding high number of monocytes (86,824 monocytes/mm3). monocytes were JUN, encoding c-Jun, and PTX3, which encodes the Although the percentage of intermediate monocytes was low (5%), it acute phase protein pentraxin. Both genes are myeloid–monocytic amounted to 4241 intermediate monocytes/mm3 in the BM of this lineage–related genes and involved in inflammatory processes. particular patient, a much higher number when compared with Downloaded from MDS-BM monocytes express less TLR4 and CD14, and MDS disease-free individuals and other patients. monocyte-derived DCs have low expression of HLA-DR and Thus, it was found that both CD562 and CD56+ BM monocytes costimulatory molecules from MDS patients have reduced phagocytic capacity when compared with monocytes from the control individuals. Moreover, Because essential molecules such as CD14, MyD88, and MAPK14 most patients have reduced numbers of intermediate monocytes, from the LPS pathway were downregulated in MDS monocytes in which was correlated with higher phagocytosis index and en- comparison with monocytes from disease-free controls, we analyzed http://www.jimmunol.org/ hanced expression of HLA-DR. the expression of TLR4 and CD14 at the protein level; these proteins are the main ligands for LPS at the cell membrane. We observed that DCs derived from MDS monocytes have altered morphology monocytes isolated from MDS patients’ BM downregulated TLR4 BM monocytes from MDS patients and controls had similar and CD14 at the protein level. The percentage of monocytes morphology when examined by light microscopy. However, the expressing TLR4 was decreased, and CD14 mean of fluorescence iDCs and mDCs derived from BM MDS monocytes were smaller intensity was reduced, meaning that there were fewer CD14 epitopes and had fewer membrane projections, and the nuclei were larger on the cell membrane, as shown in Fig. 9. These results indicate a and more centrally located than in control-derived DCs (Fig. 7). dysfunctional LPS pathway in MDS monocytes that can be linked to the reduced internalization of Gram-negative bacteria by these cells. by guest on October 2, 2021 TLRs and cytokine genes are downregulated in BM monocytes Moreover, the downregulation of MHC class II transactivation factor from MDS patients (CIITA)andMHCclassII(HLA-DP, HLA-DQ, and HLA-DR)atthe We compared global gene expression levels by BM monocytes gene expression level correlates with lower levels of HLA-DR and obtained from two MDS patients (samples 38 and 39, Table I) with CD86 by iDCs and mDCs derived from MDS monocytes when BM monocytes from two disease-free samples (samples 49 and compared with the cells derived from disease-free individuals (Fig. 10). 50, Table I). Fig. 8 depicts similarities between monocytes from MDS patients and from disease-free samples. The number of genes Discussion differing at the gene expression level (2-fold change, p , 0.05) MDS is a complex and heterogeneous group of clonal diseases between the two groups was 1029 (Supplemental Table I). that share manifestations such as BM hypercellularity, peripheral

FIGURE 6. Differences in intermediate monocytes percentages observed in patients and control samples. (A) There is no difference between classical monocytes from patients and controls (B). There is signiﬁcant decrease in the percentage of intermediate monocyte population (CD14hiCD16hi) in MDS patients compared with controls. Figure representative of ﬁve independent experiments (MDS patients = 5, and controls = 5). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 7. Morphological analysis of monocytes and DCs from peripheral blood control sample. (A) Monocyte, (B) monocyte-derived iDCs, and (C) monocyte-derived mDCs; BM control sample. (D) Monocyte, (E) monocyte-derived iDCs, and (F) monocyte-derived mDCs; BM from MDS patient. (G) Monocyte CD56+,(H) monocyte CD56+-derived iDCs, and (I) monocyte CD56+-derived mDCs; BM from MDS patient. (J) Monocyte CD562, (K) monocyte CD562-derived iDCs, and (L) monocyte CD562-derived mDCs (original magniﬁcation 3100). (M) Evaluation of MDS iDCs alterations per 100 cells compared to controls. (N) Evaluation of MDS mDCs alterations per 100 cells compared to controls. Figure representative of ﬁve experiments (peripheral blood controls = 5, BM from MDS patients = 5, and BM from controls = 5). 8 MOLECULAR ALTERATIONS IN MDS PATIENTS’ MONOCYTES

Table I. Patient disease classiﬁcation

Patient Sex Age Classiﬁcation Genetic Alterations Ogata Score 10 F 69 CMML Trisomy 8 and 13 3 14 F 44 RAEB II No alterations 3 34 F 34 RAEB II No alterations 4 38 F 62 CMML No alterations 3 39 M 30 CMML No alterations 3 37 F 82 Control Not investigated 0 40 F 67 Control Not investigated 0 48 M 30 Control Not investigated 0 49 M 42 Control Not investigated 0 50 M 37 Control Not investigated 0 CMML, chronic myelomonocytic leukemia; Control, disease-free individual; RAEB II, refractory anemia with excess of blast. cytopenia, and ineffective hematopoiesis. We veriﬁed that MDS individuals (16–18). The intermediate-type monocyte population is patients included in this study had lower frequency of intermediate- considered important to infection control, and when compared with type monocytes (CD14highCD16lhigh) in the BM in comparison classical and nonclassical monocytes, this population expresses the with disease-free subjects; the frequencies of classical monocyte highest levels of MHC class II accessory molecules and displays the high low (CD14 CD16 ) were similar to those described for disease-free highest phagocytosis index (14). Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 8. Hierarchical clustering of gene expression changes between monocytes from MDS patients and disease-free individuals. Columns represent sample types, and rows represent genes. Values are represented in fold change, and the tree view indicates the degree of correlation between genes or samples. The Journal of Immunology 9

Table II. Functional analysis of genes presenting downregulation in MDS monocytes compared with monocytes from free-disease individuals (p , 0.05)

Term Count Genes FDR GO:0006955: immune response 55 HLA-DPB1, HLA-DPA1, HLA-DQA2, 3.34 3 10211 HLA-DQA1, HLA-DQB1, HLA-DRB1, CIITA, C1QC, MBP, IL-4R, FCGR1A, FCGR1B, FCGR3A, FCGR3B, FCAR, GTPBP1, PTGER4, CMKLR1, TLR1, ZEB1, PNP, SERPINB9, CCR7, CD4, CD22, CD27, CD36, CD96, TNFSF13B, CCR1, CCR4, CCR5, IL-7R, IL2RG, TGFBR3, CST7, GPR183, ENPP2, VPREB1, JCHAIN, IL-32, CCL5, CXCL12, SLC11A1, TNFRSF1B, HRH2, FYB, SAMHD1, ETS1, TCF7, and AQP9 GO:0002250: adaptive immune response 27 CD4, CLEC6A, CLEC10A, FCGR1B, 2.54 3 1027 HAVCR2, IFNK, GPR183, ERAP2, JAK3, SLAMF7, LILRB1, LILRB4, CAMK4, FYN, TXK, PRDM1, CD79A, SKAP1, SH2D1A, SH2D1B, ITK, THEMIS, JCHAIN, and TRAT1 GO:0007166: cell surface receptor signaling 33 CD4, CD14, CD36, CD151, ADGRE2, 7.82 3 1025 Downloaded from pathway ADGRA3, TSPAN13, TSPAN17, TSPAN18, CCR1, CCR5, MYD88, MAPK14, LEPR, IFNAR2I, L7R, CLEC2D, and PTPRC GO:0045087: innate immune response 41 CD14, CD180, CD300E, TLR1, TLR7, 8.89 3 1024 TLR8, CLEC5A, CLEC6A, CLEC10A,

C1QC, C1QB, CR1, NAIP, JAK3, http://www.jimmunol.org/ HAVCR2, MYD88, CLU, JCHAIN, SRC, SERINC5, SH2D1A, FCER1G, BLK, IGLL5, SH2D1B, ITK, ADARB1, SLAMF6, MALT1, SERPING1, PADI4, S100A12, and FYN GO:0050776: regulation of immune 24 ITGAL, CD300E, SELL, SLAMF6, 0.001 response SLAMF7, LILRB1, VCAM1, CD96, SH2D1A, FCGR1A, ITGB7, CD81, CLEC2D, TREML2, FCGR3A, and SH2D1B GO:0006954: inﬂammatory response 37 CD14, CD5L, CD180, ITGAL, CIITA, 0.002 by guest on October 2, 2021 PTGS2, CRHBP, TLR1, TLR7, TLR8, CCL5, CXCL12, CCRL2, CCR1, CCR4, CCR5, CCR7, SLC11A1, TNFRSF1B, MYD88, ADAM8, CD27, HAVCR2, LIPA, ADGRE2, NFKBID, OLR1, LY96, HCK, S100A12, PRKCQ, CAMK4, NAIP, and CAMK1D GO:0006968: cellular defense response 13 CLEC5A, CD5L, PRF1, GNLY, TRAT1, 0.008442794 SH2D1A, CCR5, FCMR, CX3CR1, FOSL1, and ITK GO:0071222: cellular response to LPS 17 CD14, CD36, CD180, MAPK14, CDC73, 0.01 HAVCR2, LILRB1, SBNO2, PPARD, LITAF, PDE4D, SRC, TNFRSF1B, CCR5, CXCL16, and CX3CR1 GO:0032729: positive regulation of IFN-g 11 CD14, HLA-DPA1, HLA-DPB1, HAVCR2, 0.01 production SLC11A1, SLAMF6, PDE4D, TXK, and CD226 GO:0050900: leukocyte migration 17 ITGAL, ITGAX, ITGAV, FCER1G, THBD, 0.04 ATP1B3, OLR1, SELL, GRB2, SLC7A5, SRC, CD47, CD58, SIRPG, KRAS, FYN, and PIK3CA FDR, false discovery rate.

Monocytic lineage morphological alterations are difﬁcult to such as fewer dendritic-like projections in MDS monocyte-derived identify and classify; in fact, monocytes do not display any visible DCs may signal ineffective or suboptimal performance and poor in- morphological changes in MDS (1). Additionally, CD56+ and teractions with lymphocytes, an essential feature for Ag presentation CD562 monocytes from MDS patients and from normal controls and T cell activation. Furthermore, MDS monocytes also showed were not distinguishable when examined by light microscopy, in diminished phagocytosis, which would lead to defective internali- agreement with prior observations (7). However, we found that zation of pathogens or molecules. We also noticed that the mor- DCs derived from MDS patients’ BM monocytes presented altered phological alterations occurred independently of CD56 expression. morphological characteristics when compared with disease-free– Han et al. (18) have also described more monocytes in peripheral derived iDCs and mDCs. The observed morphological differences, blood of MDS patients, with reduced ability of differentiation into 10 MOLECULAR ALTERATIONS IN MDS PATIENTS’ MONOCYTES

Table III. Upregulated genes in MDS monocytes compared with in the course of this disease (3, 20, 21). The reduction of the monocytes from disease-free individuals (p , 0.05) intermediate-type monocytes that we found in our study could be one of the causes of the heightened susceptibility to infection. As Fold Change Control we sought to understand the cellular and molecular causes of this Gene Symbol versus Patient p Values susceptibility, we observed, in MDS monocytes, downregulation JUN 216.48 0.0006 of several pathways involved in phagocytosis, and in immune and 2 PTX3 15.25 0.004 inflammatory responses, including the response to LPS. NCAM1 213.51 0.0158 CCL3 210.93 0.0061 Among genes important to the internalization and processing NRG4 29.3 0.0167 of microorganisms and also to effector monocyte/macrophages MS4A14 28.91 0.0068 responses, it is noteworthy to mention the downregulation of genes AMPH 28.64 0.0145 FCGR1A, FCGR3A,andFCGR3B, which encode the Fc receptors MYLIP 28.15 0.0253 UACA 28.05 0.0028 FcgRI (CD64), FcgRIIIa (CD16a), and FcgRIIIb (CD16b), respec- EREG 27.88 0.0157 tively. Studies in vitro on Fc IgG receptors have established their role in triggering effector responses, such as macrophage/monocytes phagocytosis of IgG-coated bacteria or other pathogens (22, 23). monocyte-induced macrophages. These cells also presented re- In addition, we found that cytokines, chemokines, and TLRs duced phagocytic ability, confirming defective function, although, genes, such as TLR1, TLR7, TLR8, IL32, CCL5, CXCL12, and morphologically, they were identical to disease-free donors (18). CXCL16, were also downregulated in BM monocytes from MDS Miraki-Moud et al. (19) demonstrated, using an animal model, patients. The reduced expression of TLR1, TLR7,andTLR8 con- Downloaded from that during the development of acute myeloid leukemia, the pool tributes to functional impairment of internalization and processing of hematopoietic stem cells is comparable to the stem cell population of bacterial and virus molecules (13). CXCL16 in macrophages of a healthy individual, but cellular differentiation is malfunctioning and DCs mediates the adhesion and phagocytosis of bacteria, and leads to peripheral blood cytopenia (19). We did not observe such as E. coli and Staphylococcus aureus, because bacterial reduction in the total number of BM monocytes in samples from recognition is mediated by this chemokine (24). MDS patients compared with healthy BM donors but, instead, a We also observed downregulation of genes implicated in response higher number of monocytes with impaired differentiation capability. to LPS, comprising genes such as CD14, MyD88, and MAPK14. http://www.jimmunol.org/ It is known that ∼40% of MDS patients have recurrent bacterial This finding reinforces the lower capacity of these cells in rec- infections, which often constitute one of the main causes of death ognizing Gram-negative bacteria. The TLR4 downregulation in by guest on October 2, 2021

FIGURE 9. Downregulation of TLR4 in monocytes derived from MDS patients’ BM. (A) Monocytes (CD14) derived from disease-free donors BM express very low percentages of CD56 and high levels of TLR4. (B) Monocyte (CD14)–derived MDS patient BM express high percentages of CD56 and low levels of TLR4. (C) Comparative graph that displays low levels of TLR4 in patients monocytes compared with health individuals. (D) Comparative graph that displays low levels of CD14 (mean of ﬂuorescence intensity [MFI]) in patient monocytes compared with health individuals (MDS patients = 3, and controls = 3). The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 10. HLA-DR and CD86 expression profile by iDCs and mDCs in MDS patients and controls. (A and B) HLA-DR profile of expression on iDCs and mDCs CD562 and CD56+ from MDS patients compared with control. (C and D) CD86 profile expression on iDCs and mDCs CD562 and CD56+ from MDS patients compared with control. Figure representative of five independent experiments (MDS patients = 5, and controls = 5). humans can be correlated with reduced phagocytosis. TRAM, a Adding up to possible impaired T cell activation in MDS the CD14/TLR4 and MyD88, downstream molecules in the LPS expression of the costimulatory molecule CD86 (B7-2) was de- pathway, have recently been described as essential for phagocytosis creased in mDCs derived from MDS monocytes. Previous studies of Gram-negative bacteria (25). This signaling is essential to in- have also demonstrated lower expression of CD86 in peripheral duce actin cytoskeleton remodeling and regulates the extension blood monocyte-derived DCs from MDS patients (26, 27). of membrane protrusions that surround the particle to form a Among the genes that were upregulated in monocytes from phagocytic cup. MDS patients, CD56 was an expected result (28). The two other Recruitment of leukocytes to inflammatory sites would also be most upregulated genes in monocytes from MDS patients were impaired because of the downregulation of CCL5, CXCL12 syn- JUN and PTX3; the former acts as an inducer of myeloid differ- thesis by MDS monocytes. Because IL-32 is a proinflammatory entiation to monocytes (via c-Jun), and the latter regulates mac- cytokine, reduction of its expression could be at the root of reduced rophage differentiation in the blood and tissues (29, 30). secretion of inflammatory cytokines, such as TNF-a, and affect Transcription factors work either synergistically or antago- infection control. nistically (31–33), suggesting that absolute levels (34), as well Besides impairments related to internalization of bacteria and as specific combinations of them (35), are needed for normal inflammatory responses, we found that class II MHC molecules and hematopoietic differentiation. The c-Jun, also known as a proto- CIITA (which regulates class II expression) were downregulated in oncogene, has been shown to function as a coactivator of PU.1 MDS monocytes, possibly explaining the downregulation of MHC in promoting monocyte-specific gene expression (36, 37), and class II on DCs derived from these monocytes. This finding a previous report has shown that increased c-Jun expression suggests that Ag presentation and initiation of specific immune can promote aberrant monocytic differentiation (38). In contrast, responses are compromised in these patients. The reduction in pentraxins whose production can be induced by proinflammatory HLA-DR expression found in MDS monocyte-derived DCs could microenvironments also affect macrophage responses; either diminish activation of T cell responses. HLA-DR downregulation by regulating complement activation or by directly binding to in monocyte-derived DCs are supported by a previous study showing receptors and altering macrophage differentiation and polarization decreased HLA-DR expression in MDS monocyte-derived DCs (26). (39, 40). 12 MOLECULAR ALTERATIONS IN MDS PATIENTS’ MONOCYTES

These altered factors, in combination, might be important at 17. Cros, J., N. Cagnard, K. Woollard, N. Patey, S. Y. Zhang, B. Senechal, A. Puel, S. K. Biswas, D. Moshous, C. Picard, et al. 2010. Human CD14dim monocytes inducing impaired or abnormal monocyte differentiation and patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Im- should be further investigated. munity 33: 375–386. We have shown that BM monocytes from MDS patients have 18. Han, Y., H. Wang, and Z. Shao. 2016. Monocyte-derived macrophages are impaired in myelodysplastic syndrome. J. Immunol. Res. 2016: 5479013. defective phagocytosis and have reduced expression of genes and 19. Miraki-Moud, F., F. Anjos-Afonso, K. A. Hodby, E. Griessinger, G. Rosignoli, corresponding cell surface molecules that participate in phago- D. Lillington, L. Jia, J. K. Davies, J. Cavenagh, M. Smith, et al. 2013. Acute cytosis and inflammation. The reduced phagocytosis activity by myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation. Proc. Natl. Acad. Sci. USA 110: MDS monocytes was independent of CD56 expression. The DCs 13576–13581. derived from MDS monocytes have morphologic and phenotypic 20. Dayyani, F., A. P. Conley, S. S. Strom, W. Stevenson, J. E. Cortes, G. Borthakur, S. Faderl, S. O’Brien, S. Pierce, H. Kantarjian, and G. Garcia-Manero. 2010. abnormalities, such as reduced dendritic-like projections and lower Cause of death in patients with lower-risk myelodysplastic syndrome. Cancer expression of HLA-DR and CD86, both essential to Ag handling 116: 2174–2179. and presentation. These alterations may be related to the higher 21. Leone, G., and L. Pagano. 2018. Infections in myelodysplastic syndrome in relation to stage and therapy. Mediterr. J. Hematol. Infect. Dis. 10: e2018039. susceptibility to infections observed in MDS patients. 22. Ravetch, J. V., and S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19: 275–290. 23. Bournazos, S., T. T. Wang, and J. V. Ravetch. 2016. The role and function of Fcg Acknowledgments receptors on myeloid cells. Microbiol. Spectr. DOI: 10.1128/microbiolspec. We are grateful to Sociedade Beneficente Israelita Brasileira Hospital MCHD-0045-2016. Albert Einstein, Clinical Pathology Laboratory, and National Counsel 24. Shimaoka, T., T. Nakayama, N. Kume, S. Takahashi, J. Yamaguchi, M. Minami, K. Hayashida, T. Kita, J. Ohsumi, O. Yoshie, and S. Yonehara. 2003. Cutting of Technological and Scientific Development (CNPq) for support. We edge: SR-PSOX/CXC chemokine ligand 16 mediates bacterial phagocytosis by also thank Dr. Ises de Almeida Abrahamsohn for insightful suggestions APCs through its chemokine domain. J. Immunol. 171: 1647–1651. andforreviewingthisarticle. 25. Skjesol, A., M. Yurchenko, K. Bo¨sl,C.Gravastrand,K.E.Nilsen, Downloaded from L. M. Grøvdal, F. Agliano, F. Patane, G. Lentini, H. Kim, et al. 2019. The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria Disclosures by interacting with the Rab11-family interacting protein 2. PLoS Pathog. The authors have no financial conflicts of interest. 15: e1007684. 26. Davison, G. M., N. Novitzky, and R. Abdulla. 2013. Monocyte derived dendritic cells have reduced expression of co-stimulatory molecules but are able to stimulate autologous T-cells in patients with MDS. Hematol. Oncol. Stem Cell

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