Intracellular concentration of ADA2 is a marker for monocyte differentiation and activation

Abstract

Adenosine, a critical molecule regulating cellular function both inside and outside cells, is controlled by two human adenosine deaminases: ADA1 and ADA2. While ADA1 primarily resides in the cytoplasm, ADA2 can be transported to lysosomes within cells or secreted outside the cell. Patients with ADA2 deficiency (DADA2) often suffer from systemic vasculitis due to elevated levels of TNF-α in their blood. Monocytes from DADA2 patients exhibit excessive TNF-α secretion and differentiate into pro-inflammatory M1-type macrophages. Our findings demonstrate that ADA2 localizes to endolysosomes within macrophages, and its intracellular concentration decreases in cells secreting TNF-α. This suggests that ADA2 may function as a lysosomal adenosine deaminase, regulating TNF-α expression by the cells. Interestingly, pneumonia patients exhibit higher ADA2 concentrations in their bronchoalveolar lavage (BAL), correlating with elevated pro-inflammatory cytokine levels. Conversely, cord blood has low ADA2 levels, creating a more immunosuppressive environment. Additionally, secreted ADA2 can bind to apoptotic cells, activating immune cells by reducing extracellular adenosine levels. These findings imply that ADA2 release from monocytes during inflammation, triggered by growth factors, may be crucial for cell activation. Targeting intracellular and extracellular ADA2 activities could pave the way for novel therapies in inflammatory and autoimmune disorders.

Keywords

adenosine deaminase 2 (ADA2) / TNF-α / adenosine deaminase 2 deficiency (DADA2) / monocyte subsets / macrophage polarization / pneumonia

1 Introduction

Extracellular adenosine is a critical signaling molecule that regulates cellular responses to changes in the extracellular environment during inflammation or stress conditions [1]. Adenosine binds to four adenosine receptors (ADORs), which are expressed differently on nearly all human cells and exhibit varying affinities for adenosine [2]. Research studies have demonstrated that adenosine binding to ADORs suppresses inflammatory responses triggered by activating signals. Additionally, many cancer cells express 5′-nucleotidase (CD73), an enzyme that converts AMP to adenosine, creating an immunosuppressive environment [1]. Adenosine can enter or exit cells via equilibrative nucleoside transporters (ENTs) [3]. Adenosine is typically converted to AMP or inosine by adenosine kinase (AK) or adenosine deaminase inside the cells [4]. If not removed, adenosine can bind to S-adenosylhomocysteine hydrolase (SAH), inhibiting DNA methylation, which in turn regulates gene expression [5]. In humans, adenosine and deoxyadenosine levels are regulated by two adenosine deaminases: ADA1 and ADA2. Interestingly, ADA1, despite lacking a signal sequence, is found inside and outside cells, where it binds to its receptor dipeptidyl peptidase IV (DPPIV/CD26) [6]. In contrast, ADA2, which possesses a signal sequence, can be secreted by myeloid lineage cells and activated T cells. Notably, the concentration of ADA2 in biological fluids of patients with infectious diseases and cancer makes it a convenient diagnostic and prognostic marker for tuberculosis and oral cancers [7,8]. It has been suggested that ADA2 regulates the activity of specific cell subsets by reducing the local concentration of extracellular adenosine. However, during evolution from flies to humans, the K_m value for adenosine deamination by ADA2 has increased 40-fold (K_m = 2 mmol/L), compared to the K_m value of 50 μmol/L for ADA1. Consequently, ADA2 functions as a much less efficient adenosine deaminase at low adenosine concentrations [9]. The proposed role for secreted ADA2 is to regulate extracellular adenosine levels in sites of inflammation and tumor growth where adenosine concentrations are significantly elevated [10]. Additionally, post-translational modifications and trafficking of ADA2 to lysosomes have been observed [11]. This suggests that ADA2 may also modulate adenosine (deoxyadenosine) concentration within endolysosomes, where the adenosine concentration is reported to be close to the K_m value of ADA2 [12]. Another potential function of ADA2 is its ability to bind DNA, which can occur independently of its ADA activity [13]. Consequently, intracellular ADA2 may play a role in controlling cellular responses to activation signals. Notably, ADA2 in NK cells and B cells may serve a purely intracellular function.

Autosomal recessive germline mutations of ADA2 can cause ADA2 deficiency (DADA2), which manifests with various symptoms, including cytopenia, pure red cell aplasia, lacunar strokes, polyarthritis nodosa (PAN phenotype), and large granulocyte lymphocyte leukemia (LGLL phenotype) [14,15]. In DADA2 patients, the expression of ADA2 is dramatically decreased. Intriguingly, the diverse phenotypes may result from mutations affecting the unknown intracellular function of the enzyme. Specifically, patients with PAN DADA2 present with systemic vasculitis [16]. The analysis of monocytes from these patients reveals excessive production of TNF-α, IL-6, and IL-1β within the cells, along with overexpression of genes related to NF-κB and IFN inflammatory pathways. Additionally, these monocytes exhibit an inability to differentiate into M2-type macrophages [17]. The primary treatment strategies for DADA2 involve allogeneic hematopoietic stem cell transplantation (HSCT) and anti-TNF-α inhibitors. Blocking TNF-α with these inhibitors resolves inflammation and restores monocyte differentiation into M2-type macrophages. Therefore, it is likely that the ADA2 deficiency is primarily associated with excessive TNF-α expression by monocytes, leading to immune responses via activation of TNF-α receptors.

Interestingly, knocking out the ADA2 gene in monocytic THP1 and UP37 cell lines results in excessive TNF-α secretion during M1 polarization in the presence of IFN-γ and LPS [18,19]. These results are challenging to explain solely based on the absence of the secreted form of ADA2, which would theoretically result in increased adenosine concentration in the cell culture. If the extracellular adenosine concentration is high, activation of adenosine receptors should decrease rather than increase TNF-α secretion [20].

In our study, we examined ADA2 concentration and localization in monocyte subsets and monocytes undergoing differentiation into macrophages. We propose that intracellular ADA2 may modulate the activity of immune cells by regulating the level of adenosine (deoxyadenosine) inside endolysosomes.

2 Materials and methods

2.1 Ethics statement

The study was undertaken in compliance with the principles of the Helsinki Declaration and was approved by the ethics committee of Helsinki University Central Hospital, Finland. All patients were informed of the study, and they gave written consent before sample collection. The Medical Ethics Committee of Guangzhou Women and Children’s Medical Center (GWCMC) in Guangzhou, China, approved the study of patients with pneumonia under Approval No. 2016111853. The study adhered to ethical standards and guidelines. The serum from cord blood and healthy women was obtained from GWCMC Biobank.

2.2 Cell culture experiments with LPS-activated monocytes

The analysis of PBMCs isolated from fresh blood and detection of the concentration of ADA2 and TNF-α in human plasma from healthy donors and PAN DADA2 patient, described in Trotta et al. [15], were performed as shown previously [20]. CD14⁺ monocytes were isolated from the fresh blood of healthy donors and DADA2 patients, as previously described [20], and cultured in suspension in 5 mL polypropylene tubes (Falcon) at 0.5 × 10⁶ cells/ mL in 0.5 mL of the RPMI culture medium [20]. The monocytes were activated with 10 ng/mL LPS in the presence or absence of adenosine, as indicated. The cells were then separated by centrifugation (300× g, 5 min) after 3–20 h of culture in the tubes at 37 °C, 5% CO₂, and the supernatants were collected to measure the concentration of cytokines by ELISA (BioLegend).

2.3 Cell isolation, sorting, and culture

Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of healthy donors, following IRB-approved protocols. The isolation was performed using 50 mL Leucosep™ tubes (Greiner Bio-One), and after two washes with 50 mL of PBS buffer, CD14⁺ monocytes were purified using anti-CD14-conjugated magnetic microbeads (Miltenyi). In the cell cultures, RPMI 1640 was used as a complete medium supplemented with 1% nonessential amino acids, 1% sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mmol/L L-glutamine, and 10% FBS. Monocytes were cultured in 96 well plates with 200 µL of RPMI medium containing 40 ng/mL GM-CSF or 20 ng/mL M-CSF (PeproTech), respectively. The complete medium with the growth factor was replaced every three days. Monocytes were differentiated for 4–8 days and then incubated overnight with 0.5 µmol/L CpG ODN 2006 PTO (phosphothioate) or CpG ODN 2006 G5 PD (phosphodiester) (Invivogen). To sort the monocyte subsets and other immune cells, PBMCs were stained with fluorescent antibodies as indicated (BD Bioscience) and sorted using BD FACS Aria SORP. The cells were lysed with 0.5% Triton X-100 in 1× PBS and analyzed by ELISA [8]. The concentration of ADA2 in the cells lysates of M-CSF and GM-CSF differentiated monocytes and in the cell culture medium was analyzed similarly and normalized to the total protein concentration determined by BCA protein assay (Thermo Fisher). The cytokines concentration in the cell culture medium was measured by ELISA (BioLegend).

2.4 Immunostainings and confocal microscopy

Monocytes were cultured in 8 well chamber slides (Thermofisher) with 400 µL of RPMI medium with 40 ng/mL GM-CSF or 20 ng/mL M-CSF (PeproTech) as indicated. In some experiments, the cells were treated with 0.5 µmol/L CpG ODN2006 PTO, CpG ODN2006 FITC PTO or CpG ODN 2006 G5 PD (Invivogen) for 24 h. Cells were fixed for 10 min in 10% formaldehyde or 4% PFA, washed three times in PBS, permeabilized for 5 min in PBS supplemented with 0.3% Triton X-100 (PBS-T), and washed three times with PBS. Cells were incubated in the primary antibody overnight diluted in PBS containing 1% BSA at 4 °C, followed by three washes with PBS and an incubation with a secondary antibody in PBS containing 1% BSA for 0.5 h at room temperature. After three further washes, cells were mounted in 80% glycerol. Dilutions for the antibodies against LAMP2 (Sino Biological, 13555-MM05), Rab7 (Cell Signaling Technology), and ADA1 (Abcam, ab34677) were 1:100. Dilutions for the antibodies against ADA2 were 1:50. Fluorescent stains were captured on the Leica SP8 confocal microscope. Polyclonal anti-ADA2 antibodies were purified from the serum of rabbits immunized with recombinant ADA2 (the anti-serum was generated by Sino Biological) as described before [8]. To validate the specificity of the antibodies, we included polyclonal rabbit antibodies as a negative control, which did not result in any observable staining. Additionally, nuclear and endoplasmic reticulum (ER) staining was conducted via DAPI (Cell Signaling Technology) and ER-tracker (Invitrogen) following the manufacturer’s instructions.

2.5 Patients with pneumonia

The participants in this study were children with community-acquired pneumonia as a primary diagnosis without other outstanding co-morbid conditions (known or suspected active tuberculosis, primary immunodeficiency, acquired immunodeficiency syndrome, and immunosuppressive medications taken before admission). Community-acquired pneumonia was diagnosed by clinical doctors based on the chest radiograph or CT. Major criteria for severe pneumonia include (1) invasive mechanical ventilation; (2) fluid refractory shock; (3) acute need for non-invasive positive-pressure ventilation; and (4) hypoxemia requiring fractional inspired oxygen (FiO₂) > inspired concentration or flow feasible in the general-care area. Minor criteria: (1) respiratory rate > World Health Organization (WHO) classification for the patient’s age; (2) apnea; (3) increased work of breathing (e.g., retractions, dyspnea, nasal flaring and grunting); (4) arterial-oxygen partial pressure (PaO₂)/FiO₂ ratio < 250; (5) multilobar infiltrates; (6) Pediatric Early Warning Score > 6; (7) altered mental status; (8) hypotension; (9) presence of effusion; (10) comorbidities; and (11) unexplained metabolic acidosis. The acute phase was defined as patients first enrolled in the hospital with significant clinical symptoms of pneumonia.

2.6 Bronchoalveolar lavage collection and processing

Before administering corticosteroid treatment, bronchoalveolar lavage samples were obtained during the patient’s acute phase. All collected BAL samples were found to be negative for SARS-CoV-2. Flexible fiberoptic bronchoscopy was performed to retrieve additional BAL samples. During the procedure, warm sterile saline (2–3 mL/kg body weight) was injected into each affected area and then aspirated into a suction trap for recovery. The collected samples were processed simultaneously and stored at 4 °C for subsequent cytokine assays. Prior to centrifugation, BAL samples were filtered through double-layered gauze. The supernatants were then collected after centrifugation.

2.7 Quantification of inflammatory cytokines levels in bronchoalveolar lavage

The levels of cytokines in BAL supernatants were measured using Bio-Plex Pro™ Human Cytokine Standard 27-Plex, Group I-kit from Bio-Rad (Hercules, CA) with magnetic bead-based multiplex immunoassay (LX1000; Luminex, Austin, TX), according to the manufacturer’s instructions. The kit included the following cytokines: IL-1β; IL-1RA; IL-2; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-12 (p70); IL-13; IL-15; IL-17A; basic fibroblast growth factor (bFGF); eotaxin; granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF); interferon γ (IFN-γ); interferon-gamma inducible protein 10 kD (IP-10); monocyte chemoattractant protein 1 (MCP-1); macrophage inflammatory proteins 1α and 1β (MIP-1α, MIP-1β); regulated on activation, normal T cell expressed, and secreted (RANTES); tumor necrosis factor-α (TNF-α); platelet-derived growth factor with two B subunits (PDGF-BB); and vascular endothelial growth factor (VEGF).

2.8 Quantification of ADA2 levels in bronchoalveolar lavage

The experiment employs 96-well round-bottom microplates containing a buffer solution with 50 mmol/L Tris pH 6.8, 10 µmol/L ZnCl₂, and 0.02% NaN₃. Each well contains 50 μL of BAL sample or ADA2 standard (5 μL), 5 mmol/L adenosine, and 0.1 mmol/L erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, ADA1 inhibitor). The reaction is initiated by adding a pre-mix comprising adenosine and EHNA to BAL samples or ADA2 standards. The plate is then incubated at 37 °C for 18 h. Subsequently, 8 µL of the reaction mix is transferred to a UV microplate (Corning) with 192 µL water (0.2 mmol/L adenosine final concentration), and the ratio between the absorbances at 245 and 265 nm is determined as described previously [8].

2.9 ADA2 binding to apoptotic cells

THP-1 cells were cultured in a complete RPMI medium with 2 mmol/L adenosine for 3 days. The cells were then collected and incubated with 50 μg/mL ADA2 in 40 μL of FACS buffer (2% FBS, 2 mmol/L EDTA in PBS) for 10 min at room temperature in 96-well round-bottom plates. The cells were washed with 200 μL of FACS buffer and stained with Alexa Fluor 647-labeled anti-ADA2 antibodies for 10 min. The stained cells were then washed and analyzed using flow cytometry (FACS Canto, BD), and the results were analyzed using FlowJo software. The staining with ADA2-SA-ADA2 was performed as described previously [20]. Apoptotic cells were stained with 1 μg/mL propidium iodide (PI) from Sigma, anti-MPO antibodies with IgG isotype control (Abcam), and Annexin V/7-AAD Apoptosis Detection Kit (Sino Biological).

2.10 Statistical analysis

Statistical analysis was conducted using GraphPad Prism software. An unpaired Student’s t-test was used to compare two data sets, while a one-way analysis of variance (ANOVA) was employed for multiple data sets.

3 Results

Systemic polyarteritis nodosa (PAN), immunodeficiency, and ischemic or hemorrhagic strokes are common symptoms observed in PAN DADA2 patients. In our study, we analyzed monocyte responses from a PAN DADA2 patient who carries the ADA2 transcript with a c.G506A (p.Arg169Glu) mutation (Tab.1). The TNF-α level in the PAN DADA2 patient’s blood was elevated compared to healthy individuals. This TNF-α increase could be attributed to cytokine release from monocytes or macrophages. Intracellular staining of monocytes from DADA2 patients revealed enhanced TNF-α expression in unstimulated and LPS-stimulated monocytes [17]. To validate this finding, we isolated monocytes from DADA2 patient and activated them with LPS (Fig.1). As expected, the patient’s LPS-activated monocytes (LAMs) released significantly higher TNF-α levels than healthy donors (Fig.1). In contrast, MCP-1 secretion from the cells remained similar to healthy controls (Fig.1). Interestingly, the release of TNF-α from LAMs of PAN DADA2 patients was less inhibited by a high adenosine concentration compared to healthy donors (Fig.1–1F). Therefore, even in the presence of adenosine, the TNF-α level in the cell culture of LAMs from the PAN DADA2 patient remained persistently high. This suggests that at elevated adenosine concentrations, the cells from the PAN DADA2 patient exhibit reduced sensitivity to adenosine. This phenomenon could be attributed to downregulation of A2 receptors’ expression or alterations in the adenosine receptor pathway.

3.1 Analysis of ADA2 expression inside the cells

Interestingly, the addition of recombinant ADA2 to the cell culture medium does not affect the level of TNF-α secretion by the patient’s monocytes (Fig.1), questioning the extracellular role of ADA2. It was demonstrated that activated THP1 cells knocked out for the ADA2 gene expression show excessive TNF-α secretion compared to wild-type cells [19]. Our experiments further demonstrated that ADA2 knockdown in THP1 cells slows cell proliferation (Fig. S1A), which is not compensated by addition of recombinant ADA2 into the culture medium (Fig. S1C). Collectively, this data strongly suggests that ADA2 may have an intracellular function. Our analysis of blood cells using confocal microscopy revealed high levels of ADA2 expression in monocytes compared to CD3⁺ T cells (Fig. S2). Furthermore, ADA2 forms high-density clusters within monocytes and is partially co-localized with the lysosomal marker LAMP2 (Fig. S3). Next, we analyzed the expression of ADA2 and its mutants inside the cells. As depicted in Fig. S4A, the unmodified enzyme (ADA2wt) is secreted by 293T cells in its active form. It can also be extracted from the cells via cell lysis with Triton X-100. Consistent with prior research, ADA2 mutants found in DADA2 patients were poorly expressed by 293T cells compared to ADA2wt (Fig. S4B). The concentration of ADA2 mutants in cell culture varied based on the type of mutation. Among all the variants tested, the M453 mutant exhibited the highest expression. When cells were lysed with Triton X-100, the concentration of ADA2 mutants in the obtained cell lysates was also significantly lower than ADA2wt (Fig. S4C). Notably, the ADA2 mutant M47 and M109 showed higher relative concentrations inside the cells compared to their secreted form. However, when cells were lysed with SDS and analyzed by Western blot, no difference in expression between ADA2wt and mutants was observed (Fig. S4D). This suggests that the mutants are well expressed inside 293T cells, but only a small fraction of mutant ADA2 proteins can be folded appropriately and secreted outside the cells. The analysis of ADA2wt expression inside the cells by confocal microscopy showed that ADA2wt is localized in the endoplasmic reticulum and Golgi apparatus, which is required for protein secretion outside the cells. In accordance with our previous results, ADA2wt was also found to be partially co-localized with lysosomal marker LAMP2. The mutant variants of ADA2 were also found to be expressed inside 293T cells; also, their expression pattern was different from ADA2wt (Figs. S5–S7). These results indicate that the concentration of ADA2 mutants inside the cells and their distribution may depend on the type of mutation.

3.2 The CD16⁺ subset of monocytes has lower concentrations of ADA2 inside the cells than the classical monocyte subset

Our study confirms that the CD16⁺ subset of monocytes is the primary source of TNF-α (Fig. S8). We observed that monocytes depleted from the CD16⁺ subset produced significantly less TNF-α after activation with LPS. To determine the correlation between the intracellular ADA2 concentration and TNF secretion by the cells, we sorted cell subsets and analyzed the concentration of ADA2 in the cell lysates using ELISA. We found that the concentration of ADA2 inside the CD16⁺ subset was significantly lower than the classical CD16^– monocytes (Fig.2). Our results suggest that the decreased intracellular concentration of ADA2 in CD16⁺ monocytes could be associated with the cells’ ability to produce a higher amount of TNF-α, similar to the monocytes from PAN DADA2 patients (Fig.1). Moreover, the concentration of ADA1 in the monocyte subsets showed the opposite trend, increasing in the non-classical CD16⁺ subset (Fig.2). This result is consistent with previously published data that revealed an increase in ADA1 gene expression in CD16⁺ monocytes [21]. In tuberculosis, the expansion of CD16⁺ monocytes is linked to an increase in concentration of ADA2 in pleural fluid [22,23]. Conversely, cord blood has a lower frequency of CD16⁺ monocytes compared to adult blood, and monocytes isolated from cord blood secrete less TNF-α than those isolated from adult blood [24]. Analysis of ADA2 concentration in the serum from cord blood shows that the concentration of ADA2 is significantly reduced compared to the serum from adult blood (Fig.2). Our findings support the hypothesis that the intracellular concentration of ADA2 may be linked to the activation and differentiation of monocytes.

3.3 Both the levels of secreted and intracellular ADA2 are decreased in GM-CSF differentiated monocytes

It was found that DADA2 monocytes differentiate into M1-type macrophages when exposed to GM-CSF, but they cannot differentiate into M2-type macrophages when exposed to M-CSF [16,17]. This suggests that ADA2 deficiency may influence macrophage polarization toward the inflammatory M1 type, which may contribute to the pathogenesis of DADA2. Our findings indicate that the polarization of macrophages and cell activation may be influenced by the concentration of ADA2 within the cells. We analyzed monocytes differentiated into macrophages in the presence of GM-CSF or M-CSF for four days. It was found that ADA2 expression was present but showed little co-localization with the lysosomal marker LAMP2 (Figs. S9A and S10A). However, when the cells were treated with CpG ODN 2006 PTO (phosphorothioate), it was observed that the density of ADA2 increased inside the lysosomes (Figs. S9B and S10B). Contrary, we did not observe a similar increase in ADA2 density when the cells were treated with CpG ODN 2006 G5 PD (phosphodiester), which has a lower affinity for ADA2 and is more sensitive to DNases compared to PTO ODNs (Figs. S9C and S10C). Our observations suggest that the binding of ADA2 to CpG ODN 2006 PTO in endosomes may lead to the subsequent translocation of the complex into lysosomes. Interestingly, the concentration of ADA2 is increased in cells treated with CpG ODN 2006 PTO (Fig. S11), indicating that the binding of ADA2 to ODN may protect it from degradation inside the lysosomes.

However, the result varied when monocytes were differentiated into macrophages for 8 days (Fig. S14). When M-CSF macrophages were treated with CpG ODN 2006 PTO, the density of ADA2 co-localized to lysosomal and late endosomal markers LAMP2 and Rab7 was increased (Fig.3 and 3D). This was consistent with previous observations (Fig. S10). The enzyme was found to be co-localized with fluorescein-labeled CpG ODN 2006 PTO in the same cell compartments. However, treatment of GM-CSF-derived macrophages with CpG ODN 2006 PTO did not show a similar increase in ADA2 density nor co-localization with the markers. This difference could be due to a lower level of ADA2 expression inside GM-CSF differentiated macrophages, as evidenced by the analysis of ADA2 concentration inside the macrophages. The concentration of ADA2 in GM-CSF differentiated monocytes was significantly lower than in M-CSF differentiated cells (Fig.4). The level of ADA2 secreted by GM-CSF macrophages was also much lower than that of M-CSF macrophages (Fig.4). Additionally, M-CSF differentiated macrophages produced less TNF-α after their treatment with LPS than GM-CSF differentiated macrophages, which aligns with previously published data [25]. These results indicate that M-CSF and GM-CSF regulate the expression level of ADA2 and that the cells expressing less ADA2 secrete more TNF-α in response to the activation of cells with LPS.

3.4 The concentration of ADA2 is increased in the bronchoalveolar lavage of patients with pneumonia

Studies have shown that the activity of ADA increases in the bronchoalveolar lavage (BAL) of patients suffering from pulmonary tuberculosis [26]. We decided to investigate the concentration of ADA2 in BAL samples of children with pneumonia. BAL has a low concentration of proteins that does not interfere with measuring of ADA activity at high adenosine concentration. To quantify the concentration of ADA2 in BAL, we developed a simplified assay that involved incubating a reaction mixture containing BAL samples or standards with known ADA2 concentrations with adenosine and ADA1 inhibitor EHNA in a 96-well plate overnight (Fig.5). The reaction mixture was then transferred to a UV-transparent plate, diluted with water, and read in a plate reader. The concentration of ADA2 in BAL samples was determined from a standard curve where the ratio between absorbances at 245 and 265 nm was plotted against the known concentration of ADA2 standards (Fig.5). Alternatively, ADA2 concentration in BAL and serum can be determined by ADA2 ELISA [8]. Both assays give similar results for BAL (Fig.5). Surprisingly, the analysis of plasma samples from 10 patients with pneumonia at different stages of the treatment did not reveal a significant difference in ADA2 concentration in serum compared to the healthy donors (Tab.1, Fig.5). However, there was a substantial increase in ADA2 concentration in BAL samples from two patients (Fig.5). These findings suggest that ADA2 is secreted by activated immune cells in the lungs of pneumonia patients and accumulates at high concentrations in BAL.

3.5 The concentration of ADA2 in BAL of pneumonia patients correlates with a high concentration of pro-inflammatory cytokines

ADA2 levels were analyzed in 166 pneumonia patients at different admission times and with different treatments. The results revealed an increase in ADA2 concentration (> 3.8 ng/mL) in 98 patients (Fig.6). However, a lower concentration of ADA2 in BAL could indicate successful treatment of the disease. Interestingly, a very high ADA2 concentration (> 50 ng/mL) was found in patients with severe pneumonia but not with mild or moderate infection. To further investigate this, the concentration of ADA2 was compared with the concentration of cytokines in the samples from 77 patients (Table S1). The samples with higher ADA2 concentrations (> 20 ng/mL) contained significantly higher concentrations of pro-inflammatory cytokines IL-6, TNF-α, and other factors (Fig.6C–6E). These results suggest that ADA2 could be a new marker for pneumonia, and the increased concentration of ADA2 is associated with an increased concentration of cytokines, chemokines, and other factors found in patients with pneumonia. The analysis of ADA2 concentration in BAL from patients with severe pneumonia on different days after treatment showed a decrease in ADA2 concentration after the treatment (Fig.6). All the patients showed significant improvement in clinical symptoms and were discharged from the hospital after treatment, with no fatal cases reported. Therefore, ADA2 in BAL could be used as a prognostic marker and an indicator of successful treatment of the disease.

3.6 ADA2 binds to early apoptotic cells

Cells that undergo apoptosis produce extracellular traps made of DNA that can bind to positively charged proteins [27]. In our study, we induced apoptosis in THP1 monocytic cells using adenosine, a molecule known to be toxic to cells at high concentrations [28]. We investigated ADA2 binding to apoptotic cells by incubating the cells with adenosine, which resulted in the emergence of two populations: early apoptotic cells (Apo 1) and late apoptotic cells (Apo 2). These populations were successfully stained using a DNA binding dye propidium iodide (PI) (Fig.7–7C). We observed that the early apoptotic Apo 1 population dominated when THP1 cells were grown in fresh media at a low density of cells in the presence of adenosine (Fig.7D–7F). To further confirm apoptosis, we stained the cells with anti-MPO (myeloperoxidase), an enzyme associated with extracellular traps (Fig.7). Additionally, staining with annexin V, which binds to phosphatidylserine on the outer plasma membrane of apoptotic cells, and 7-aminoactinomycin D (7-AAD), which binds to DNA, provided further evidence supporting the identification of Apo 1 and Apo 2 as cells undergoing apoptosis (Fig.7). To analyze ADA2 binding to THP1 cells undergoing apoptosis, we added the recombinant enzyme to the cells, and the bound ADA2 was detected with anti-ADA2 antibodies after removing the unbound protein during the cell washing step. We found that both live and late apoptotic cells were negative for ADA2, and only early apoptotic cells specifically bound ADA2 (Fig.7). Previously, we have shown that two molecules of ADA2 linked via streptavidin (SA) bind to the surface of immune cells [20]. Accordingly, ADA2-SA-ADA2 dimer binds both to the viable and early apoptotic cells due to its strong affinity to proteoglycans expressed on the cell surface and possibly the DNA in apoptotic cells (Fig.7). The binding of ADA2-SA-ADA2 to the cells could be inhibited with CpG ODN 2006 PTO (Fig.7), confirming that ADA2 binds CpG ODN 2006 PTO.

Extracellular adenosine can bind to adenosine receptors or enter the cells through equilibrative nucleoside transporters (ENT). Though activation of adenosine receptors (ADOR) with a non-selective adenosine receptor agonist NECA does not result in apoptosis of THP1 cells, it is more likely that adenosine is transported into the cells via ENT, where it triggers apoptosis (Fig. S15A and S15B). Inhibition of adenosine deaminase activity in THP1 cells with the ADA1 inhibitor EHNA or blocking ENT1 transporter also leads to apoptosis, suggesting that intracellular adenosine or its derivatives are responsible for the cell’s death (Fig. S15C and S15D). Moreover, ADA1 or ADA2 added to the cell culture medium efficiently converts adenosine to inosine and protects the cells from apoptosis (Fig. S15E and S15F), suggesting an additional function of extracellular ADAs.

4 Discussion

Humans have two types of adenosine deaminases, ADA1 and ADA2. While ADA2 has a signal sequence, ADA1 does not, which led to the suggestion that ADA1 is mainly an intracellular ADA and ADA2 functions as an extracellular adenosine deaminase [6]. However, the low affinity of ADA2 for adenosine makes it inefficient at low adenosine concentrations, and the absence of ADA1 activity due to genetic mutations is not compensated by the presence of ADA2. In this work, we propose a possible intracellular function of ADA2 as a lysosomal adenosine deaminase. We suggest that ADA2 has an anti-inflammatory function inside endolysosomes and that the release of ADA2 from the cells in response to growth factors may be required for cell activation, resulting in increased secretion of cytokines and cell polarization. At the same time, the extracellular enzyme could bind to apoptotic cells and concentrate in sites of inflammation in the lungs, such as bronchial liquid, where it acts as an ADA at high concentrations of adenosine and activates immune cells by removing adenosine.

According to multiple reports, patients with PAN DADA2 exhibit elevated levels of TNF-α in their plasma [20]. TNF-α, released excessively from macrophages, natural killer (NK) cells, and T cells, binds to TNF-α receptors, triggering an inflammatory response [29]. This chronic inflammation may contribute to the development of autoimmune diseases. Notably, both stimulated and unstimulated PAN DADA2 monocytes secrete higher TNF-α, IL-6, and IL-1β levels than healthy donors [17]. However, subsequent treatment of PAN DADA2 patients with anti-TNF-α inhibitors has shown promising results. These inhibitors downregulate the expression of pro-inflammatory cytokines and interferons while also restoring the ability of monocytes to differentiate in the presence of M-CSF. Despite these findings, the precise mechanism linking ADA2 deficiency to the excessive release of TNF-α from macrophages remains unclear.

In this study, we confirmed the published data by demonstrating that LPS-stimulated monocytes from DADA2 patients release more TNF-α than healthy donors (Fig.1). Additionally, monocytes from DADA2 patients have lower sensitivity to extracellular adenosine (Fig.1 and 1E), which can also lead to higher levels of TNF-α in the patients’ plasma (Tab.1). The addition of extracellular ADA2 did not decrease the TNF-α secretion from the activated monocytes (Fig.1), which does not support the idea that ADA2 may have extracellular ADA or growth factor activity that regulates TNF-α secretion. This observation also indicates that ADA2 replacement therapy may not give positive results. However, it was shown previously that the level of TNF-α and IL-6 release from DADA2 monocytes decreased after the overexpression of ADA2 using the lentivirus transfection of monocytes [19]. Furthermore, the level of cytokine secretion was even lower than that of untreated cells from healthy donors. These results support the hypothesis that intracellular rather than extracellular ADA2 downregulates the level of cytokines secretion by activated monocytes.

The study on human leukemia monocytic cells THP1 has revealed that knocking out the ADA2 gene results in slower cell proliferation, indicating the enzyme’s intracellular function (Fig. S1A). The addition of extracellular recombinant ADA2 did not restore the proliferation rate (Fig. S1C). These results suggest that intracellular ADA2 is essential for the efficient proliferation of human leukemia monocytic cells. This finding is in line with previously published observations showing that CD34⁺ hemopoietic stem progenitor cells (HSPC) from a patient with DADA2 have a slower rate of proliferation compared to the healthy controls, which can be restored by overexpressing ADA2 in DADA2 HSPC cells [18]. Therefore, targeting ADA2 activity with specific inhibitors inside the cells could be a potential drug target for leukemia treatment.

Analysis of CD3⁺ T cells and CD14⁺ monocytes by confocal microscopy shows that ADA2 is expressed inside the cells, and the density of ADA2 staining is much higher in monocytes than T cells (Fig. S2). ADA2 was found to be partially localized in the endosomal reticulum (ER) and lysosomes (Fig. S3), in line with the previous studies showing that ADA2 is a secreted protein and it can also be trafficked into lysosomes [30–32]. HEK293 transfected with lentiviruses carrying the ADA2 gene express fully active ADA2 inside the cells and secreted in the culture medium (Fig. S4A). Inside the cells, the enzyme is partially co-localized with the marker for ER (Fig.5), Golgi apparatus (Fig.6), and lysosomes (Fig.7), indicating that both secretion and lysosome trafficking pathways are activated in 293T cells. Interestingly, the analysis of the cells expressing ADA2wt and ADA2 mutants found in DADA2 patients [16] by Western blot did not reveal a significant difference between ADA2wt and the mutant protein level (Fig. S4D). However, the concentration of active ADA2 molecules carrying mutations was dramatically reduced outside and inside the cells compared to ADA2wt (Fig. S4B and S4C). This result suggests that mutations in ADA2 destabilize the protein structure, leading to protein aggregation and subsequent degradation. Analysis of the cells expressing ADA2 mutants shows the protein distribution different from the wild-type ADA2, indicating that both secretion and lysosomal trafficking are affected by the mutations. Interestingly, the remaining active intracellular ADA2 in DADA2 patients may explain the difference between the disease phenotype [33].

Recently, we have found that monocytes differentiated into macrophages in the presence of GM-CSF and treated with phosphothioated (PTO) CpG oligonucleotides, which display strong affinity for ADA2, drive ADA2 into the lysosomes [13] (Fig.8). This study confirms this finding, showing that both GM-CSF and M-CSF differentiated monocytes treated with CpG ODN 2006 PTO have increased ADA2 density in the lysosomes (Figs. S8–S10). The analysis of the cell lysates by ELISA reveals the increased concentration of ADA2 in the cells treated with CpG ODN 2006 PTO (Fig. S11). The latter could be explained by reduced degradation of ADA2 bound to CpG ODN 2006 PTO in the lysosomes, which is absent when the cells are treated with natural phosphodiester (PD) CpG ODNs. Interestingly, GM-CSF differentiated monocytes treated with CpG ODN 2006 PTO secrete less TNF-α in response to IFN-γ and LPS (M1) activation (Fig. S11), suggesting that the increase in ADA2 concentration inside the lysosomes may downregulate TNF-α expression by macrophages. It was shown that lysosomes contain up to 3 mmol/L adenosine, a product of ATP hydrolysis [12]. Adenosine can be transported into the cytoplasm via NTP3 [3], inhibiting methyltransferase and hence, methylation of the genes [5]. Hypomethylation of the genes could increase TNF-α secretion in response to macrophage activation [34].

Our previous studies have demonstrated that ADA2 is released from monocytes in response to monocyte activation by growth factors [31]. In this study, we further investigate monocytes undergoing differentiation into macrophages over 8 days in the presence of M-CSF and GM-CSF. We found that M-CSF macrophages express higher levels of ADA2 inside and outside the cells than GM-CSF-differentiated monocytes (Fig.4). When probing the cells with CpG ODN 2006 PTO, we observed that ADA2 colocalizes with the lysosomal marker and CpG ODN 2006 in M-CSF macrophages but not in GM-CSF macrophages (Fig.3). This suggests that the concentration of ADA2 within endolysosomes of GM-CSF macrophages is significantly lower than in M-CSF macrophages. Interestingly, LPS-treated GM-CSF macrophages produce more TNF-α than M-CSF macrophages (Fig.4), which aligns with previous observations [25]. While it might imply that the low concentration of ADA2 in endolysosomes is linked to higher TNF-α secretion, it is also possible that ADA2 expression is downregulated in both M-CSF and GM-CSF macrophages albeit at different rates [35].

Here, we propose a new mechanism according to which ADA2 controls inflammation by regulating the sensitivity of cells to activating signals, which induce TNF-α expression and secretion by the cells. According to our hypothesis, ADA2 can be either secreted outside the cells or trafficked into lysosomes. It has been shown that ADA2 secretion requires the enzyme glycosylation in the endoplasmic reticulum [30]. To transport ADA2 into lysosomes, the enzyme is modified with mannose-6 phosphate (M6P) and binds to an M6P receptor in trans-Golgi [36]. These two pathways can be regulated by growth factors such as GM-CSF or M-CSF produced by cells activated during the inflammation. The decrease in ADA2 concentration in the endosomes of DADA2 patients may explain the excessive release of TNF-α by the patient’s monocytes and macrophages and the inhibition of M2 macrophage polarization [37]. As proposed in Fig.8, ADA2 may control the concentration of adenosine in the lysosomes that could be released in the cytoplasm and modulate DNA methylation. This mechanism should be further investigated.

Intracellular infections such as tuberculosis or HIV are associated with increased concentrations of ADA2 in pleural fluid and plasma, respectively [38,39]. Here we show that the concentration of ADA2 is increased in the bronchioalveolar fluid of patients with pneumonia (Fig.5), and it is correlated with elevated levels of proinflammatory cytokines (Fig.6–6E). This observation supports our hypothesis that the release of ADA2 from monocytes initiated by growth factors results in the increased cells’ sensitivity to the activation signals and the release of pro-inflammatory cytokines from the cells. The concentration of ADA2 in BAL is decreasing following the treatment of patients with severe pneumonia (Fig.6). Therefore, the level of ADA2 in BAL is a new biomarker for diagnosing pneumonia and follow-up of the treatment.

We and the others show that CD16⁺ monocytes secrete a higher amount of TNF-α upon activation with LPS compared to the classical CD16^– monocytes (Fig. S11) [40]. At the same time, the concentration of ADA2 decreases in CD16⁺ monocytes (Fig.2). This observation provides evidence of another link between intracellular ADA2 concentration and TNF-α secretion by monocytes, suggesting that the differentiation of CD16^– monocytes into CD16⁺ subset is associated with a decrease in the intracellular concentration of ADA2. Indeed, patients with pleural tuberculosis have an increased number of CD16⁺ monocytes and extracellular ADA2 concentration [41]. This observation supports our hypothesis that the activation of monocytes is associated with the release of ADA2 from the cells and increased secretion of proinflammatory cytokines. On the contrary, the decreased number of CD16⁺ monocytes in cord blood is associated with a decreased concentration of ADA2 (Fig.2). Interestingly, the expression of the ADA2 gene was not found to be significantly changed in CD16⁺ monocytes [21], indicating that the activated CD16^– subset could release ADA2 stored in the cells upon the cells’ differentiation into the CD16⁺ subset.

We also demonstrated that contrary to ADA2, the concentration of ADA1 increases in the CD16⁺ monocyte subset. This result is in line with the previously published gene expression data showing that the expression of ADA1 is downregulated in the classical CD16^– monocytes [21,42]. Therefore, CD16^– monocytes with low levels of ADA1 gene expression could be more sensitive to the intracellular adenosine concentration. Hence, a decrease in ADA2 concentration in endosomes would result in the release of adenosine into the cytoplasm and subsequent inhibition of the gene methylation and increased cytokines secretion (Fig.8). Interestingly, the level of ADA1 expression was not found to change in monocytes differentiated into macrophages or DC cells [35], indicating that these cells are also sensitive to fluctuations in the intracellular concentration of adenosine. However, the precise role of ADA1 and ADA2 in regulating monocyte/macrophage activity should be further investigated.

We also show that THP1 cells incubated with adenosine undergo apoptosis, indicating that high levels of adenosine entering the cells through the ENT channels are not removed by the intracellular ADA1 or ADK (adenosine kinase), preventing the cells from dying (Figs. S2A and 8). Inhibition of ADA1 with an ADA1-specific inhibitor EHNA and blocking the ENT channel induces the cell’s apoptosis (Fig. S2C and S2D). All these results show that the cells are sensitive to both intracellular and extracellular concentrations of adenosine. We demonstrate that ADA2 binds to apoptotic cells (Fig.7), suggesting that ADA2 secreted by differentiating monocytes in the sites of inflammation can protect the immune cells from apoptosis by decreasing adenosine concentration in the sites of inflammation (Fig. S2F). The decrease in adenosine concentration would also restore the activity of the immune cells and potentiate cytokine secretion, which is inhibited by A_2A receptors bound to adenosine (Fig.1 and 8) [20].

5 Conclusions

In summary, our study has shown that in addition to being extracellular adenosine deaminase, ADA2 is present in endolysosomes, and its concentration decreases in cells that secrete TNF-α, specifically the CD16⁺ subset of monocytes and GM-CSF differentiated macrophages. This suggests a new mechanism where growth factors regulate the expression level of ADA2 within cells and can affect the cells’ sensitivity to activation signals. Therefore, inhibiting ADA2 activity in the endolysosome could be a promising therapeutic strategy for controlling monocyte differentiation and macrophage polarization in cases of inflammation or cancer.

References

[1] Boison D, Yegutkin GG. Adenosine metabolism: emerging concepts for cancer therapy. Cancer Cell 2019; 36(6): 582–596

[2] Fredholm BB. Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ 2007; 14(7): 1315–1323

[3] Boswell-Casteel RC, Hays FA. Equilibrative nucleoside transporters—a review. Nucleosides Nucleotides Nucleic Acids 2017; 36(1): 7–30

[4] Zhulai G, Oleinik E, Shibaev M, Ignatev K. Adenosine-metabolizing enzymes, adenosine kinase and adenosine deaminase, in cancer. Biomolecules 2022; 12(3): 418

[5] Xu Y, Wang Y, Yan S, Zhou Y, Yang Q, Pan Y, Zeng X, An X, Liu Z, Wang L, Xu J, Cao Y, Fulton DJ, Weintraub NL, Bagi Z, Hoda MN, Wang X, Li Q, Hong M, Jiang X, Boison D, Weber C, Wu C, Huo Y. Intracellular adenosine regulates epigenetic programming in endothelial cells to promote angiogenesis. EMBO Mol Med 2017; 9(9): 1263–1278

[6] Antonioli L, Colucci R, La Motta C, Tuccori M, Awwad O, Da Settimo F, Blandizzi C, Fornai M. Adenosine deaminase in the modulation of immune system and its potential as a novel target for treatment of inflammatory disorders. Curr Drug Targets 2012; 13(6): 842–862

[7] Delemarre EM, van Hoorn L, Bossink AWJ, Drylewicz J, Joosten SA, Ottenhoff THM, Akkerman OW, Goletti D, Petruccioli E, Navarra A, van den Broek BTA, Paardekooper SPA, van Haeften I, Koenderman L, Lammers JWJ, Thijsen SFT, Hofland RW, Nierkens S. Serum biomarker profile including CCL1, CXCL10, VEGF, and adenosine deaminase activity distinguishes active from remotely acquired latent tuberculosis. Front Immunol 2021; 12: 725447

[8] Luo W, Dong L, Chen F, Lei W, He L, Zhou Q, Lamy T, Zavialov AV. ELISA based assays to measure adenosine deaminases concentration in serum and saliva for the diagnosis of ADA2 deficiency and cancer. Front Immunol 2022; 13: 928438

[9] Zavialov AV, EngströmÅ. Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity. Biochem J 2005; 391(1): 51–57

[10] Driver AG, Kukoly CA, Ali S, Mustafa SJ. Adenosine in bronchoalveolar lavage fluid in asthma. Am Rev Respir Dis 1993; 148(1): 91–97

[11] Sleat DE, Zheng H, Qian M, Lobel P. Identification of sites of mannose 6-phosphorylation on lysosomal proteins. Mol Cell Proteomics 2006; 5(4): 686–701

[12] Zhong XZ, Zou Y, Sun X, Dong G, Cao Q, Pandey A, Rainey JK, Zhu X, Dong XP. Inhibition of transient receptor potential channel mucolipin-1 (TRPML1) by lysosomal adenosine involved in severe combined immunodeficiency diseases. J Biol Chem 2017; 292(8): 3445–3455

[13] Dong L, Luo W, Skaldin M, Robson CS, Zavialov AV. Adenosine deaminase 2 regulates the activation of the Toll-like receptor 9 in response to nucleic acids. Front Med 2024; 18(5): 814–830

[14] Lee PY, Aksentijevich I, Zhou Q. Mechanisms of vascular inflammation in deficiency of adenosine deaminase 2 (DADA2). Semin Immunopathol 2022; 44(3): 269–280

[15] Trotta L, Martelius T, Siitonen T, Hautala T, Hämäläinen S, Juntti H, Taskinen M, Ilander M, Andersson EI, Zavialov A, Kaustio M, Keski-Filppula R, Hershfield M, Mustjoki S, Tapiainen T, Seppänen M, Saarela J. ADA2 deficiency: clonal lymphoproliferation in a subset of patients. J Allergy Clin Immunol 2018; 141(4): 1534–1537.e8

[16] Zhou Q, Yang D, Ombrello AK, Zavialov AV, Toro C, Zavialov AV, Stone DL, Chae JJ, Rosenzweig SD, Bishop K, Barron KS, Kuehn HS, Hoffmann P, Negro A, Tsai WL, Cowen EW, Pei W, Milner JD, Silvin C, Heller T, Chin DT, Patronas NJ, Barber JS, Lee CCR, Wood GM, Ling A, Kelly SJ, Kleiner DE, Mullikin JC, Ganson NJ, Kong HH, Hambleton S, Candotti F, Quezado MM, Calvo KR, Alao H, Barham BK, Jones A, Meschia JF, Worrall BB, Kasner SE, Rich SS, Goldbach-Mansky R, Abinun M, Chalom E, Gotte AC, Punaro M, Pascual V, Verbsky JW, Torgerson TR, Singer NG, Gershon TR, Ozen S, Karadag O, Fleisher TA, Remmers EF, Burgess SM, Moir SL, Gadina M, Sood R, Hershfield MS, Boehm M, Kastner DL, Aksentijevich I. Early-onset stroke and vasculopathy associated with mutations in ADA2. N Engl J Med 2014; 370(10): 911–920

[17] Deuitch NT, Yang D, Lee PY, Yu X, Moura NS, Schnappauf O, Ombrello AK, Stone D, Kuehn HS, Rosenzweig SD, Hoffmann P, Cudrici C, Levy DM, Kessler E, Soep JB, Hay AD, Dalrymple A, Zhang Y, Sun L, Zhang Q, Tang X, Wu Y, Rao K, Li H, Luo H, Zhang Y, Burnham JM, Boehm M, Barron K, Kastner DL, Aksentijevich I, Zhou Q. TNF inhibition in vasculitis management in adenosine deaminase 2 deficiency (DADA2). J Allergy Clin Immunol 2022; 149(5): 1812–1816.e6

[18] Hong Y, Casimir M, Houghton BC, Zhang F, Jensen B, Omoyinmi E, Torrance R, Papadopoulou C, Cummins M, Roderick M, Thrasher AJ, Brogan PA, Eleftheriou D. Lentiviral mediated ADA2 gene transfer corrects the defects associated with deficiency of adenosine deaminase type 2. Front Immunol 2022; 13: 852830

[19] Zoccolillo M, Brigida I, Barzaghi F, Scala S, Hernández RJ, Basso-Ricci L, Colantuoni M, Pettinato E, Sergi LS, Milardi G, Capasso P, Lombardo A, Gregori S, Sanvito F, Schena F, Cesaro S, Conti F, Pession A, Benedetti F, Gattorno M, Lee PY, Naldini L, Cicalese MP, Aiuti A, Mortellaro A. Lentiviral correction of enzymatic activity restrains macrophage inflammation in adenosine deaminase 2 deficiency. Blood Adv 2021; 5(16): 3174–3187

[20] Kaljas Y, Liu C, Skaldin M, Wu C, Zhou Q, Lu Y, Aksentijevich I, Zavialov AV. Human adenosine deaminases ADA1 and ADA2 bind to different subsets of immune cells. Cell Mol Life Sci 2017; 74(3): 555–570

[21] Wong KL, Tai JJY, Wong WC, Han H, Sem X, Yeap WH, Kourilsky P, Wong SC. Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets. Blood 2011; 118(5): e16–e31

[22] Sampath P, Moideen K, Ranganathan UD, Bethunaickan R. Monocyte subsets: phenotypes and function in tuberculosis infection. Front Immunol 2018; 9: 1726

[23] Zeng T, Ling B, Hu X, Wang S, Qiao W, Gao L, Shen Y, Li D. The value of adenosine deaminase 2 in the detection of tuberculous pleural effusion: a meta-analysis and systematic review. Can Respir J 2022; 2022: 7078652

[24] Pedraza- Sánchez S, Hise AG, Ramachandra L, Arechavaleta-Velasco F, King CL. Reduced frequency of a CD14⁺CD16⁺ monocyte subset with high Toll-like receptor 4 expression in cord blood compared to adult blood contributes to lipopolysaccharide hyporesponsiveness in newborns. Clin Vaccine Immunol 2013; 20(7): 962–971

[25] Schneider A, Weier M, Herderschee J, Perreau M, Calandra T, Roger T, Giannoni E. IRF5 is a key regulator of macrophage response to lipopolysaccharide in newborns. Front Immunol 2018; 9: 1597

[26] Kayacan O, Karnak D, Delibalta M, Beder S, Karaca L, Tutkak H. Adenosine deaminase activity in bronchoalveolar lavage in Turkish patients with smear negative pulmonary tuberculosis. Respir Med 2002; 96(7): 536–541

[27] Carmona-Rivera C, Khaznadar SS, Shwin KW, Irizarry-Caro JA, O’Neil LJ, Liu Y, Jacobson KA, Ombrello AK, Stone DL, Tsai WL, Kastner DL, Aksentijevich I, Kaplan MJ, Grayson PC. Deficiency of adenosine deaminase 2 triggers adenosine-mediated NETosis and TNF production in patients with DADA2. Blood 2019; 134(4): 395–406

[28] Puchalowicz K, Tarnowski M, Tkacz M, Chlubek D, Kłos P, Dziedziejko V. Extracellular adenine nucleotides and adenosine modulate the growth and survival of THP-1 leukemia cells. Int J Mol Sci 2020; 21(12): 4425

[29] van Loo G, Bertrand MJM. Death by TNF: a road to inflammation. Nat Rev Immunol 2023; 23(5): 289–303

[30] Lee PY, Huang Y, Zhou Q, Schnappauf O, Hershfield MS, Li Y, Ganson NJ, Sampaio Moura N, Delmonte OM, Stone SS, Rivkin MJ, Pai SY, Lyons T, Sundel RP, Hsu VW, Notarangelo LD, Aksentijevich I, Nigrovic PA. Disrupted N-linked glycosylation as a disease mechanism in deficiency of ADA2. J Allergy Clin Immunol 2018; 142(4): 1363–1365.e8

[31] Zavialov AV, Gracia E, Glaichenhaus N, Franco R, Lauvau G. Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages. J Leukoc Biol 2010; 88(2): 279–290

[32] Sleat DE, Wang Y, Sohar I, Lackland H, Li Y, Li H, Zheng H, Lobel P. Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins. Mol Cell Proteomics 2006; 5(10): 1942–1956

[33] Lee PY, Davidson BA, Abraham RS, Alter B, Arostegui JI, Bell K, Belot A, Bergerson JRE, Bernard TJ, Brogan PA, Berkun Y, Deuitch NT, Dimitrova D, Georgin-Lavialle SA, Gattorno M, Grimbacher B, Hashem H, Hershfield MS, Ichord RN, Izawa K, Kanakry JA, Khubchandani RP, Klouwer FCC, Luton EA, Man AW, Meyts I, Van Montfrans JM, Ozen S, Saarela J, Santo GC, Sharma A, Soldatos A, Sparks R, Torgerson TR, Uriarte IL, Youngstein TAB, Zhou Q, Aksentijevich I, Kastner DL, Chambers EP, Ombrello AK, Makley MK, Hayner KL, Kling BE, Cowsert LM, Williams JS. Evaluation and management of deficiency of adenosine deaminase 2: an international consensus statement. JAMA Netw Open 2023; 6(5): e2315894

[34] Sullivan KE, Reddy ABM, Dietzmann K, Suriano AR, Kocieda VP, Stewart M, Bhatia M. Epigenetic regulation of tumor necrosis factor alpha. Mol Cell Biol 2007; 27(14): 5147–5160

[35] Sander J, Schmidt SV, Cirovic B, McGovern N, Papantonopoulou O, Hardt AL, Aschenbrenner AC, Kreer C, Quast T, Xu AM, Schmidleithner LM, Theis H, Thi Huong LD, Sumatoh HRB, Lauterbach MAR, Schulte-Schrepping J, Günther P, Xue J, Baßler K, Ulas T, Klee K, Katzmarski N, Herresthal S, Krebs W, Martin B, Latz E, Händler K, Kraut M, Kolanus W, Beyer M, Falk CS, Wiegmann B, Burgdorf S, Melosh NA, Newell EW, Ginhoux F, Schlitzer A, Schultze JL. Cellular differentiation of human monocytes is regulated by time-dependent interleukin-4 signaling and the transcriptional regulator NCOR2. Immunity 2017; 47(6): 1051–1066.e12

[36] Coutinho MF, Prata MJ, Alves S. Mannose-6-phosphate pathway: a review on its role in lysosomal function and dysfunction. Mol Genet Metab 2012; 105(4): 542–550

[37] Murray PJ. Macrophage polarization. Annu Rev Physiol 2017; 79(1): 541–566

[38] Keng LT, Shu CC, Chen JYP, Liang SK, Lin CK, Chang LY, Chang CH, Wang JY, Yu CJ, Lee LN. Evaluating pleural ADA, ADA2, IFN-γ and IGRA for diagnosing tuberculous pleurisy. J Infect 2013; 67(4): 294–302

[39] Khodadadi I, Abdi M, Ahmadi A, Wahedi MS, Menbari S, Lahoorpour F, Rahbari R. Analysis of serum adenosine deaminase (ADA) and ADA1 and ADA2 isoenzyme activities in HIV positive and HIV-HBV co-infected patients. Clin Biochem 2011; 44(12): 980–983

[40] Belge KU, Dayyani F, Horelt A, Siedlar M, Frankenberger M, Frankenberger B, Espevik T, Ziegler-Heitbrock L. The proinflammatory CD14⁺CD16⁺DR⁺⁺ monocytes are a major source of TNF. J Immunol 2002; 168(7): 3536–3542

[41] Lastrucci C, Bénard A, Balboa L, Pingris K, Souriant S, Poincloux R, Al Saati T, Rasolofo V, González-Montaner P, Inwentarz S, Moraña EJ, Kondova I, Verreck FAW, Sasiain MC, Neyrolles O, Maridonneau-Parini I, Lugo-Villarino G, Cougoule C. Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16⁺ monocyte population via the IL-10/STAT3 axis. Cell Res 2015; 25(12): 1333–1351

[42] Ancuta P, Liu KY, Misra V, Wacleche V, Gosselin A, Zhou X, Gabuzda D. Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16⁺ and CD16^– monocyte subsets. BMC Genomics 2009; 10(1): 403

RIGHTS & PERMISSIONS

Higher Education Press