Biomarker for diagnosing pneumonia

Abstract

The invention relates to the field of biological medicine, in particular to a biomarker for diagnosing pneumonia. The invention provides application of a biomarker in preparation of a kit for diagnosing pneumonia and / or evaluating the curative effect of pneumonia, and is characterized in that the biomarker comprises ADA2 in bronchoalveolar lavage fluid, and the kit is used for quantitatively detecting the content of ADA2 in a bronchoalveolar lavage fluid sample of a subject. By detecting the content of ADA2 in a bronchoalveolar lavage fluid sample of a subject, whether the subject is a severe pneumonia patient or not can be quickly and accurately diagnosed.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a biomarker for diagnosing pneumonia. Background Technology

[0002] Extracellular adenosine is a key signaling molecule that regulates cellular responses to changes in the extracellular environment under inflammatory or stress conditions. In humans, adenosine and deoxyadenosine levels are regulated by two adenosine deaminases: ADA1 and ADA2. Although ADA1 lacks a signaling sequence, it is present both inside and outside the cell and binds to its receptor, dipeptidyl peptidase IV (DPPIV / CD26). In contrast, ADA2, which possesses a signaling sequence, can be secreted by myeloid cells and activated T cells. Studies have shown that ADA2 regulates the activity of specific cell subpopulations by reducing local concentrations of extracellular adenosine. However, during the evolution from flies to humans, the Km value for adenosine deamination by ADA2 increased 40-fold (Km = 2 mM), while the Km value for ADA1 is 50 μM. Therefore, at low adenosine concentrations, ADA2's function as an adenosine deaminase is much lower.

[0003] Autosomal recessive germline mutations in ADA2 can lead to ADA2 deficiency (DADA2), manifested by a variety of symptoms, including cytopenia, impaired pure red cell aplasia, lacunar stroke, polyarthritis nodosa (PAN phenotype), and large myeloid lymphocytic leukemia (LGLL phenotype). In DADA2 patients, ADA2 expression is dramatically decreased. PAN DADA2 patients present with systemic vasculitis. Analysis of monocytes from these patients revealed excessive intracellular production of TNF-α, IL-6, and IL-1β, as well as overexpression of genes associated with the NF-κB and IFN inflammatory pathways. Furthermore, these monocytes fail to differentiate into M2 macrophages. The primary treatment strategies for DADA2 include allogeneic hematopoietic stem cell transplantation (HSCT) and TNF-α inhibitors. Blocking TNF-α with these inhibitors can eliminate inflammation and restore monocyte differentiation into M2 macrophages. Summary of the Invention

[0004] In a first aspect, the present invention provides the use of a biomarker in the preparation of a kit for diagnosing pneumonia and / or evaluating the efficacy of pneumonia treatment, characterized in that the biomarker includes ADA2 in bronchoalveolar lavage fluid, and the kit is used to quantitatively detect the content of ADA2 in the bronchoalveolar lavage fluid sample of a subject.

[0005] In some embodiments, the kit includes a detection reagent for quantitatively detecting the content of ADA2 in bronchoalveolar lavage fluid samples.

[0006] In some embodiments, the kit is used to distinguish between patients with severe pneumonia and patients with mild / moderate pneumonia.

[0007] In some embodiments, the subject is assessed as a patient with severe pneumonia when the ADA2 content in the bronchoalveolar lavage fluid sample is >50 ng / ml.

[0008] In some embodiments, the subject is SARS-CoV-2 negative.

[0009] In some embodiments, the pneumonia is caused by one or more of bacterial, viral, and fungal infections.

[0010] In some embodiments, the levels of one or more of the following factors are increased in the pneumonia patients: IL-1ra, IL-4, IL-6, IL-9, IL-10, IL-17, Eotaxin, FGF, IFN-γ, IP-10, MCP-1, MIP-1α, PDGFB, MIP-1β, RANTES, and TNF-α.

[0011] The current diagnostic criteria for severe pneumonia are cumbersome and complex. Even with some simplification, the simplified diagnostic criteria published in the 2015 Chinese Adult CAP Guidelines require that patients meet one of the following major criteria or ≥3 minor criteria to be diagnosed with severe pneumonia. The major criteria include: ① endotracheal intubation requiring mechanical ventilation; ② continued use of vasoactive drugs after aggressive fluid resuscitation for septic shock. The minor criteria include: ① respiratory rate ≥30 breaths / min; ② PaO2 / FiO2 ≤250 mmHg; ③ multilobar infiltration; ④ altered mental status and / or disorientation; ⑤ blood urea nitrogen ≥7 mmol / L; ⑥ hypotension requiring aggressive fluid resuscitation. Furthermore, current technologies also struggle to effectively assess the treatment efficacy for severe pneumonia. Surprisingly, this invention discovered that the ADA2 content in bronchoalveolar lavage fluid (BAL) samples from subjects is related to the concentrations of cytokines, chemokines, and other factors in pneumonia patients. By detecting the ADA2 content in BAL samples, a rapid and accurate diagnosis of whether a subject has severe pneumonia can be achieved, whereas the ADA2 content in plasma (serum) samples cannot distinguish between different stages of pneumonia (i.e., serum ADA2 levels are relatively similar in patients at different stages of pneumonia). Furthermore, the ADA2 content in BAL samples can be used to evaluate the treatment efficacy in pneumonia patients. Therefore, ADA2 in BAL can serve as a biomarker for diagnosing pneumonia and / or for assessing pneumonia treatment efficacy (or prognosis).

[0012] Secondly, the present invention provides the use of oligodeoxynucleotides in the preparation of TNF-α inhibitors, characterized in that the oligodeoxynucleotides have a phosphate thioester backbone.

[0013] In some embodiments, the TNF-α inhibitor is used to reduce the secretion of TNF-α in monocytes.

[0014] In some embodiments, the monocytes include CD16+ monocytes, M-CSF-differentiated monocytes, and / or GM-CSF-differentiated monocytes.

[0015] In some embodiments, the TNF-α inhibitor is used to reduce the secretion of TNF-α in macrophages.

[0016] In some embodiments, the oligodeoxynucleotide is a class B CpG oligodeoxynucleotide.

[0017] In some embodiments, the oligodeoxynucleotide comprises ODN 2006PTO.

[0018] In some embodiments, the TNF-α inhibitor is used to treat ADA2 deficiency.

[0019] In some embodiments, the ADA2 deficiency is the PAN phenotype.

[0020] In some embodiments, the ADA2 deficiency patient carries the c.G506A mutation.

[0021] In some embodiments, the sequence of the oligodeoxynucleotide is shown in SEQ ID NO:1.

[0022] This invention provides the use of oligodeoxynucleotides in the preparation of TNF-α inhibitors. Experiments of this invention demonstrate that intracellular ADA2 can downregulate the secretion levels of cytokines (e.g., TNF-α) in activated monocytes, and unexpectedly, it was found that oligodeoxynucleotides with a phosphate thioester backbone (e.g., CpG ODN 2006PTO) can reduce TNF-α secretion in monocytes. Therefore, they can serve as a novel TNF-α inhibitor to specifically downregulate TNF-α expression in monocytes (e.g., CD16+ monocytes, M-CSF-differentiated monocytes, and / or GM-CSF-differentiated monocytes), making them particularly suitable for treating immunodeficiency diseases (e.g., ADA2 deficiency). Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A graph showing the results of LPS-activated monocytes (LAMs) in PAN DADA2 patients exhibiting excessive TNF-α production and limited response to high adenosine concentrations;

[0025] Figure 2 The graph shows the results of the analysis of the intracellular concentrations of ADA2 and ADA1 in lymphocytes and monocyte subsets, as well as the concentration of ADA2 in umbilical cord blood.

[0026] Figure 3 The image shows the results of confocal microscopy analysis of macrophages differentiated from GM-CSF and M-CSF.

[0027] Figure 4 The figure shows the results of the analysis of extracellular and intracellular concentrations of ADA2 in monocytes differentiated into macrophages 8 days in the presence of M-CSF or GM-CSF.

[0028] Figure 5 Methods and results for quantitative determination of ADA2 concentration in bronchoalveolar lavage fluid;

[0029] Figure 6 Figure showing the results of ADA2 and cytokine concentration analysis in bronchoalveolar lavage fluid;

[0030] Figure 7 This is a diagram showing the binding of ADA2 to apoptotic THP1 cells.

[0031] Figure 8 A schematic diagram illustrating the potential functions of ADA2 being transported to the lysosomes of monocytes / macrophages and secreted from outside the cell;

[0032] Figure 9 The image shows the localization of ADA2 in the lysosomes of monocytes differentiated from GM-CSF on day 4.

[0033] Figure 10 The image shows the localization of ADA2 in the lysosomes of monocytes differentiated from M-CSF on day 4.

[0034] Figure 11 The effect of ODN 2006PTO incubation on ADA2 density in macrophage lysosomes after 4 days of differentiation in the presence of GM-CSF is shown in the figure.

[0035] Figure 12 The graph shows the intracellular concentration of ADA2 in monocytes differentiated from M-CSF and GM-CSF.

[0036] Figure 13 The figure shows the results of CpG 2006ODN inhibiting TNF-α secretion by monocytes differentiating from GM-CSF.

[0037] Figure 14 The figure shows the results of TNF-α secretion analysis in total monocytes and monocytes lacking the CD16+ subset. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0040] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0041] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0042] As used in this specification, the term "about" typically means + / - 5% of the value, more typically + / - 4%, more typically + / - 3%, more typically + / - 2%, even more typically + / - 1%, even more typically + / - 0.5% of the value.

[0043] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0044] Detailed description of the attached figures

[0045] Figure 1 (A, B) TNF-α and MCP-1 concentrations in the culture medium of monocytes from PAN-phenotyped DADA2 patients and six healthy donors after incubation with 10 ng / ml LPS for 20 hours. (C) Adding recombinant ADA2 to LAM does not affect TNF-α release in cells. TNF-α concentration in the culture medium of monocytes from PAN-phenotyped DADA2 patients after incubation with 10 ng / ml LPS for 20 hours in the presence or absence of 5.8 μg / ml ADA2. (D, E, F) TNF-α concentrations in the culture medium of monocytes from PAN DADA2 patients (D) and two healthy donors (healthy donor 1 (E), healthy donor 2 (F)) after incubation with 10 ng / ml LPS for 20 hours with progressively increasing adenosine concentrations. Each data point represents the mean of three replicates.

[0046] Figure 2 PBMCs were isolated from fresh blood and labeled with specific cell surface markers. Cell subpopulations were sorted using flow cytometry. (A) Monocyte subpopulation: (1) CD16-CD14+ monocytes, (2) CD16-low CD14+ monocytes, (3) CD16+CD14+ monocytes, (4) CD16+CD14-low monocytes. (B) ADA2 concentration in cell lysates was analyzed by ELISA. (C) ADA1 concentration in cell lysates was analyzed by ELISA. Similar results were obtained using cells from three healthy donors. Each column in the figure represents the mean of three measurements. (D) ADA2 concentration in adult serum and cord blood was analyzed by ELISA. The mean and standard deviation are shown in the figure. Data were analyzed using an unpaired t-test.

[0047] Figure 3 Monocytes were isolated from PBMCs and differentiated into macrophages for 8 days in the presence of 20 ng / ml GM-CSF (A, B) or 40 ng / ml M-CSF (C, D). Cells were then incubated with 0.5 μM CpG ODN 2006FITC for 24 hours and stained with LAMP2 (lysosomal marker) or Rab7 (late endosome marker) antibodies.

[0048] Figure 4(A) Concentration of ADA2 secreted in cell culture medium. (B) Concentration of ADA2 in cells. The concentration of ADA2 in cell lysates was normalized to 100 μg / ml total protein. (C) Concentration of TNF-α in cell culture medium 24 hours after activation with 1000 ng / ml LPS. Each column in the figure represents the mean of eight independent experiments. P < 0.0001.

[0049] Figure 5 (A) Describe the assay protocol for quantifying ADA2 in BAL. (B) Plot a standard curve by comparing the concentration of ADA2 standard with the absorbance ratio at 245 and 265 nm (x-axis: absorbance ratio at 245 and 265 nm; y-axis: ADA2 standard concentration). Each point represents the average of two replicates. (C) Correlation between results obtained using the new assay (A) and results obtained using ADA2 ELISA (x-axis: ADA2 content obtained using the new assay (ng / ml); y-axis: ADA2 content obtained using ADA2 ELISA (ng / ml)). (D, E) ADA2 concentrations in the serum of ten pneumonia patients (D, x-axis: patient number) and BAL (E, x-axis: patient number). Error bars represent standard deviations, and results are from two independent measurements.

[0050] Figure 6 (A) ADA2 concentration in 166 BAL samples from pneumonia patients. (B) Time-varying ADA2 concentration in BAL fluid from pneumonia patients after treatment. Horizontal axis: Number of days since hospitalization. Each point represents the mean of two replicates. (C, D) Concentrations of TNF-α and IL-6 in BAL fluid samples with low (<20 ng / ml) and high (>20 ng / ml) ADA2 levels. (E) Table showing statistically significant differences in the concentrations of various factors in BAL fluid samples from pneumonia patients with low and high ADA2 levels. Data were analyzed using unpaired t-tests.

[0051] Figure 7(A, B, C) Flow cytometry analysis of THP1 cells (A) incubated with 2 mM adenosine for 1 day (B) and 2 days (C). Gated apoptotic cell populations (Apo 1 and Apo 2). (D, E, F) Live and apoptotic THP1 cells stained with IgG isotype control antibody (E) or anti-myeloperoxidase (anti-MPO) (F) and PI antibody. (G, H, J) Live and apoptotic cells stained with the apoptosis marker Annexin V and DNA-binding reagent 7-aminoactinomycin D (7-AAD), anti-ADA2 antibody without (H), and anti-ADA2 antibody with added recombinant ADA2 (J). (K, L, M) Live and apoptotic THP1 cells stained with streptavidin (SA) (K) and ADA2-SA-ADA2 complex in the absence of (L) or in the presence of 5 μM ODNCpG 2006PTO (M).

[0052] Figure 9 Mononuclear cells (0.4 x 10⁻⁶) were isolated from PBMCs. 6 Macrophages were differentiated into macrophages using 40 ng / ml GM-CSF for 3 days. Macrophages were either untreated (A) or treated with 0.5 μM CpG ODN 2006PTO (B) or CpG ODN 2006G5 PD (C) and fixed after 24 hours. ADA2 (blue) and the lysosomal marker LAMP2 (red) were analyzed in stained cells. Increased ADA2 density and colocalization of ADA2 and LAMP2 were observed in macrophages treated with CpG ODN 2006PTO (B).

[0053] Figure 10 Mononuclear cells (0.4 x 10⁻⁶) were isolated from PBMCs. 6 Macrophages were differentiated into macrophages using 20 ng / ml M-CSF for 3 days. Macrophages were either untreated (A) or treated with 0.5 μM CpG ODN 2006PTO (B) or CpG ODN 2006G5 PD (C) and fixed after 24 hours. ADA2 (blue) and the lysosomal marker LAMP2 (red) were analyzed in stained cells. Increased ADA2 density and colocalization of ADA2 and LAMP2 were observed in macrophages treated with CpG ODN 2006PTO (B).

[0054] Figure 11 Compared with the control group, macrophages treated with ODN 2006PTO showed increased ADA2 density (p<0.0001). Error bars represent standard deviations, and results were derived from six independent measurements. Data were analyzed using unpaired t-tests.

[0055] Figure 12 Mononuclear cells (0.15 x 10⁻⁶) were isolated from PBMCs. 6 Cells were differentiated into macrophages (cells / mL) using 20 ng / mL M-CSF or 40 ng / mL GM-CSF for five days. Afterward, cells were washed and treated with 2 μM CpG ODN 2006PTO or CpG ODN 2006G5 PD for 24 hours or untreated. The concentration of ADA2 in cell lysates was then analyzed using ELISA. Error bars represent standard deviations, and results were derived from four independent measurements. Data were analyzed using one-way ANOVA.

[0056] Figure 13 Mononuclear cells (0.25 x 10⁻⁶) were isolated from PBMCs. 6 Cells were cultured (number of cells / mL) and differentiated into macrophages for 5 days using 40 ng / mL GM-CSF. Cells were washed with fresh culture medium and then treated either untreated or with 2 μM CpG ODN2006PTO or CpG ODN2006G5PD for 24 hours. Subsequently, cells were activated for 24 hours with 10 ng / mL IFN-γ and 100 ng / mL LPS. TNF-α concentration in the cell culture medium was analyzed by ELISA. Error bars represent standard deviation, and results were derived from four independent measurements. Data were analyzed using one-way ANOVA.

[0057] Figure 14 (A) Flow cytometry analysis of blood monocytes before (A) and after (B) depletion of CD16-positive monocyte subsets. (C) Concentrations of TNF-α released in cell culture medium by total monocytes (A) and monocytes lacking CD16+ subsets (B). (D) Calculation of 1x10 6 The amount of TNF-α produced by CD16+ and CD16- monocytes in each cell. Error bars represent standard deviations, and results are from four independent measurements. Unpaired t-tests were used to analyze the data.

[0058] Example 1: Materials and Methods

[0059] Cell culture experiments using LPS-activated monocytes

[0060] PBMCs isolated from fresh blood were analyzed, and the concentrations of ADA2 and TNF-α in the plasma of healthy donors and PAN DADA2 patients were determined. Monocytes were isolated from fresh blood of healthy donors and DADA2 patients and cultured in 5 ml polypropylene tubes (Falcon) at a concentration of 0.5 x 10⁻⁶ cells / mL. 6Cells were cultured in suspension at a concentration of 0.5 ml RPMI medium. Monocytes were activated with 10 ng / mL LPS, with or without adenosine. Cells were then cultured in test tubes at 37°C and 5% CO2 for 3–20 hours, separated by centrifugation (300 g, 5 min), and the supernatant was collected for cytokine concentration measurement using ELISA (BioLegend).

[0061] Cell isolation, sorting and culture

[0062] Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of healthy donors according to the IRB-approved protocol. 50 mL of Leucosep... TM CD14+ monocytes were isolated using tubes (Greiner Bio-One), washed twice with 50 ml PBS buffer, and purified using anti-CD14 binding magnetic beads (Miltenyi). For cell culture, RPMI 1640 was used as the complete medium, supplemented with 1% non-essential amino acids, 1% sodium pyruvate, 100 U / ml penicillin, 100 μg / ml streptomycin, 2 ml M-glutamine, and 10% FBS. Monocytes were cultured in 96-well plates with 200 μl RPMI medium containing either 40 ng / ml GM-CSF or 20 ng / ml M-CSF (PeproTech). The complete medium containing growth factors was changed every three days. Monocytes differentiated for 4–8 days, then incubated overnight with 0.5 μM CpG ODN2006 PTO or CpG ODN2006 G5 PD (Invivogen). To sort monocyte subsets and other immune cells, PBMCs were stained with fluorescent antibodies (BD Bioscience) and then sorted using BD FACS Aria SORP. Cells were lysed with 0.5% Triton X-100 in 1x PBS and analyzed by ELISA. Similar analyses were performed on cell lysates and ADA2 concentrations in cell culture medium from M-CSF and GM-CSF differentiated monocytes, and these were normalized to total protein concentrations determined by the BCA protein assay (ThermoFisher). Cytokine concentrations in cell culture medium were measured using ELISA (BioLegend).

[0063] Immunostaining and confocal microscopy

[0064] Mononuclear cells were cultured in 8-well slides (Thermofisher) in 400 μl RPMI medium containing 40 ng / ml GM-CSF or 20 ng / ml M-CSF (PeproTech). In some experiments, cells were treated with 0.5 μL CpG ODN2006 PTO, CpG ODN2006 FITC PTO, or CpG ODN 2006 G5 PD (Invivogen) for 24 hours. Cells were fixed in 10% formaldehyde or 4% PFA for 10 minutes, washed three times in PBS, permeabilized in PBS supplemented with 0.3% Triton X-100 (PBS-T) for 5 minutes, and washed three times with PBS. Cells were incubated overnight at 4°C with primary antibody diluted in PBS containing 1% BSA, washed three times with PBS, and incubated at room temperature with secondary antibody in PBS containing 1% BSA for 0.5 hours. After washing three times, the cells were mounted in 80% glycerol. Antibodies against LAMP2 (Sino Biological, 13555-MM05), Rab7 (Cell Signaling Technology), and ADA1 (Abcam, ab34677) were diluted 1:100. The antibody against ADA2 was diluted 1:50. Fluorescent staining was captured using a Leica SP8 confocal microscope. Polyclonal anti-ADA2 antibody was purified from rabbit serum immunized with recombinant ADA2 (antiserum produced by Sino Biological). To verify antibody specificity, a polyclonal rabbit antibody was added as a negative control, which did not produce any observable staining. Furthermore, nuclear and endoplasmic reticulum (ER) staining was performed using DAPI (Cell Signaling Technology) and an ER-tracker (Invitrogen) according to the manufacturer's instructions.

[0065] Bronchoalveolar lavage collection and treatment

[0066] Bronchoalveolar lavage (BAL) samples were collected during the acute phase of the patient's treatment prior to corticosteroid administration. All collected BAL samples were negative for SARS-CoV-2. Flexible fiberoptic bronchoscopy was performed to obtain additional BAL samples. During this procedure, warm sterile saline (2–3 ml / kg body weight) was injected into each affected area and then aspirated into a suction device for recovery. The collected samples were simultaneously processed and stored at 4°C for subsequent cytokine assays. BAL samples were filtered through double-layered gauze before centrifugation. The supernatant was collected after centrifugation.

[0067] Quantification of inflammatory cytokine levels in bronchoalveolar lavage fluid

[0068] Cytokine levels in BAL supernatant were measured using the following method. Bio-Plex Pro from Bio-Rad was used according to the manufacturer's instructions. TM Human cytokine standard 27-Plex (Group I) kit and magnetic bead-based multiplex immunoassay (LX1000; Luminex). This kit includes 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); eosinophil chemokine (Eotaxin); granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF). CSF); interferon-γ (IFN-γ); interferon-γ inducible protein 10kD (IP-10); monocyte chemoattractant protein 1 (MCP-1); macrophage inflammatory proteins 1α and 1β (MIP-1α, MIP-1β); activation regulation, normal T cell expression and secretion (RANTES); tumor necrosis factor-α (TNF-α); platelet-derived growth factor with two B subunits (PDGF-BB); and vascular endothelial growth factor (VEGF).

[0069] Quantification of ADA2 levels in bronchoalveolar lavage fluid

[0070] The experiment used 96-well round-bottom microplates containing a buffer solution of 50 mM Tris pH 6.8, 10 μM ZnCl2, and 0.02% NaN3. Each well contained 50 μl of BAL sample or ADA2 standard (5 μl), 5 mM adenosine, and 0.1 mM racemic-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, an ADA1 inhibitor). The reaction was initiated by adding a premixture of adenosine and EHNA to the BAL sample or ADA2 standard. The plate was then incubated at 37 °C for 18 hours. Subsequently, 8 μl of the reaction mixture was transferred to a UV microplate (Corning) containing 192 μl of water (0.2 mM adenosine final concentration), and the absorbance ratio at 245 nm and 265 nm was measured.

[0071] ADA2 binds to apoptotic cells

[0072] THP-1 cells were cultured for 3 days in complete RPMI medium containing 2 mM adenosine. Cells were then collected and incubated for 10 minutes at room temperature in 96-well round-bottom plates with 50 μg / ml ADA2 in 40 μl FACS buffer (2% FBS, 2 mM EDTA in PBS). Cells were washed with 200 μl FACS buffer and stained with Alexa Fluor 647-labeled anti-ADA2 antibody for 10 minutes. The stained cells were then washed and analyzed by flow cytometry (FACS Canto, BD), and the results were analyzed using FlowJo software. ADA2-SA-ADA2 staining was performed. Apoptotic cells were stained with 1 μg / ml propidium iodide (PI) (Sigma), anti-MPO antibody with IgG isotype control (Abcam), and Annexin V / 7-AAD apoptosis detection kit (Sino Biological).

[0073] The sequence of CpG ODN 2006PTO is 5'-tcgtcgttttgtcgttttgtcgtt-3' (SEQ ID NO:1)

[0074] The sequence of CpG ODN 2006G5 PD is 5'-TCGTCGTTTTGTCGTTTTGTCGTTGGGGG-3' (SEQ ID NO:2).

[0075] Example 2:

[0076] Systemic polyarteritis nodosa (PAN), immunodeficiency, and ischemic or hemorrhagic stroke are common symptoms in PAN DADA2 patients. This study analyzed the monocyte responses of PAN DADA2 patients carrying the c.G506A (p.Arg169Glu) mutant ADA2 transcript (see Table 1). As shown in Table 1, the cell subset distribution in PAN patients was very similar to that in healthy donors. However, compared to healthy individuals, PAN DADA2 patients had elevated levels of TNF-α in their blood. This increase in TNF-α may be attributed to cytokines released by monocytes or macrophages. Intracellular staining of monocytes from DADA2 patients showed enhanced TNF-α expression in both unstimulated and LPS-stimulated monocytes. To validate this finding, monocytes were isolated from DADA2 patients and activated with LPS (…). Figure 1 The level of TNF-α released by LPS-activated monocytes (LAMs) in patients was significantly higher than that in healthy donors. Figure 1 A). Conversely, MCP-1 secretion in cells was similar to that in healthy controls. Figure 1B). Interestingly, compared to healthy donors, high adenosine concentrations slightly inhibited the release of TNF-α (α-alpha) from LAM in PAN DADA2 patients. Figure 1 D、 Figure 1 E and Figure 1 (F). Therefore, even in the presence of adenosine, TNF-α levels remained persistently high in LAM cell cultures from PAN DADA2 patients. This indicates that cells from PAN DADA2 patients are less sensitive to adenosine in the presence of elevated adenosine concentrations. This phenomenon may be attributed to downregulation of A2 receptor expression or alterations in the adenosine receptor pathway.

[0077] Table 1: Analysis of lymphocyte subsets and ADA2 concentration in patients with ADA2 deficiency (Arg169Gln / c.973-2A>G). Cell subsets were gated and expressed as a percentage of total cells. Values ​​other than control levels were highlighted with an underline.

[0078] Example 3: The intracellular ADA2 concentration of the CD16 monocyte subset was lower than that of the classic monocyte subset.

[0079] like Figure 14 As shown, the CD16+ monocyte subset is the main source of TNF-α. It was observed that after activation with LPS, the production of TNF-α by depleted monocytes in the CD16+ subset was significantly reduced. To determine the correlation between intracellular ADA2 concentration and cellular TNF secretion, cell subsets were classified, and the concentration of ADA2 in cell lysates was analyzed using ELISA.

[0080] This embodiment found that the ADA2 concentration in the CD16+ subset was significantly lower than that in classic CD16- monocytes ( Figure 2 A, Figure 2 B). The results indicate that the decreased intracellular concentration of ADA2 in CD16+ monocytes may be related to the cells' ability to produce more TNF-α, similar to the monocytes of PAN DADA2 patients ( Figure 1 A). Furthermore, the concentration of ADA1 in monocyte subsets showed the opposite trend, increasing in the non-classical CD16+ subset ( Figure 2 C). This indicates increased ADA1 gene expression in CD16+ monocytes. The frequency of CD16+ monocytes in cord blood is lower than in adult blood, and monocytes isolated from cord blood secrete less TNF-α than those isolated from adult blood. Analysis of ADA2 concentration in cord blood serum showed a significantly lower concentration compared to adult serum. Figure 2D). Based on the above results, this embodiment draws the following conclusion: the intracellular concentration of ADA2 may be related to the activation and differentiation of monocytes.

[0081] Example 4: In GM-CSF differentiated monocytes, both secreted and intracellular ADA2 levels were reduced.

[0082] DADA2 monocytes differentiate into M1 macrophages upon exposure to GM-CSF, but fail to differentiate into M2 macrophages upon exposure to M-CSF. This suggests that ADA2 deficiency may affect macrophage polarization toward the inflammatory M1 type, potentially contributing to the pathogenesis of DADA2.

[0083] The results of this embodiment suggest that macrophage polarization and cell activation may be influenced by intracellular ADA2 concentration. Analysis of monocytes differentiated into macrophages four days in the presence of GM-CSF or M-CSF revealed ADA2 expression, but with almost no colocalization with lysosomal markers. Figure 9 A and Figure 10 A). However, when cells were treated with CpG ODN 2006PTO (phosphothiophosphate), an increase in ADA2 density was observed in lysosomes ( Figure 9 B and Figure 10 B). Conversely, no similar increase in ADA2 density was observed when cells were treated with CpG ODN2006G5 PD (phosphodiester). Figure 9 C and Figure 10 C). The concentration of ADA2 increased in cells treated with CpGODN 2006PTO. Figure 11 This indicates that the binding of ADA2 to ODN can protect it from degradation in lysosomes.

[0084] However, the results differed when monocytes differentiated into macrophages for 9 days. Treatment of M-CSF macrophages with CpG ODN 2006PTO increased the density of ADA2, which co-localized with lysosomal and late endosome markers LAMP2 and Rab7. Figure 3 C Figure 3 D), which is consistent with previous observations. Figure 10 ADA2 was found to co-localize with fluorescein-labeled CpG ODN 2006PTO in the same cellular compartment. However, treatment of GM-CSF-derived macrophages with CpG ODN 2006PTO did not show a similar increase in ADA2 density or co-localization with the marker. This difference may be due to the lower ADA2 expression level in GM-CSF-differentiated macrophages, as evidenced by analysis of ADA2 concentrations within macrophages. The concentration of ADA2 in GM-CSF-differentiated monocytes was significantly lower than that in M-CSF-differentiated cells. Figure 4 B). The level of ADA2 secreted by GM-CSF macrophages is also much lower than that of M-CSF macrophages. Figure 4 A). Furthermore, macrophages differentiated from M-CSF produced less TNF-α after LPS treatment than macrophages differentiated from GM-CSF. These results indicate that M-CSF and GM-CSF regulate ADA2 expression levels, and that cells with lower ADA2 expression secrete more TNF-α after LPS activation.

[0085] like Figure 12 As shown, compared with CpG ODN 2006G5 PD, the concentration of ADA2 in macrophages differentiated in the presence of GM-CSF and M-CSF after treatment with 2 μM CpG ODN 2006PTO was significantly increased.

[0086] like Figure 13 As shown, compared with CpG ODN 2006G5 PD, the TNF-α concentration of macrophages treated with 2 μM CpG ODN 2006PTO was significantly reduced, indicating that oligodeoxynucleotides with a phosphate thioester backbone can serve as a novel TNF-α inhibitor.

[0087] Example 5: Increased ADA2 concentration in bronchoalveolar lavage fluid of pneumonia patients

[0088] This embodiment studies the concentration of ADA2 in BAL samples from children with pneumonia. BAL has a low protein concentration, which does not interfere with the measurement of ADA activity at high adenosine concentrations. To quantify the concentration of ADA2 in BAL, this invention employs a simplified detection method, which involves incubating a reaction mixture of a BAL sample or standard containing a known ADA2 concentration with adenosine and the ADA1 inhibitor EHNA overnight in a 96-well plate. Figure 5 A). The reaction mixture was then transferred to a UV-coated plate, diluted with water, and read using a microplate reader. The concentration of ADA2 in the BAL sample was determined based on a standard curve, which plotted the absorbance ratio at 245 nm and 265 nm against a known ADA2 standard concentration (A). Figure 5 B). Alternatively, BAL and serum ADA2 concentrations can be measured using an ADA2 ELISA. The two methods yield similar results for BAL. Figure 5 C). Surprisingly, analysis of plasma samples from 10 pneumonia patients at different stages of treatment did not show a significant difference in serum ADA2 concentrations. Figure 5 D). However, the concentration of ADA2 in the BAL samples of both patients was significantly increased ( Figure 5 E). These results suggest that ADA2 in BAL may serve as a biomarker for diagnosing pneumonia.

[0089] Example 6: The concentration of ADA2 in the blood albumin (BAL) of pneumonia patients is correlated with high concentrations of pro-inflammatory cytokines.

[0090] ADA2 levels were analyzed in 166 pneumonia patients (including those with bacterial, viral, fungal, and co-infections) who received different treatments at different times of hospitalization. The results showed that ADA2 concentrations were elevated (>3.8 ng / ml) in 98 patients. Figure 6 A). However, lower ADA2 concentrations in the BAL may indicate successful treatment. It is important to note that ADA2 concentrations in the BAL of patients with severe pneumonia are very high (>50 ng / ml), but not as high in patients with mild / moderate pneumonia.

[0091] To further investigate this, the concentration of ADA2 in BAL was compared with the concentrations of cytokines in samples from 77 patients. Samples with higher ADA2 concentrations (>20 ng / ml) showed significantly higher concentrations of pro-inflammatory cytokines IL-6, TNF-α, and other factors. Figure 6 C Figure 6 D、 Figure 6 E). The above results suggest that ADA2 in BAL may become a new biomarker for pneumonia, and the increase in ADA2 concentration is related to the increase in the concentration of cytokines, chemokines, and other factors in pneumonia patients. Analysis of ADA2 concentration in BAL at different days after treatment in patients with severe pneumonia showed that ADA2 concentration decreased after treatment ( Figure 6 B). Therefore, ADA2 in BAL can serve as a prognostic biomarker for pneumonia and an indicator of treatment success.

[0092] Example 7: ADA2 binding to early apoptotic cells

[0093] Cells undergoing apoptosis produce extracellular traps made of DNA that can bind to positively charged proteins. This example uses adenosine to induce apoptosis in THP1 monocytes; adenosine is a molecule known to be cytotoxic at high concentrations. The binding of ADA2 cells to apoptotic cells was studied by incubating cells with adenosine, resulting in two cell populations: early apoptotic cells (Apo 1) and late apoptotic cells (Apo 2). These cell populations were successfully stained with the DNA-binding dye propidium iodide (PI). Figure 7 A, Figure 7 B. Figure 7 C). When THP1 cells were grown at low cell density in fresh culture medium in the presence of adenosine, an early-apoptotic Apo1 cell population dominated. Figure 7 D、 Figure 7 E, Figure 7 F).

[0094] To further confirm apoptosis, cells were stained with anti-MPO (myeloperoxidase), an enzyme associated with extracellular traps. Figure 7 F). Furthermore, staining with Annexin V (which binds to phosphatidylserine on the extracellular membrane of apoptotic cells) and 7-aminoactinomycin D (which binds to DNA) provided further evidence supporting the identification of Apo1 and Apo2 as cells undergoing apoptosis. Figure 7 G). To analyze the binding of ADA2 to apoptotic THP1 cells, a recombinase was added to the cells. After removing unbound proteins during cell washing, the binding of ADA2 was detected using an anti-ADA2 antibody. It was found that both live cells and late-apoptotic cells were negative for ADA2; only early-apoptotic cells specifically bound ADA2. Figure 7 H, Figure 7 J). Previously, it has been demonstrated that two ADA2 molecules linked by streptavidin (SA) can bind to the surface of immune cells. Therefore, the ADA2-SA-ADA2 dimer has a strong affinity for proteoglycans expressed on the cell surface and DNA in apoptotic cells, and thus can bind to both surviving and early apoptotic cells. Figure 7 K, Figure 7 L). CpG ODN 2006PTO can inhibit the binding of ADA2-SA-ADA2 to cells (L). Figure 7 M), thus confirming the binding of ADA2 with CpG ODN 2006PTO.

[0095] like Figure 8 As shown, this invention proposes a novel mechanism in which ADA2 can be secreted extracellularly and transported into lysosomes. ADA2 can control the concentration of adenosine in lysosomes, modulating cellular sensitivity to activation signals to control inflammation, thereby inducing TNF-α expression and cellular secretion. Furthermore, ADA2 secretion requires glycosylation of an enzyme in the endoplasmic reticulum. To transport ADA2 into lysosomes, this enzyme needs to be modified with mannose-6-phosphate (M6P) and bind to the M6P receptor in the Golgi apparatus. Both pathways can be regulated by growth factors (such as GM-CSF or M-CSF) produced by cells activated during inflammation. The decreased ADA2 concentration in the body of DADA2 patients may explain the excessive release of TNF-α from monocytes and macrophages and the inhibition of M2 macrophage polarization. The TNF-α inhibitor provided by this invention can inhibit the secretion of TNF-α by monocytes / macrophages, thereby achieving more targeted treatment of DADA2.

[0096] Summarize:

[0097] Humans possess two types of adenosine deaminases: ADA1 and ADA2. ADA2 has a signaling sequence, while ADA1 does not; therefore, existing technology considers ADA1 primarily an intracellular adenosine deaminase, while ADA2 is an extracellular adenosine deaminase. However, ADA2 has a low affinity for adenosine, making it inefficient at low adenosine concentrations, and the loss of ADA1 activity due to gene mutations cannot be compensated for by the presence of ADA2. This invention creatively discovers that ADA2 can function as a lysosomal adenosine deaminase within cells, a breakthrough in the existing understanding of ADA2. Furthermore, this invention reveals that ADA2 possesses anti-inflammatory functions within lysosomes, and that the release of ADA2 by cells under the influence of growth factors may be essential for cell activation, leading to increased cytokine secretion and cell polarization. Simultaneously, ADA2 can bind to apoptotic cells and concentrate at sites of lung inflammation (e.g., bronchial fluid), acting as ADA at high adenosine concentrations, and activating immune cells by removing adenosine.

[0098] Elevated TNF-α levels in the plasma of PAN DADA2 patients. Excessive release of TNF-α from macrophages, natural killer cells, and T cells binds to TNF-α receptors, triggering an inflammatory response. This chronic inflammation may contribute to the development of autoimmune diseases. Notably, both stimulated and unstimulated PAN DADA2 monocytes secrete higher levels of TNF-α, IL-6, and IL-1β than healthy donors. Treatment of PAN DADA2 patients with TNF-α inhibitors has shown therapeutic efficacy. This invention demonstrates that LPS-stimulated monocytes of DADA2 patients release more TNF-α than healthy donors. Figure 1 Furthermore, monocytes in DADA2 patients have lower sensitivity to extracellular adenosine, which also leads to higher plasma TNF-α levels (Table 1). Adding extracellular ADA2 did not reduce TNF-α secretion from activated monocytes. Figure 1 C). In other words, this invention creatively discovers that intracellular, rather than extracellular, ADA2 can downregulate cytokine secretion levels in activated monocytes. In other words, ADA2 may not have the function of regulating extracellular adenosine deaminase or growth factor activity in TNF-α secretion, and ADA2 replacement therapy may not produce positive results.

[0099] This invention has discovered that monocytes differentiating into macrophages in the presence of GM-CSF, when treated with phosphate-thiocyanate (PTO) CpG oligonucleotides that have a strong affinity for ADA2, will drive ADA2 into the lysosomes. Figure 8For example, GM-CSF and M-CSF-differentiated monocytes treated with CpG ODN2006PTO showed increased ADA2 density in lysosomes. This did not occur when cells were treated with the natural phosphodiester (PD) CpG ODN. Surprisingly, this invention found that GM-CSF-differentiated monocytes treated with CpG ODN2006PTO secreted less TNF-α after IFN-γ and LPS (M1) activation. Figure 11 This suggests that oligodeoxynucleotides with a thiophosphate backbone (e.g., CpG ODN 2006PTO) can serve as a novel TNF-α inhibitor to downregulate TNF-α expression in monocytes (e.g., CD16+ monocytes, M-CSF-differentiated monocytes, and / or GM-CSF-differentiated monocytes).

[0100] The experiments of this invention further investigated monocytes that differentiated into macrophages within 8 days in the presence of M-CSF and GM-CSF, and found that the levels of ADA2 expressed intracellularly and extracellularly in M-CSF-differentiated macrophages were higher than those in GM-CSF-differentiated monocytes (e.g., Figure 4 (As shown). When cells were probed with CpG ODN 2006PTO, ADA2 was observed to colocalize with lysosomal markers and ODN 2006 in M-CSF macrophages, but not in GM-CSF macrophages. Figure 3 A, Figure 3 B). This indicates that the concentration of ADA2 in lysosomes within GM-CSF macrophages is significantly lower than that in M-CSF macrophages. Furthermore, LPS-treated GM-CSF macrophages produce more TNF-α than M-CSF macrophages. Figure 4 C), while oligodeoxynucleotides with a thiophosphate backbone (e.g., CpG ODN 2006PTO) can downregulate increased TNF-α secretion.

[0101] Furthermore, this invention has found that the concentration of ADA2 in the bronchoalveolar fluid of pneumonia patients is elevated ( Figure 5 ), and is associated with elevated levels of pro-inflammatory cytokines ( Figure 6 C- Figure 6 E). This result supports the conclusion that growth factors induce monocytes to release ADA2, leading to increased cellular sensitivity to activation signals and the release of pro-inflammatory cytokines. The experiments of this invention demonstrate that serum (plasma) ADA2 concentrations do not accurately reflect different stages of pneumonia (i.e., Figure 5(In group D, serum ADA levels were high and relatively similar across patients.) ADA2 in BAL can differentiate between patients with severe pneumonia and those with mild / moderate pneumonia. After treatment, ADA2 concentrations in BAL decreased in patients with severe pneumonia. Therefore, ADA2 levels in BAL are a novel biomarker for diagnosing pneumonia and monitoring treatment.

[0102] In summary, this invention demonstrates that ADA2, in addition to being an extracellular adenosine deaminase, also exists in lysosomes, and its concentration is reduced in cells that secrete TNF-α, particularly the CD16+ subset of monocytes and macrophages differentiated from GM-CSF cells. Furthermore, oligodeoxynucleotides with a phosphate thioester backbone (e.g., CpG ODN 2006PTO) can downregulate TNF-α secretion from monocytes, thereby enabling targeted treatment of immunodeficiency diseases (e.g., DADA2 deficiency).

[0103] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. The use of a biomarker in the preparation of a kit for diagnosing pneumonia and / or evaluating the efficacy of pneumonia treatment, characterized in that, The biomarker includes ADA2 in bronchoalveolar lavage fluid, and the kit is used to quantitatively detect the content of ADA2 in the bronchoalveolar lavage fluid sample of the subject.

2. The use as described in claim 1, characterized in that, The kit is used to differentiate between patients with severe pneumonia and those with mild / moderate pneumonia.

3. The use as described in claim 1, characterized in that, The subjects had increased levels of one or more of the following factors: IL-1ra, IL-4, IL-6, IL-9, IL-10, IL-17, Eotaxin, FGF, IFN-γ, IP-10, MCP-1, MIP-1α, PDGFB, MIP-1β, RANTES, and TNF-α.

4. The use as described in claim 2, characterized in that, When the ADA2 content in the bronchoalveolar lavage fluid sample is >50 ng / ml, the subject is assessed as a patient with severe pneumonia.

5. The use of an oligodeoxynucleotide in the preparation of a TNF-α inhibitor, characterized in that, The oligodeoxynucleotide has a phosphate thioester backbone.

6. The use as described in claim 5, characterized in that, The TNF-α inhibitor is used to reduce the secretion of TNF-α in monocytes.

7. The use as described in claim 6, characterized in that, The monocytes include CD16+ monocytes, M-CSF-differentiated monocytes, and / or GM-CSF-differentiated monocytes.

8. The use as described in claim 5, characterized in that, The oligodeoxynucleotide includes ODN 2006PTO.

9. The use as described in claim 5, characterized in that, The TNF-α inhibitor is used to treat ADA2 deficiency.

10. The use as described in claim 9, characterized in that, The ADA2 deficiency is the PAN phenotype.

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