CN112526143B - Application of myeloid cell trigger receptor 2 as novel coronavirus pneumonia diagnosis or treatment target - Google Patents
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Abstract
The invention belongs to the technical field of biology, and discloses application of a myeloid cell trigger receptor 2 as a novel coronavirus pneumonia diagnosis or treatment target, wherein researches show that TREM-2 is highly expressed in peripheral blood CD4+ and CD8+ T cells of a novel coronavirus pneumonia patient and is positively correlated with the disease severity. The invention also finds that TREM-2-Fc fusion protein (the action effect is that TREM-2 signals are blocked) obviously reduces the levels of cell factors IFN-g, TNF, IL-2 and GranzymeB, and the TREM-2 expression has obvious correlation with severe indexes of old age, reduction of lymphocyte count and increase of C-reactive protein and D-dimer, which are provided by the novel diagnosis and treatment scheme of coronavirus pneumonia. The intervention of TREM-2 expression can be prompted to relieve and even hopefully treat the novel coronavirus pneumonia.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a myeloid cell triggering receptor 2 as a novel coronavirus pneumonia diagnosis or treatment target.
Background
The novel coronavirus pneumonia is an acute infectious pneumonia, and the pathogen is a novel coronavirus which is not found in human before, namely severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2).
According to the existing case data, the novel coronavirus pneumonia mainly shows fever, dry cough, hypodynamia and the like, and a few patients are accompanied with upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Some patients have mild onset symptoms and no fever, and recover after 1 week. The prognosis is good for most patients, and the disease is critical or even death for a few patients. In severe cases, respiratory distress syndrome occurs after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, hemorrhagic coagulation dysfunction, multiple organ failure and the like. Therefore, early diagnosis, early intervention and development of novel therapeutic strategies are key means for the prevention and control of novel coronavirus pneumonia.
The current clinical definite diagnosis detection method is to detect the positive of novel coronavirus nucleic acid by real-time fluorescence RT-PCR; or viral gene sequencing, with high homology to known novel coronaviruses. A novel diagnosis and treatment scheme (trial seventh edition) for coronavirus pneumonia lists heavy and critical clinical early warning indexes, including: 1. progressive decline in peripheral blood lymphocytes; 2. progressive increase in peripheral blood inflammatory factors such as IL-6, C-reactive protein; 3. progressive increase in lactic acid; 4. the lung lesions progress rapidly in a short period of time. However, the above indexes have no specificity and the prediction accuracy is not high, and new diagnostic indexes are urgently needed. At present, the clinical novel coronavirus pneumonia has no specific medicine, and generally adopts broad-spectrum antiviral treatment, including ribavirin, interferon, lopinavir, ritonavir, arbidol and chloroquine phosphate. Although the medicines have certain effects on most patients, the treatment effect on serious patients is still poor, and the death rate of diseases is high.
Disclosure of Invention
The invention aims to solve the technical problem that an effective diagnosis and treatment means for novel coronavirus pneumonia is lacked in the prior art, and provides application of a reagent for detecting the expression level of a myeloid cell trigger receptor 2 in preparation of a novel coronavirus pneumonia diagnosis or treatment reagent.
The invention mainly aims to provide application of a reagent for detecting the expression level of a myeloid cell trigger receptor 2 in preparing a novel diagnostic and/or prognostic reagent for coronavirus pneumonia.
The second object of the present invention is to provide a drug for alleviating inflammatory reactions caused by the novel coronavirus pneumonia.
The third purpose of the invention is to provide the application of the substance for specifically inhibiting the expression of the myeloid cell trigger receptor 2 in the preparation of the medicine for relieving the inflammatory reaction caused by the novel coronavirus pneumonia.
The purpose of the invention is realized by the following technical scheme:
use of an agent for detecting the level of expression of myeloid cell-triggered receptor 2 in the preparation of a diagnostic and/or prognostic agent for novel coronavirus pneumonia.
Preferably, the agent is an antibody capable of specifically binding to myeloid cell-triggering receptor 2.
Myeloid cell-Triggered Receptor (TREM) 2 is a member of the immunoglobulin superfamily expressed predominantly on the surface of neutrophils and monocytes, and consists of an extracellular immunoglobulin-like region, a transmembrane region, and an intracellular region. The inventor researches and finds that the expression of TREM-2 in peripheral blood CD4+ and CD8+ T lymphocytes of a novel coronavirus pneumonia patient is obviously higher than that of a healthy control, and the expression of TREM-2 in a severe patient is obviously higher than that of a non-severe patient by adopting flow cytometry. Further, the trend of the TREM-2 in the early disease stage (pre-ICU), the critical illness stage (ICU) and the recovery stage (post-ICU) of the novel coronavirus pneumonia patient is detected to be firstly increased and then reduced by adopting flow cytometry, and the TREM-2 is prompted to be closely related to the occurrence of the critical illness state, so that the TREM-2 can be used as a marker for diagnosis and prognosis of the novel coronavirus pneumonia.
Preferably, the antibody may be conjugated or carry a detection label, and more preferably, the antibody is a monoclonal antibody or a polyclonal antibody of the myeloid cell-triggering receptor 2.
Preferably, the myeloid-lineage cell trigger receptor 2 is derived from CD4+ T cells, CD8+ T cells.
The expression abundance of TREM-2 in CD4+ and CD8+ T cells is much higher in patients with novel coronavirus pneumonia than in healthy people and is associated with increased disease. Therefore, the expression accuracy and the sensitivity for detecting TREM-2 in the CD4+ and CD8+ T cells are high. Therefore, the novel coronavirus pneumonia can be diagnosed by detecting the content of TREM-2 in peripheral blood, and whether the progress of the disease condition of a patient is changed to severe disease or not can be predicted.
Specifically, when used as a diagnostic reagent, the diagnostic process includes: detecting the expression level of TREM-2 in a blood sample (or biopsy tissue sample) of a subject by using an antibody specifically binding to TREM-2, comparing the expression level with a reference level, and when the expression level of TREM-2 is remarkably up-regulated, the diagnosis result is positive; the diagnosis is negative when the expression level of TREM-2 is not significantly changed, wherein the reference level is the expression level of the TREM-2 in the peripheral blood sample of the healthy population.
Specifically, when used as a prognostic agent, the prognostic procedure includes: the expression level of TREM-2 in a subject's blood sample (or biopsy tissue sample) at different disease stages is detected using an antibody that specifically binds to TREM-2 and compared to a reference level to curve the expression level of TREM-2 at different disease stages, thereby determining the patient's prognosis. Wherein the reference level is the expression level of said TREM-2 in a peripheral blood sample from a healthy human.
The present invention also provides a medicament for reducing an inflammatory response caused by novel coronavirus pneumonia, the medicament comprising a substance specifically inhibiting the expression of myeloid cell-triggered receptor 2.
The co-immunoprecipitation experiment proves that TREM-2 is combined with SARS-CoV-2 virus membrane protein (M protein) and is not combined with nucleoprotein (N protein) N protein and angiotensin converting enzyme 2(ACE 2). TREM-2 promotes the secretion of cytokines including interferon-gamma (IFN-gamma), Tumor Necrosis Factor (TNF), interleukin-2 (IL-2) and granzyme B (granzyme B) by CD4+ and CD8+ T cells under the condition of SARS-CoV-2M protein stimulation. Further treatment with TREM-2-Fc (TREM-2 extracellular domain linked immunoglobulin constant region) fusion protein reduced the levels of the above 4 cytokines secreted by CD4+ and CD8+ T cells.
At the cellular level, blocking TREM-2 receptor signaling inhibits secretion of interferon- γ (IFN- γ), Tumor Necrosis Factor (TNF), interleukin-2 (IL-2), and granzyme b (granzyme b) by CD4+ and CD8+ T cells; in addition, the seventh trial proposal suggests that old Age, decreased lymphocyte (lymphocyte) count, increased C-reactive protein (CRP) and D-dimer (D-dimer) are indicators of severe acute coronavirus pneumonia, that TREM-2 expression is positively correlated with Age (Age), CRP and D-dimer respectively, that the correlation coefficients (r) are 0.385, 0.5368 and 0.323 respectively, and that statistical P values are less than 0.01, i.e. the two indicators have significant difference compared. TREM-2 expression is inversely related to the number of the lymphocytes, the correlation coefficient (r) is-0.4084, and the P value is less than 0.001, namely the significant difference exists.
The results prove that TREM-2 has correlation with clinical severe indexes of the novel coronavirus pneumonia, so that the inflammatory response of a patient with the novel coronavirus pneumonia can be relieved by triggering the expression of the receptor 2 through targeting myeloid cells in a stem precursor, and the novel coronavirus pneumonia can be treated.
Preferably, the drug comprises fusion protein targeting myeloid cell triggering receptor 2, specific antibody, siRNA, anti-sense RNA.
The invention also provides application of a substance for specifically inhibiting the expression of the myeloid cell trigger receptor 2 in preparing a medicament for relieving inflammatory reaction caused by the novel coronavirus pneumonia.
Preferably, the drug comprises fusion protein targeting myeloid cell triggering receptor 2, specific antibody, siRNA, anti-sense RNA.
More preferably, the drug is a TREM-2-Fc fusion protein.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the TREM-2 is found to be highly expressed in peripheral blood CD4+ and CD8+ T cells of a novel coronavirus pneumonia patient through research, and is positively correlated with the severity of the disease. The TREM-2 can be used as a novel marker for diagnosis and prognosis of coronavirus pneumonia, the marker can be from peripheral blood, and compared with complex procedures such as biochemistry, blood coagulation and multiple-index detection of blood sample saturation, the marker can directly take a blood sample for detection, the pain of a patient is relieved, and the novel coronavirus pneumonia diagnosis and prognosis marker is small in wound and high in sensitivity.
The invention also researches the intervention of TREM-2-Fc fusion protein on CD4+ and CD8+ T cells of a patient, finds the level of TREM-2-Fc fusion protein (the action effect is to block TREM-2 signals) obvious cytokines IFN-g, TNF, IL-2 and GranzymeB, and prompts that the intervention of the expression of TREM-2 can reduce the inflammatory reaction caused by the novel coronavirus pneumonia, thereby being expected to treat the novel coronavirus pneumonia.
The novel molecular marker for diagnosis and prognosis of coronavirus pneumonia, provided by the invention, has the advantages of simplicity, convenience, rapidness, small wound, easiness in rechecking and the like, and has a wide application prospect.
Drawings
FIG. 1 is a graph showing the expression level of TREM-2 on the surface of CD4+ and CD8+ T cells in peripheral blood of healthy examiners and patients with novel coronavirus pneumonia;
FIG. 2 is a graph showing the expression levels of TREM-2 in the disease progression stages including Pre-stage (Pre-ICU), intensive stage (ICU) and remission stage (Post-ICU) of patients with novel coronavirus pneumonia;
FIG. 3 is a graph of TREM-2 binding to SARS-CoV-2 virus M protein and no binding to other virus proteins;
FIG. 4 is a graph showing the expression levels of IFN-g, TNF, IL-2 and granzyme B secreted from TREM-2+ CD4+ and CD8+ T cells under the condition of SARS-CoV-2 virus M protein stimulation;
FIG. 5 is a graph of the expression levels of IFN-g, TNF, IL-2, and granzyme B secreted from CD4+ and CD8+ T cells using TREM-2-Fc fusion protein to block TREM-2 receptor signaling; wherein ns represents a statistical difference P > 0.05 compared to the control group; represents a statistical difference P < 0.05 compared to the control group; statistical difference P < 0.01 compared to control group; statistical difference P < 0.001 compared to control;
FIG. 6 is a correlation analysis of TREM-2 expression with four indices (age, C-reactive protein, D-dimer, lymphocytes).
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
All subjects in the trial signed informed consent, and the general data differences between patients were not statistically significant.
Example 1
Selecting 15 cases of Severe cases (Severe) and 20 cases of Non-Severe cases (Non-Severe) of patients with the novel coronavirus pneumonia as experimental groups; 20 healthy examinees were selected and set as a control group.
The health physical examiners: subjects who had no clinical symptoms.
Novel patients with coronavirus pneumonia: SARS-CoV-2 virus nucleic acid detection is positive and clinical symptoms are manifested in pneumonia of the patients.
Collecting 5mL of peripheral blood of experimental group and control group subjects, respectively, lysing erythrocytes to prepare single cell suspension, and adjusting total cell amount to 5 × 106Adding TREM-2 antibody (R) into 100 μ L system&Company D, cat 1278P) and antibodies to CD4 and CD8 (both from Biolegend), were mixed and protected from light for 30 minutes, and flow cytometry was performed to determine the proportion of TREM-2 positive cells to CD4 and CD8 positive cells, respectively, by FCS/SSC gating.
FIG. 1 is a graph of the expression levels of TREM-2 on the surface of CD4+ and CD8+ T cells in the peripheral blood of healthy examiners, patients with novel coronavirus pneumonia, and patients with non-severe coronavirus pneumonia. Setting the gates as CD4+ and CD8+ cells respectively, it can be seen that the proportion of TREM-2 positive cells of the novel coronavirus pneumonia patient is far higher than that of a healthy physical examiner, and the proportion of TREM-2 positive cells of an intensive patient is obviously higher than that of a non-intensive patient, which indicates that TREM-2 is highly expressed in peripheral blood CD4+ and CD8+ T cells of the novel coronavirus pneumonia patient and is related to the severity of the disease.
Example 2
5 patients with severe and severe pneumonia of the novel coronavirus are selected, and the expression conditions of TREM-2 of patients in disease development stages including a Pre-stage (Pre-ICU), a severe stage (ICU) and a remission stage (Post-ICU) are respectively detected.
Reference to diagnosis and treatment prescription for New coronary pneumonia for critical stageIn a case (trial seventh edition), 5mL of peripheral blood of the patient was collected at 3 times and tested as follows. Separately lysing erythrocytes from the above samples to prepare single cell suspensions, and adjusting the total amount of cells to 5X 106Adding TREM-2 antibody (R) into 100 μ L system&Company D, cat # FAB1278P), mixing, keeping out of the sun for 30 minutes, detecting by flow cytometry, and analyzing the proportion of TREM-2 positive cells in CD4 and CD8 positive cells by FCS/SSC gating.
FIG. 2 shows the expression level of TREM-2 on the surface of T cells in the peripheral blood CD4+ and CD8+ in the early stage (Pre-ICU), in the severe stage (ICU) and in the remission stage (Post-ICU) of novel coronavirus pneumonia. Setting the gates as CD4+ and CD8+ cells; it can be seen that TREM-2 is expressed less in the early stage of the disease, and is expressed more after the disease develops into severe disease, and has statistical differences; when the treated patient enters remission stage, the TREM-2 expression is reduced, and the statistical difference exists.
Example 3
As a result, as shown in FIG. 3, 5 kinds of plasmids were constructed in total, including Vector blank plasmid, HA-tagged TREM-2 expression plasmid (TREM-2-HA), FLAG-tagged SARS-CoV-2 membrane protein expression plasmid (M-FLAG), FLAG-tagged SARS-CoV-2 nuclear protein expression plasmid (N-FLAG), and FLAG-tagged angiotensin-converting enzyme 2(ACE2) expression plasmid (ACE 2-FLAG).
The above 5 plasmids were transferred into 293T cells in combination as shown in the figure, indicating that the plasmid was not transferred and indicating that the plasmid was transferred. And (3) after transferring for 24 hours, collecting cells, cracking to obtain protein, and detecting the binding condition of TREM-2 and virus protein by co-immunoprecipitation. The Input on the left side indicates the expression of the plasmids in the cell lysis protein detected by the immunoblotting after transfer, the results show that the plasmids can effectively express the protein when transferred into 293T cells, and black bands are respectively expressed by ACE2-FLAG, N-FLAG, M-FLAG and TREM-2-HA from top to bottom. And IP on the right side: FLAG shows that cell lysis protein is treated by agarose beads targeting a FLAG label, the FLAG labeled protein (ACE2-FLAG, N-FLAG and M-FLAG in the experiment) and the binding protein thereof are pulled down by a co-immunoprecipitation method, the rest of the protein without the FLAG label or the protein not bound with the protein is removed, and finally the co-precipitated protein is detected by immunoblotting. The result shows that the black bands are ACE2-FLAG, N-FLAG and M-FLAG from top to bottom respectively, and the TREM-2-HA can detect the bands only under the condition of being transferred together with the M-FLAG, thus prompting that the TREM-2 and the M have interaction; no band appears in the co-transformation of TREM-2-HA and N-FLAG or ACE2-FLAG, which indicates that TREM-2 HAs no interaction with N and ACE 2.
Example 4
10 patients with the novel coronavirus pneumonia were selected and Peripheral Blood Mononuclear Cells (PBMC) were isolated using lymphocyte isolate. Stimulation of PBMC with VSV-G viral vectors containing SARS-CoV-2M protein, detection and analysis of IFN-G, TNF, IL-2 and granzyme B for CD4+ negative and positive expression in TREM-2 (i.e., TREM-2) by flow cytometry-CD4 and TREM-2+CD4) or CD8+ T cells (i.e., TREM-2)-CD8 and TREM-2+CD 8).
The results are shown in FIG. 4, which is the expression level of IFN-g, TNF, IL-2 and granzyme B on CD4+ and CD8+ T cells. Mock is a negative control, i.e., no stimulus is applied; VSV-G was a blank control of viral vectors to stimulate PBMC; VSV-G SARS-CoV-2M is the experimental group, i.e., the VSV-G viral vector containing SARS-CoV-2M stimulates PBMC.
As can be seen from FIG. 4, IFN-G, TNF, IL-2 and granzyme B4 cytokines were not substantially expressed in the Mock group, 4 cytokines were not significantly increased after VSV-G blank virus stimulation, and TREM-2-And TREM-2+There was no significant difference between the two groups. IFN-G, TNF, IL-2, and granzyme B expression are significantly increased following VSV-G virus stimulation of SARS-CoV-2M, wherein TREM-2+The expression of the above 4 cytokines is obviously higher than that of TREM-2-And (4) grouping. The above phenomenon is homogeneous in CD4+ and CD8+ T cells, i.e., TREM-2M under stimulation of VSV-G virus of SARS-CoV-2M+Promote the secretion of IFN-g, TNF, IL-2 and granzyme B by CD4+ and CD8+ T cells.
Example 5
10 patients with the novel coronavirus pneumonia were selected and Peripheral Blood Mononuclear Cells (PBMC) were isolated using lymphocyte isolate. PBMC were stimulated with VSV-G viral vectors containing SARS-CoV-2M protein, with the addition of TREM-2-Fc fusion protein (which effects blocking of TREM-2 signal) or isotype control IgG, and IFN-G, TNF, IL-2 and granzyme B expression levels at CD4+ and CD8+ T cells were detected and analyzed separately using flow cytometry.
The results are shown in FIG. 5, where Mock is a negative control without any stimulation. Control is VSV-G virus stimulation with SARS-CoV-2M protein. IgG was VSV-G virus stimulation with SARS-CoV-2M protein, and IgG isotype control was added. TREM-2-Fc is VSV-G virus stimulation added with SARS-CoV-2M protein, and TREM-2-Fc fusion protein is added at the same time, namely an experimental group.
As can be seen from FIG. 5, the addition of TREM-2-Fc fusion protein to PBMC of patients with novel coronavirus pneumonia results in significant reduction of IFN-g, TNF, IL-2 and granzyme B secretion by CD4+ and CD8+ T cells, and has statistical significance. Whereas addition of isotype Control IgG did not affect IFN-g, TNF, IL-2 and granzyme B expression levels (compared to Control). It is suggested that blocking TREM-2 receptor signaling inhibits IFN-g, TNF, IL-2, and granzyme B secretion by CD4+ and CD8+ T cells.
Example 6
103 patients with the novel coronavirus pneumonia are selected, peripheral blood of the patients is collected, the method of the embodiment 1 is adopted to detect the expression of TREM-2 on the surface of the T cell, and the correlation between the TREM-2 expression and clinical indexes is analyzed.
"diagnosis and treatment of novel coronavirus pneumonia" (trial seventh edition), old age, decreased lymphocyte (lymphocyte) count, and increased C-reactive protein (CRP) and D-dimer (D-dimer) are suggested as indicators of severe pneumonia of novel coronavirus. Here, the correlation of TREM-2 expression with the above four indices is analyzed here.
The results are shown in fig. 6, TREM-2 expression is positively correlated with Age (Age), CRP, and D-dimer, the correlation coefficients (r) are 0.385, 0.5368, and 0.323, respectively, and the statistical P values are all less than 0.01, i.e. the two indexes have significant difference. TREM-2 expression is inversely related to the number of the lymphocytes, the correlation coefficient (r) is-0.4084, and the P value is less than 0.001, namely the significant difference exists. The results prove that TREM-2 has correlation with clinical severe indexes of the novel coronavirus pneumonia, and the TREM-2 is suggested to be a diagnosis and treatment target of the novel coronavirus pneumonia.
It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.
Claims (6)
1. Use of an agent for detecting the expression level of peripheral blood myeloid lineage cell-triggered receptor 2 for the preparation of a diagnostic agent for novel coronavirus pneumonia; the myeloid cell triggering receptor 2 is from CD4+ T cells, CD8+ T cells.
2. The application of the reagent for detecting the expression level of the peripheral blood marrow cell trigger receptor 2 in preparing a reagent for detecting the severity of the novel coronavirus pneumonia; the myeloid cell triggering receptor 2 is from CD4+ T cells, CD8+ T cells.
3. Use according to claim 1 or 2, wherein the agent is an antibody capable of specifically binding to the myeloid cell-triggering receptor 2.
4. The use according to claim 3, wherein the antibody is provided with or conjugated to a detection label.
5. The use according to claim 4, wherein the antibody is a monoclonal or polyclonal antibody to myeloid cell-triggered receptor 2.
Use of a TREM-2-Fc fusion protein in the preparation of a medicament for reducing inflammatory response factors caused by novel coronavirus pneumonia, wherein the inflammatory response factors are IFN-g, TNF, IL-2 and granzyme B, and the inflammatory response factors are from CD4+ T cells and CD8+ T cells.
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