CN114966057A

CN114966057A - Immune marker for tuberculosis and application thereof

Info

Publication number: CN114966057A
Application number: CN202210662455.4A
Authority: CN
Inventors: 沈洪波; 王菲菲; 彭影; 沙巍
Original assignee: Shanghai Pulmonary Hospital
Current assignee: Shanghai Pulmonary Hospital
Priority date: 2022-06-13
Filing date: 2022-06-13
Publication date: 2022-08-30

Abstract

The invention relates to the technical field of biological medicines, in particular to an immune marker for tuberculosis and application thereof. Experiments prove that the expression level of CD355 (also known as ClassI-restrictedTcell-associated bacterium, CRTAM for short) causes corresponding change of the bactericidal effect of human peripheral lymphocytes on intracellular infection of mycobacterium tuberculosis. Animal experiments show that: compared with a wild mouse, the mouse with the deletion of the CD355 coding gene is more sensitive to the infection of mycobacterium tuberculosis, namely the CD355 is an important action target of host targeted immune-assisted therapy of tuberculosis, and the research can be used for preparation of products related to combined screening or diagnosis, curative effect evaluation and prognosis recurrence risk evaluation, and has good application prospect.

Description

Immune marker for tuberculosis and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an immune marker for tuberculosis and application thereof.

Background

Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium Tuberculosis (Mtb) and its complex flora. At present, the treatment of tuberculosis mainly depends on chemical drugs, and the pathogenic bacteria are very easy to generate drug resistance in the treatment process due to the influence of factors such as slow growth of mycobacterium tuberculosis, immune microenvironment in vivo and the like. At present, all clinical chemical drugs have found clinical isolates with resistance to them.

In recent years, the emergence and prevalence of drug-resistant tuberculosis, especially multi-drug resistant tuberculosis, have led to a very severe situation in the prevention and control of tuberculosis. Multi-drug resistant tuberculosis has developed drug resistance to the most effective first-line clinical drugs isoniazid and rifampicin, so the treatment of multi-drug resistant tuberculosis is very difficult, even no drug is available. Therefore, there is an urgent need to develop new therapeutic strategies.

The occurrence, development and prognosis of tuberculosis are closely related to the immune state of human body. The immune adjuvant therapy for enhancing the immune function of human bodies plays an important role in the treatment process of multi-drug resistant tuberculosis.

The prior art is as follows: the molecular mechanism of interaction of NK cell receptors with ligands of the Nectin/Necl family (2014.5.1, Zhang Yong et al) describes: .. recognition of CRTAM and Necl-2 promotes the killing of NK cells on tumor cells on one hand, and inhibits the adhesion, aggregation and metastasis of tumor cells to tissues on the other hand.

The Chinese patent application: CN105247075A discloses a biomarker for diagnosing lung diseases and a method of using the same, providing a method for diagnosing Interstitial Lung Diseases (ILD). Methods are provided for differentiating diagnostic (differential diagonsis) idiopathic pulmonary fibrosis from other ILDs. Also provided are compositions and kits useful for performing the subject methods. Interstitial Lung Disease (ILD), also known as Diffuse Parenchymal Lung Disease (DPLD), represents a variety of disorders that can lead to diffuse remodeling (diffuse remodelling), structural damage (architectural damage) to normal lung tissue, and inflammation that can lead to progressive loss of lung function. In ILD, in addition to inflammation and fibrosis often seen in the lung parenchyma, the trachea and vessels are also significantly affected, and the relationship of CRTAM expression levels to Interstitial Lung Disease (ILD) is mentioned in the application documents.

However, although the prior art (molecular mechanism study of interaction between NK cell receptor and ligand of the Nectin/Necl family (2014.5.1, zhangyongjun et al)) and the patent literature (CN105247075A) all relate to the relationship between CRTAM and immunity, those skilled in the art also know that two major pillars of immunotherapy are to enhance the offensive power of immune cells and to release the suppressive power of cancer cells on immunity. The treatment method comprises the following steps: it is not obvious that one is to increase the offensive power of immune cells, and the other is to restore the offensive state of immune cells to cancer (various immune checkpoint inhibitors) by releasing the suppression of immunity by cancer cells, so that the purpose of treating diseases is achieved by increasing the immunity of the body organism in any case, and the offensive state of cancer is restored by suppressing the immunity of the body organism in any case.

In the invention, the inventor firstly discovers that CD355, namely Class I-restricted T cell-associated polypeptide (CRTAM), is an important target for regulating host protective immunity, can be used as a tuberculosis immune marker, and provides a new idea for treating multi-drug resistant tuberculosis. The immune marker for tuberculosis and the application thereof are not reported in documents at present.

Disclosure of Invention

The invention aims to provide an immune marker for tuberculosis and application thereof aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the present invention provides an immune marker for tuberculosis, wherein the immune marker is: CD355 expression level.

In a second aspect, the invention provides an application of the immune marker in the preparation of a kit for the differential diagnosis of tuberculosis and/or the prognosis of curative effect.

Further, the invention provides application of the immune marker in preparation of a kit for differential diagnosis of multi-drug resistant tuberculosis and/or prognosis of curative effect monitoring.

Preferably, the only effective components of the kit are: and (3) a reagent for detecting the expression level of CD 355.

Preferably, the kit is used for monitoring the prognosis of the treatment effect by the following indexes: the CD355 expression level in peripheral blood is obviously increased, which indicates that the medicament has curative effect and low recurrence risk after prognosis.

In a third aspect, the present invention provides a kit for differential diagnosis of multi-drug resistant tuberculosis and/or prognosis of therapeutic effect, the kit comprising: the kit comprises a detection reagent and an instruction, wherein the detection reagent comprises a substance for detecting the expression quantity of CD355, and the instruction records the procedures and indexes for identifying the multi-drug resistant tuberculosis and monitoring the curative effect prognosis.

In a fourth aspect, the invention provides the use of a CD355 promoter in the preparation of a medicament for the treatment of tuberculosis.

Further, the invention provides application of the CD355 promoter in preparing a medicine for treating multi-drug resistant tuberculosis.

Furthermore, the invention provides application of the Necl2 promoter in preparing a medicine for treating multi-drug resistant tuberculosis.

Preferably, the promoter is a substance for promoting the expression level of CD355 and/or Necl2, and the promoter is selected from small molecule compounds or biological macromolecules.

The invention has the advantages that:

the invention proves that CD355 (also called Class I-restricted T cell-associated molecule, CRTAM for short) is an important protective cell factor of a host in anti-tuberculosis immune response for the first time. The gene encoding CD355 is present in both humans and mice and functions similarly. In human peripheral lymphocytes, the effect of killing the intracellular mycobacterium tuberculosis infection by lymphocytes can be enhanced by increasing the expression quantity of CD355, and the effect of killing the intracellular mycobacterium tuberculosis infection by the lymphocytes is obviously reduced by inhibiting the expression of the CD 355. Moreover, the number of colonies in the lung of mice with the deletion of the gene encoding CD355 is significantly higher after infection with Mycobacterium tuberculosis than in the control wild-type mice, i.e., the mice with the deletion of the gene encoding CD355 are more susceptible to infection with Mycobacterium tuberculosis. Thus, CD355 is a novel target for host-targeted immune-assisted therapy of tuberculosis. The invention provides a new idea for the treatment of multi-drug resistant tuberculosis and has strong practicability.

Drawings

FIG. 1 shows the expression levels of CRTAM and its ligand, Necl 2. A. The expression levels of CRTAM and Necl2 were determined by quantitative PCR in PBMCs of healthy Humans (HC) and tuberculosis patients (TB). Expression levels of CRTAM and Necl2 following PPD/HMBPP stimulation in PBMC. C. Expression level of Necl2 in THP-1 cells before and after stimulation of BCG infection.

FIG. 2 is a graph showing the effect of CD355 positive cells on the growth of intracellular infectious Mycobacterium tuberculosis. The colony count results of PBMC of tuberculosis infection-resistant people after sorting CD355+ CD3+ cells (black bars) and CD355-CD3+ cells (gray bars) and hMDM cells infected by tubercle bacillus for 3 days.

FIG. 3 is a graph showing the effect of varying the expression level of CD355 on the inhibition of M.tuberculosis intracellulare growth in human PBMC cells. A. After transfecting human PBMC cells with lentivirus (Lenti-CD355) overexpressing a gene encoding CD355 and an empty vector virus (Lenti-Control) as controls, the cells were incubated with macrophages infected with Mycobacterium tuberculosis standard strain H37Rv, and the number of H37Rv Colonies (CFU) was counted by plating after cell lysate was diluted. B. The shRNA fragment targeting the CD355 coding gene was ligated into vector-generated lentivirus (Sh-CD355) and empty vector control virus (Sh-NC) to transfect human PBMC, and the number of H37Rv Colonies (CFU) was counted as described in A.

FIG. 4 shows the number of colonies detected in the lung of mice at various time points after infection of the mice with Mycobacterium tuberculosis H37 Rv. The colony counts in the lungs of CD 355-encoding gene-deleted mice (KO) were significantly higher than wild-type control mice (WT) after 1 week (1W, a) and 2 weeks (2W, B) of infection.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1

1 method of experiment

1.1 recruitment of Experimental populations

The first group of tuberculosis infection resistant people: close contacts are recruited from sputum-positive pulmonary tuberculosis patient partners, the partners which are continuously and closely contacted with patients after the disease occurrence are selected, specific antibodies of serum of the partners aiming at ESAT6 and CFP10 protein are detected, the antibody detection is positive individuals, and then TB.

A second group of sputum-bacteria-positive tuberculosis patients: was recruited by a hospital in the pulmonaceae department of Shanghai to diagnose tuberculosis.

A third group of healthy people not infected with tuberculosis: healthy people without a history of tuberculosis in close contact are selected.

The recruited personnel had no other infectious diseases, no chronic obstructive pulmonary disease, no history of smoking, etc.

Subjects draw peripheral blood and PBMCs are separated from peripheral blood using Ficoll centrifugation according to a technical system we have established.

PBMC cells are stimulated with PPD and HMBPP and stained on the cell surface with fluorescent antibodies such as CD3, CD107a, CRTAM (CD355), CXCR3, etc. The stained cells are analyzed or sorted using a flow cytometer.

After the cell subpopulations of interest are sorted, they are sent to companies for transcriptome sequencing analysis. Or extracting cell mRNA according to an established technical system and detecting the expression quantity of a specific gene.

1.2 transfection of human PBMC cells with lentiviral vectors and detection of PBMC bactericidal effect in vitro the expression of CD355 in lymphocytes was varied according to the technical system we have established.

Methods for increasing the expression level of CD 355: lentiviruses expressing the gene encoding CD355 were constructed and PBMCs were transfected to increase intracellular CRTAM expression.

Method for reducing expression level of CD 355: constructing a lentiviral vector to express shRNA targeting CD355, and transferring the shRNA into human PBMC to reduce the expression quantity of CD 355.

After the CD355 expression was altered by the method described above, it was stimulated with PPD and HMBPP. Meanwhile, macrophage THP-1 was infected with mycobacterium tuberculosis H37Rv at MOI ═ 10. According to the established technical system, the two types of cells are mixed and cultured for 3 days, the cells are lysed, the cell lysate is diluted and coated on a 7H10 plate, the plate is placed at 37 ℃ for culturing for 4 weeks, and the colony number is counted.

1.3 mice were challenged with M.tuberculosis H37Rv, dissected mice to detect the number of colonies in the lung

Mice with a deletion of the gene encoding CD355 (KO) and normal wild-type mice were purchased. Each mouse was infected with Mycobacterium tuberculosis 1x105 CFU by nasal drip. After 1 and 2 weeks of feeding, the mice were dissected, the lungs of the mice were isolated, disrupted and homogenized, the homogenate was diluted and plated, and the number of colonies was counted after 4 weeks of incubation at 37 ℃.

2 results

2.1 high level expression of CD355 in cells chemotactic to migrate to the lung in tuberculosis infection resistant populations

In order to search for effective immune protection response of human bodies to tubercle bacillus infection, the invention takes tuberculosis infection resistant people capable of rapidly eliminating tuberculosis infection as target people, and analyzes the immunological characteristics of cytotoxic effector cells, namely CXCR3+ CD107a +, which can rapidly chemotactic and migrate to the lung in the human bodies of the people. Peripheral blood was drawn to isolate lymphocytes, and CXCR3+ CD107a + cells and CXCR3-CD107 a-cells were sorted for transcriptome analysis. Transcriptome analysis was also performed on the same type of cells derived from tuberculosis patients. The results show that in the tuberculosis infection resistant people, the expression level of CD355 in CXCR3+ CD107a + cells is obviously higher than that of CXCR3-CD107 a-cells (Table 1). CD355 has very low expression of CXCR3+ CD107a + cells in tuberculosis patients, significantly lower than in tuberculosis infection resistant populations (table 2). The results indicate that CD355 can be expressed at high levels in cytotoxic active cells that migrate to the lung, and that CD355 may play an important role in the anti-tubercular immune response in humans.

TABLE 1 expression level of CD355 in double-positive cells of tuberculosis infection-resistant people

Note: (CXCR3+ CD107a +, double positive DP for short) and (CXCR3-CD107a-, double negative DN for short)

TABLE 2 expression level of CD355 in cells of tuberculosis infection-resistant persons

Name of Gene	Expression level in DP-R	Expression level in DP-TB	log2 (fold change)	P value	q value
						CD355	581.537	7.14369	-6.3471	0.00005	0.00322

Note: (CXCR3+ CD107a +, double positive resistance, DP-R for short) and tuberculosis patients (CXCR3+ CD107a +, double positive tuberculosis, DP-TB for short)

2.2 the expression level of CD355 and the gene coding the ligand Necl2 thereof is obviously reduced in tuberculosis patients

In the absence of tuberculosis antigen stimulation, the expression levels of the genes encoding CD355 and its ligand Necl2 in PBMC of healthy people were significantly higher than those of tuberculosis patients (fig. 1A). Both CD355 and Necl2 were also expressed in healthy human PBMC at significantly higher levels after challenge with tuberculosis antigen than in tuberculosis patients (fig. 1B). The result shows that the expression level of CD355 and the ligand Necl2 thereof in tuberculosis patients is remarkably reduced.

Human macrophage THP-1 was infected in vitro with Mycobacterium tuberculosis, and the expression level of Necl2 was determined by quantitative PCR. The results showed that the expression level of Necl2 in the cells was significantly reduced 48 hours after infection (fig. 1C).

The above results indicate that mycobacterium tuberculosis infection inhibits the expression of CD355 and its ligand Necl 2.

2.3 the positive cells of tuberculosis antigen reactivity CD355 can obviously inhibit the growth of intracellular infection mycobacterium tuberculosis

To explore the antituberculous effector function of CD355 positive cells, the inventors examined the effect of CD355+ CD3+ cells on the growth of M.tuberculosis intracellulare infection. PBMCs from tuberculosis infection resistant people (Resister) were sorted for CD355+ CD3+ cells, CD355-CD3+ cells after PPD/HMBPP stimulation. Sorting CD14+ cells from the same individual induced hMDM, which was infected with M.tuberculosis. The two types of cells were then co-cultured and counted on a plate. The results showed that the number of bacteria in the CD355+ CD3+ treated group (black bars) was significantly lower than in the CD355-CD3+ treated group (grey bars) (FIG. 2).

2.4 CD355 can regulate and control the bactericidal function of human lymphocytes

In order to explore the influence of CD355 on the anti-tuberculosis effector function of lymphocytes, the inventor changes the expression level of CD355 and then verifies the bactericidal effect of the lymphocytes on intracellular infection of mycobacterium tuberculosis. First, human lymphocytes were transfected with a lentiviral vector that overexpresses CD355, and the expression level of CD355 was increased. PBMC cells were then incubated with H37Rv infected with M.tuberculosis standard for 3 days, and the number of H37Rv Colonies (CFU) was counted after cell lysis. The results of the study showed that the colony number of the lentivirus-treated group with CD355 over-expression (Lenti-CD355) was significantly lower than that of the Control group (FIG. 3A) compared to the Control virus-transfected group (Lenti-Control). Similarly, the present invention transfected human PBMC with a lentiviral vector expressing shRNA targeting CD355 coding gene to reduce the expression level of CD355, and then examined the bactericidal effect of PBMC cells on intracellular infection H37Rv, and found that the colony number of the treatment group in which CD355 coding gene was suppressed (Sh-CD355) was significantly increased compared to the control (fig. 3B). The research result shows that the change of the expression quantity of the CD355 can influence the in-vitro bactericidal effect of the human lymphocyte, namely that the CD355 can regulate and control the anti-tuberculosis immune function of the human lymphocyte.

2.5 mice with deletion of the CD 355-encoding Gene are more susceptible to infection with Mycobacterium tuberculosis

In order to verify the role of the CD355 coding gene in the host anti-tuberculosis infection process, mice (KO) with the CD355 coding gene deleted are constructed, and then infected by Mycobacterium tuberculosis, and the colony number of the lungs of the mice is detected. As a result of the study, it was found that the colony count of mice (KO) in which the gene encoding CD355 was deleted was significantly increased at both 1 week (FIG. 4A) and 2 weeks (FIG. 4B) after infection with Mycobacterium tuberculosis H37Rv, as compared with wild-type mice (WT). The above results indicate that the mice with the deletion of the gene encoding CD355 have reduced resistance to infection with Mycobacterium tuberculosis, i.e., CD355 has an important protective effect against infection with Mycobacterium tuberculosis in the host.

3 conclusion

The invention proves that CD355 (also called Class I-restricted T cell-associated molecule, CRTAM for short) is an important protective cell factor of a host in anti-tuberculosis immune response for the first time. The gene encoding CD355 is present in both humans and mice and performs similar functions. In human peripheral lymphocytes, the effect of killing the intracellular mycobacterium tuberculosis infection by lymphocytes can be enhanced by increasing the expression quantity of CD355, and the effect of killing the intracellular mycobacterium tuberculosis infection by the lymphocytes is obviously reduced by inhibiting the expression of the CD 355. Moreover, the number of colonies in the lung of mice with the deletion of the gene encoding CD355 is significantly higher after infection with Mycobacterium tuberculosis than in the control wild-type mice, i.e., the mice with the deletion of the gene encoding CD355 are more susceptible to infection with Mycobacterium tuberculosis. Thus, CD355 is a novel target for host-targeted immune-assisted therapy of tuberculosis.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims

1. An immunological marker for tuberculosis, wherein the immunological marker is: CD355 expression level.

2. Use of the biomarker of claim 1 in the preparation of a kit for the differential diagnosis of tuberculosis and/or the prognosis of therapeutic efficacy.

3. Use of the biomarker of claim 1 in the preparation of a kit for differential diagnosis of multi-drug resistant tuberculosis and/or prognosis of therapeutic effect monitoring.

4. The use according to any one of claims 2 to 3, wherein the kit comprises the following active ingredients: and (3) a reagent for detecting the expression level of CD 355.

5. The use of claim 4, wherein the kit is used for prognostic monitoring of therapeutic effects as follows: the CD355 expression level in peripheral blood is obviously increased, which indicates that the medicament has curative effect and low recurrence risk after prognosis.

6. A kit for differential diagnosis of multi-drug resistant tuberculosis and/or prognosis of therapeutic effect, which is characterized by comprising: the kit comprises a detection reagent and an instruction, wherein the detection reagent comprises a substance for detecting the expression quantity of CD355, and the instruction records the procedures and indexes for identifying the multi-drug resistant tuberculosis and monitoring the curative effect prognosis.

Use of a CD355 promoter in the preparation of a medicament for the treatment of tuberculosis.

The application of the CD355 promoter in preparing the medicine for treating multi-drug resistant tuberculosis.

Application of the Necl2 promoter in preparing a medicament for treating multi-drug resistant tuberculosis.

10. The use according to any one of claims 7 to 9, wherein the promoter is a substance that promotes the expression of CD355 and/or Necl2, and the promoter is selected from a small molecule compound or a biological macromolecule.