WO2019041597A1

WO2019041597A1 - Application of candida in preparation of product for preventing and treating immune system disease

Info

Publication number: WO2019041597A1
Application number: PCT/CN2017/113539
Authority: WO
Inventors: 石彦
Original assignee: 清华大学
Priority date: 2017-08-31
Filing date: 2017-11-29
Publication date: 2019-03-07
Also published as: CN109419820A; CN109419820B

Abstract

Provided is an application of intestinal Candida or a lipid extract thereof in the preparation of a product for preventing and treating an immune system disease. Also provided is a product with intestinal Candida or a lipid extract thereof as an active ingredient.

Description

Application of Candida tropicalis in the preparation of products for prevention and treatment of diseases of the immune system

Technical field

The invention belongs to the field of biotechnology and relates to the application of a Candida tropicalis in preparing products for preventing and treating diseases of the immune system.

Background technique

Microorganisms enter the animal from the skin, mouth, respiratory tract, etc. They evolve with the host, affect each other, inhibit and regulate each other's functions. Studying the regulatory mechanisms between these commensal bacteria and hosts is important for human health. Among them, immunologists are most concerned about the effects of intestinal symbiotic bacteria on host metabolism and anti-infection. One of the topics is how symbiotic bacteria regulate host immune function. Most of the literature's research focuses on local immune regulation in the intestine. This may be just the tip of the iceberg, and the effect of commensal bacteria on the immune system may not stop at the intestines. For example, sterile animals have significant peripheral immunodeficiency, mainly manifested by hypoplasia of the systemic secondary lymphoid organs (ie, lymph nodes). Because lymph nodes are the origin of immune responses, how symbiotic bacteria regulate these widely distributed immune organs has become an important issue. However, the link between these commensal bacteria and the development of the peripheral immune system has not been clear so far.

The development of lymph nodes is a delicate and complex process. In the embryo, the primordium of the lymph nodes appears in the epithelial cell cluster. After being stimulated by retinoic acid from nearby nerve endings, lymphoid tissue-inducing cells (LTi) begin to initiate the development of primitive lymphoid structures. After the mouse is born, the LTi cells no longer stay, but the peripheral lymph nodes continue to grow and the number of cells contained continues to increase. One to two weeks after birth, the infiltrated lymphocytes form clear T and B cell regions, almost identical to adult mouse lymph nodes. In contrast, the development of lymph nodes in sterile animals completely ceased after birth. The above developmental problems bring about defects in the immune response. The sterile mice were not immune to the infection after being infected by the intestinal pathogen Shigella flexneri. The symptoms of Salmonella infection in sterile mice are also more severe. As the main site of the immune response, it is conceivable that defects in the structure of the lymph nodes may cause disorders in the immune response. So how does intestinal colony initiate lymph node development after birth?

Invention disclosure

It is an object of the present invention to provide a new use of Candida enteric.

The novel use provided by the present invention is specifically: the application of the lipid extract of Candida enteric or Candida enteric in any of the following (A)-(D):

(A) preparing a product for preventing and/or treating an immune disorder-related disease;

(B) preparing a product for regulating the function of the peripheral immune system;

(C) prevention and/or treatment of diseases associated with immune disorders;

(D) Regulate peripheral immune system function.

The use of a lipid extract of Candida enteric or Candida enteric in any of the following (E)-(H) is also within the scope of the present invention:

(E) preparing a product for driving the transfer of intestinal-derived dendritic cells to lymph nodes;

(F) preparing a product for promoting lymph node development and/or promoting lymph node function;

(G) driving intestinal-derived dendritic cells to lymph nodes;

(H) Promote lymph node development and/or promote lymph node function.

The invention also claims the following methods:

First, a method for preventing and/or treating an immune disorder-related disease is to prevent and/or treat an immune disorder-related disease using a lipid extract of Candida enteric or Entericococcus.

Second, a method of regulating the function of the peripheral immune system is to use a lipid extract of Candida enteric or Candida enteric to regulate peripheral immune system function.

Third, a method of driving intestinal-derived dendritic cells to lymph nodes is to use a lipid extract of Candida enteric or Candida enteric to drive intestinal-derived dendritic cells to lymph nodes.

Fourth, a method of promoting lymph node development and/or promoting lymph node function is to use a lipid extract of Candida enteric or Entericococcus to promote lymph node development and/or to promote lymph node function.

The present invention also claims a product having any of the following functions: preventing and/or treating immune disorder-related diseases, modulating peripheral immune system function, driving intestinal-derived dendritic cells to lymph node metastasis, promoting lymph node development and/or Or promote lymph nodes to function.

In the present invention, all of the aforementioned Candida albicans are specifically Candida tropicalis.

In the present invention, all of the intestinal-derived dendritic cells described above are specifically intestinal-derived dendritic cells expressing CD103, CD11b, and retinal dehydrogenase.

In the present invention, all of the lipid extracts of the aforementioned Candida of the genus Candida can be obtained by a method comprising the steps of: placing the intestinal candida in a volume ratio of 2:1 of chloroform and methanol. The mixture was sonicated, and after centrifugation, the methanol was washed with water, and the chloroform was blown dry with nitrogen to obtain the lipid extract of Candida enteric.

In the present invention, all of the aforementioned immune disorder-related diseases may be autoimmune diseases or hyperimmune inflammatory reactions. Specifically, such as rheumatism, lupus erythematosus, enteritis, multiple sclerosis or ankylosing spondylitis.

In the present invention, all of the aforementioned driving intestinal-derived dendritic cells to lymph nodes may specifically drive the transfer of the intestinal-derived dendritic cells to mesenteric lymph nodes and/or non-intestinal peripheral lymph nodes.

DRAWINGS

Figure 1 shows lymphocytes isolated from lymph nodes of adult normal mice. After labeling, normal (SPF) or sterile (GF) mice are injected through the tail vein.

Figure 2 shows the absence of RALDH positive DC cells in adult sterile mouse lymph nodes.

Figure 3 shows that intestinal microbes regulate lymph node development through retinal dehydrogenase RALDH ⁺ dendritic cells.

Figure 4 is an analysis of the source of CD103 ⁺ CD11b ⁺ RALDH ⁺ DC-like cells.

Figure 5 shows that Candida tropicalis drives intestinal CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells to lymph node metastasis. In the figure, RALDH ⁺ CD103 ⁺ at the ordinate indicates CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells.

Figure 6 shows that the lipid extract of Candida tropicalis can drive BMDC to lymph node metastasis.

The best way to implement the invention

The following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.

Example 1. Candida tropicalis and its lipid extracts drive intestinal DC cells to lymph node metastasis

1. The return of lymphocytes to lymph nodes is defective in sterile mice.

Lymphocytes isolated from lymph nodes of adult normal C57BL/6 mice were labeled with CFSE and injected into normal (SPF) or sterile (GF) C57BL/6 mice via the tail vein (dose was 2 x per mouse) 10 ⁶ cells) After 24 hours, various lymph nodes were isolated, subjected to fluorescence imaging after sectioning, or after cell suspension was obtained and analyzed by flow cytometry.

The result is shown in Figure 1. Left: Infusion of lymphocytes into the inguinal lymph nodes (iLN), mesenteric lymph nodes (mLN) and spleen (spl), and analysis of lymphocyte subtypes to iLN reflux (bottom left). Right: GF, SPF, and expression of MAdCAM-1 and PNAd on high endothelial venules (HEV) in iLN in GF mice (converted) after SPF. This experiment shows that the return of lymphocytes to lymph nodes is defective in sterile mice due to the absence of expression of PNED.

Second, adult sterile mouse lymph node loss RALDH positive DC cells

The lymph nodes of normal (SPF) and sterile (GF) C57BL/6 mice were compared for staining, and the presence or absence of RALDH-positive DC cells was observed.

The result is shown in Figure 2. Top: Dendritic cells of a class of retinal dehydrogenase (RALDH) are absent from the lymph nodes of adult sterile mice compared to normal mice. After being housed in a cage with SPF mice, RALDH-positive cells appeared in the peripheral lymph nodes of the sterile mice. Bottom: Comprehensive data analysis.

Third, intestinal microbes regulate lymph node development through RALDH ⁺ dendritic cells

RALDH ⁺ dendritic cells and RALDH ^- dendritic cells were isolated from SPF-class C57BL/6 mouse lymph nodes, and sterile (GF) C57BL/6 mice (injected at a dose of 2 × 10 ⁵ cells) were injected through the tail vein. Seven days later, lymph nodes were taken for sectioning and flow cytometry analysis.

The result is shown in Figure 3. Upper: RALDH ⁺ dendritic cells or RALDH ^- dendritic cells isolated from the lymph nodes of SPF mice, which were injected into sterile mice through the tail vein, caused changes in lymph node volume and imaging of mature T and B regions. Bottom: The size of the lymph nodes, as well as the statistical results of the area of the B and T areas. This experiment demonstrates that intestinal microbes regulate lymph node development through RALDH ⁺ dendritic cells.

Analysis of CD103 ⁺ CD11b ⁺ RALDH ⁺ DC-like cell sources

The effects of intestinal commensal bacteria on the development of immune organs may not be straightforward. There is a class of unconventional dendritic cells in the lamina propria, which are capable of expressing CD103, CD11b and retinal dehydrogenase (RALDH). They can come into contact with lymphocytes, and this process has a great impact on the fate of these lymphocytes. On the surface, lymph node development seems to have little to do with the above process. The preliminary work of our laboratory found that while the lymphocytes of newborn mice induce cell disappearance, if the intestinal colonies appear in time, a CD103 ⁺ CD11b ⁺ DC-like cell will appear in the lymph nodes. When these cells isolated from normal mice are injected into sterile mice through the tail vein, they promote the development of the latter lymph nodes and a large number of lymphocytes enter the lymph nodes. These dendritic cells highly express retinal dehydrogenase and can express a substance called peripheral lymphotropic substance on the vascular epithelium at the entrance of the lymph node. It can also be regarded as a marker for address localization. T and B cells form lymph nodes by recognizing peripheral lymphotropins into lymph nodes. This process is particularly evident when the mouse is born. However, in adult rats, there are still a small number of such dendritic cells in the lymph nodes, which maintain the long-term homeostasis of the lymph nodes. In vitamin A-deficient mice, dendritic cells such as lymph nodes disappear, causing structural damage. We have demonstrated by labeling that these dendritic cells are derived from the gut and are of the same type as the unconventional dendritic cells (expressing CD103, CD11b and retinal dehydrogenase) in the intestinal lamina propria. .

The specific method is as follows: neonatal C57BL/6 mice were intragastrically administered with FITC Dextran (FITC-dextran2000KD (Sigma) 0.3 mg/g body weight), and 6 hours later, the CD11b and CD103 single positive in the peripheral lymph nodes were detected by flow cytometry, and the double negative ( The ratio of DN) to double positive (DP) dendritic cells.

The results are shown in Fig. 4. It can be seen from the figure that the CD11b and CD103 double positive dendritic cells have the strongest FITC signal, and they are found to be derived from the intestinal tract.

5. Candida tropicalis drives intestinal CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells to lymph node metastasis

Adult SPF grade C57BL/6 mice were mixed with antibiotics in drinking water (antibacterial and antifungal only, 1 g/L of ampicillin, 1 g/L of neomycin, 1 g/L of metronidazole and 0.5 g/L) Vancomycin, the concentration indicates the final concentration in drinking water, Sigma) or the antifungal drug fluconazole (final concentration in drinking water is 0.5g/L, Sigma), and lymph nodes are taken after three weeks. The percentage of CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells in mouse mesenteric lymph nodes (mLN) and Peyer's patches (PP) was analyzed by flow cytometry.

Adult normal C57BL/6 mice were intragastrically administrated with three major intestinal fungi, Candida tropicalis, Saccharomyces cerevisiae, and Trichosporon (per mouse per mouse). After 24 hours of inoculum 10 ⁸ CFU), lymph nodes were taken for flow cytometry to analyze the percentage of CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells in mouse inguinal lymph nodes (iLN) and mesenteric lymph nodes (mLN). In the same manner, the experiment was performed on newborn C57BL/6 mice and sterile C57BL/6 mice.

The result is shown in Figure 5. Upper left: Adult SPF mice were mixed with antibiotics or the antifungal fluconazole in drinking water, CD103 ⁺ CD11b ⁺ RALDH ⁺ dendrites in mesenteric lymph nodes (mLN) and Peyer's patches (PP) after 3 weeks. The percentage of cells. Upper right: Adult normal mice were inguinal lymph nodes (iLN) and mesenteric lymph nodes (mLN) after 24 hours of intragastric administration with cultured Candida tropicalis, Saccharomyces cerevisiae and Trichosporon. Percentage of CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells. Bottom left: Similar to the upper right, the result of newborn mice. Bottom right: Similar to the results of upper right and lower left, adult sterile mice. It can be seen that Candida (C. tropicalis) in intestinal microbes drives the transfer of CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells to lymph nodes.

6. The lipid extract of Candida tropicalis can drive intestinal CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells to lymph node metastasis

The ribonucleic acids, proteins and lipids of various fungi and bacteria (the fungi have Candida tropicalis, Saccharomyces cerevisiae and Trichosporon, and the bacteria are Escherichia coli) are obtained by a separate extraction method. After these isolates were injected into mice, only Candida tropicalis lipids could drive the transfer of dendritic cells to lymph nodes. details as follows:

The fungi and bacterial lipids were extracted by chloroform-methanol extraction, wherein the fungi were Candida tropicalis, Saccharomyces cerevisiae and Trichosporon, and the bacteria were E coli. The fungus or bacteria were sonicated in chloroform-methanol (2:1 by volume), centrifuged, washed with methanol, chloroform, and dried under low temperature nitrogen to obtain a lipid extract.

The lipid extracts of the fungi and bacteria obtained above were separately subjected to liquid chromatography analysis, as follows: chromatography column: diameter 1.5 cm, length 30 cm; filler silica gel (200-300 mesh, particle size 45-75 μm, pore size 40-70A) , specific surface area of 400-600 m ² /g, pore volume of 0.60-0.85 ml / g), atmospheric pressure filling. The lipid extracted from 5 g (wet weight) fungus or bacteria was loaded with 100% chloroform (30 ml), and then the volume ratio of chloroform:methanol was 10:0, 8:2, 6:4, 5:5, 3:7. , 0:10 elution, the first 5 gradients each eluted 30ml, each flat divided into two paragraphs before and after, the last gradient (ie the sixth gradient) eluted 45ml, divided into three paragraphs. This forms 1 to 13 paragraphs. At room temperature, free fall, all samples were blown dry with nitrogen.

After the lipid extract is separated by liquid phase, a segmented component is obtained. Each segment obtained by liquid phase analysis is dissolved in the same volume of DMSO as the unsegmented total lipid, and Transwell experiment is carried out (lipid is added to the lower chamber, lipid The ratio of the culture medium to the 1:1000 assay for the activity of each segment to attract the migration of mouse bone marrow-derived dendritic cells (BMDC) (the skilled person knows that the chemotaxis of BMDC is similar to that of CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells, The reason for using BMDC instead of CD103 ⁺ CD11b ⁺ RALDH ⁺ dendritic cells is that it is difficult to obtain sufficient cell numbers for large-scale analysis. Furthermore, the latter has poor activity in culture and is not suitable for long-term use. experiment).

The results are shown in Figure 6. A: Liquid chromatography spectrum of various fungal lipids; B: Several passages in tropical yeast can induce the migration of dendritic cells in vitro, with the ninth segment being the strongest. It can be seen that the relevant activity in the lipids in the fungus can be concentrated and purified by the liquid phase.

Industrial application

Experiments have shown that Candida albicans such as Candida tropicalis and its lipid extracts can drive intestinal-derived dendritic cells (expressing CD103, CD11b, and retinal dehydrogenase) to lymph nodes. Therefore, in practical applications, it is expected to regulate the number and metabolism of Candida albicans such as Candida tropicalis. And the method of regulating the strength and type of the body's immune response and the number and function of each immune cell subtype by means of oral intake and injection of lipid extracts of such fungi, thereby achieving treatment and/or prevention of immune disorder-related diseases. Such as rheumatism, lupus erythematosus, enteritis, multiple sclerosis, and the purpose of enhancing immune function (such as immunotherapy and vaccination).

Claims

The lipid extract of Candida enteric or Enterobacter sinensis is used in any of the following (A)-(D):

(A) preparing a product for preventing and/or treating an immune disorder-related disease;

(B) preparing a product for regulating the function of the peripheral immune system;

(C) prevention and/or treatment of diseases associated with immune disorders;

(D) Regulate peripheral immune system function.
A method for preventing and/or treating an immune disorder-related disease is to prevent and/or treat an immune disorder-related disease by using a lipid extract of Candida enteric or Candida enteric.
One method of regulating the function of the peripheral immune system is to use a lipid extract of Candida enteric or Candida enteric to regulate peripheral immune system function.
The method according to claim 1 or the method according to claim 2 or 3, wherein the Candida enteric is Candida tropicalis.
Use of a lipid extract of Candida enteric or Candida enteric in any of the following (E)-(H):

(E) preparing a product for driving the transfer of intestinal-derived dendritic cells to lymph nodes;

(F) preparing a product for promoting lymph node development and/or promoting lymph node function;

(G) driving intestinal-derived dendritic cells to lymph nodes;

(H) Promote lymph node development and/or promote lymph node function.
A method for driving intestinal-derived dendritic cells to lymph nodes is to use a lipid extract of Candida enteric or Candida enteric to drive intestinal-derived dendritic cells to lymph nodes.
A method of promoting lymph node development and/or promoting lymph node function is to use a lipid extract of Candida enteric or Entericococcus to promote lymph node development and/or to promote lymph node function.
The method according to claim 5 or the method according to claim 6 or 7, wherein the Candida enteric is Candida tropicalis.
Use or method according to any one of claims 5-8, characterized in that the intestinal-derived dendritic cells express CD103, CD11b and retinal dehydrogenase.
A product for preventing and/or treating diseases associated with immune disorders, wherein the active ingredient is a lipid extract of Candida enteric or Candida enteric.
A product for regulating the function of the peripheral immune system, the active ingredient of which is a lipid extract of Candida enteric or Candida enteric.
A product for driving the transfer of intestinal-derived dendritic cells to lymph nodes, the active ingredient of which is a lipid extract of Candida enteric or Candida enteric.
A product for promoting lymph node development and/or promoting lymph node function, the active ingredient of which is a lipid extract of Candida enteric or Candida enteric.
A product according to any one of claims 10-13, wherein said intestinal rosary The fungus is Candida tropicalis.
The product according to claim 12, wherein said intestinal-derived dendritic cells express CD103, CD11b and retinal dehydrogenase.
An application or method or product according to any one of claims 1 to 15, wherein the lipid extract of Candida entericus is prepared according to a method comprising the steps of: placing the intestinal candida The mixture was sonicated in a mixture of chloroform and methanol in a volume ratio of 2:1. After centrifugation, the methanol was washed with water, and the chloroform was blown dry with nitrogen to obtain a lipid extract of the candida of the intestinal tract.
The use or method or product according to any one of claims 1 to 16, wherein the immune disorder-related disease is an autoimmune disease or an excessive immune inflammatory response.
The use or method or product of claim 17, wherein the immune disorder-related disease is rheumatism, lupus erythematosus, enteritis, multiple sclerosis or ankylosing spondylitis.
A method according to claim 5 or a method according to claim 6 or a product according to claim 12, wherein said driving intestinal-derived dendritic cells are transferred to lymph nodes to drive said intestinal source The dendritic cells metastasize to the mesenteric lymph nodes and/or non-intestinal peripheral lymph nodes.