KR101552610B1 - Animal model for the study of granuloma and method for producing the same - Google Patents

Abstract

The present invention relates to a method for preventing or treating an infectious disease by infecting an experimental animal with Mycobacterium infosus, And injecting the immature dendritic cells into the experimental animal. The present invention also relates to a method for producing an animal model of granuloma and an animal model of granuloma produced by the method. The granuloma animal model according to the present invention can be used for studying the pathological and immunological characteristics by using the granulomatous animal model because it can sustain the growth of M. occidentalisseus and form human necrotic granuloma, It can be useful for screening candidates for antimicrobial agents of granuloma treatment candidates and Mycobacterium infosus.

Description

Animal model of granuloma and method of manufacturing the same Animal model for granuloma and method for producing same

The present invention relates to a method for preventing or treating an infectious disease by infecting an experimental animal with Mycobacterium infosus, And injecting the immature dendritic cells into the experimental animal. The present invention also relates to a method for producing an animal model of granuloma and an animal model of granuloma produced by the method.

Mycobacterium genus includes not only species that cause serious diseases in humans and animals such as tuberculosis, bovine tuberculosis, and leprosy, but also microorganisms called opportunity bacteria, Around 72 species have been known to date, including saprophytic species, which are found in the environment. It is known that there are 25 species related to human diseases. Such a Mycobacterium genus is not readily stained with a commonly used stain solution but once it is stained, it is also called an auto acid bacteria because it is not easily discolored when it is treated with alcohol or hydrochloric acid.

About one-third of the world's population, including about 200 million people, is latently infected with tuberculosis, and 5 to 10% of them can be switched to active tuberculosis for a lifetime. After being infected through the respiratory tract, the tubercle bacillus is incubated for a certain period of time depending on the immune status of the person, and then undergoes active tuberculosis or long-term latent infection.

Among M. tuberculosis isolates except M. tuberculosis isolates, the incidence of M. tuberculosis is increasing in Korea, and most of them are resistant to antibiotics. Like M. tuberculosis, M. abscessus differs from intracellular parasitic bacterium or M. tuberculosis in that it is widely found in the soil such as soil and water. It is the only antibiotic resistant to all currently used antimicrotubule agents have. Mycobacterium abscessus has been recognized as an opportunistic infectious disease causing diseases in people with impaired immune function in the past, but recently it has been recognized as a true pathogen that causes severe diseases to human body regardless of whether immune function is normal or not . As a feature, the size of the genome of Mycobacterium abscessus ATCC19977T is about 5 Mb. Unlike other mycobacteria, it does not have a characteristic insertion sequence but has a full-length prophage of 81 kb have. It is also known that there are many genes that are relatively conserved compared to Mycobacterium tuberculosis and that the composition of the gene is similar to M. smegmatis, which is not pathogenic. Unlike other mycobacteria, the most distinctive feature is that they contain non-mycobacterial genes found in streptomycetes and pseudomonads, while horizontal transfer ) Are being studied.

Generally, after the infection with the antibiotics, the host has the maintenance or activity of latent infection depending on the balance of the pro-inflammatory and anti-inflammatory responses of the host. The pathogenic bacteria that infected the lungs of the host through the respiratory tract may continue to proliferate if the congenital defense immunity is not perfect, and then the acquired immunity is induced, the bacteria can be fished by the cell mediated immune response, the lesion tissue is necrotic, (hypoxia), the antibiotics do not maintain their growth any more, and the metabolism is almost stopped. The cell-mediated immune response and antioxidant bacteria are in balance, and many lymphocytic cells are called granuloma.

The granuloma usually has a massive mesenchymal cell that is matured in the middle, and the large predominant cell in the granuloma becomes a multinucleated giant cell or a foam cell that can accumulate fat. Unusually, some matured large proliferating cells are transformed into epithelioid cells and help maintain strong cell-cell tight junctions with zipper-like arrays. Granulomas include neutrophils, dendritic cells, natural killer cells, fibroblasts, B cells, and T cells as well as large predominant cells.

Unusually, granulomas are formed in the mouse due to infection with mycobacterial strains, but they are very different from necrotic granuloma formed in humans. Necrotizing granuloma differs markedly from non-necrotizing granuloma, drug permeability, and immune response. In general, necrotizing granuloma is known to be difficult to permeate drugs, and since the metabolism of bacteria is different from that of non-necrotizing granulomas, the drugs that are found to be effective by drug detection using the mouse infection model are also effective There are many cases that come out completely different.

On the other hand, dendritic cells are derived from hemopoietic bone marrow progenitor cells. These progenitor cells are initially transformed into immature dendritic cells, characterized by high endocytosis activity and T-cell activation capacity. Immature dendritic cells constantly digest surrounding pathogens such as viruses and bacteria. This is possible through pattern recognition receptors (PRRs) such as toll-like receptors (TLRs).

TLRs recognize specific chemical characteristics found on a subset of pathogens, and immature dendritic cells digest the cell membrane through a process known as nibbling from living autologous cells. When they come into contact with existing antigens, they become activated to mature dendritic cells and migrate to the lymph nodes. When immature dendritic cells mature by digesting pathogens and digesting their proteins into small pieces, these pieces appear on the cell surface using MHC (Major Histocompatibility Complex) molecules. At the same time, it increases cell surface receptors that act as co-receptors in T-cell activation such as CD (Cluster of Differentiation) 80, CD86, and CD40. They activate helper T-cells, killer T-cells, as well as B-cells by showing pathogen-derived antigens along with non-antigen specific co-stimulatory signals.

Unusually, M. mycobacterium infestus causes only a slight increase in the number of microbes at the initial stage of infection with the mouse, and most of the microbes disappear due to host immunity toward the later stage of infection. This is one of the biggest problems when developing antibiotics targeting Mycobacterium abscessus. Therefore, the development of a model capable of sustained growth during infections of Mycobacterium infosus and the development of models capable of forming human-like forms of granuloma is essential for studying preclinical drug experiments, vaccines and pathology of bacteria Element.

Accordingly, the present inventors completed the present invention by preparing a novel granulomas animal model by injecting M. tuberculosis and immature dendritic cells into an experimental animal.

It is an object of the present invention to provide a method for producing an animal model of granuloma.

Another object of the present invention is to provide an animal model of granuloma produced by the above method.

It is still another object of the present invention to provide a screening method for antibiotic candidate substances of Mycobacterium infosus or candidate substance for treating granuloma using the animal model of granuloma.

In order to achieve the above object,

(a) infecting an experimental animal with Mycobacterium abscessus; And

(b) injecting the immature dendritic cells into the experimental animal. The present invention also provides a method of producing an animal model of granuloma comprising the steps of:

The present invention also provides an animal model of granuloma produced by the above method.

The present invention also provides a method for screening candidates for antimicrobial agents of Mycobacterium infosus or candidate substances for treating granuloma using the animal model of granuloma.

The granuloma animal model according to the present invention can be used for studying the pathological and immunological characteristics by using the granulomatous animal model because it can sustain the growth of M. occidentalisseus and form human necrotic granuloma, It can be useful for screening candidates for antimicrobial agents of granuloma treatment candidates and Mycobacterium infosus.

1 is a diagram showing an overall experimental schedule for developing an animal model of necrotizing granuloma of the present invention.
Figure 2 shows the effect of TLR2 deficient or immature dendritic cell infusion on the survival rate of mice infected with mycobacterial hyphae.
Figure 3 shows the effect of TLR2 deficiency or immature dendritic cell infusion on the growth of mycobacterial hyphae in vivo.
Figure 4 shows the effect of TLR2 deficiency or immature dendritic cell infusion on the number and distribution of mycobacterial hyphae in lung tissue using acid-fast staining.
Figure 5 shows the effect of TLR2 deficient or immature dendritic cell infusion on the level of inflammation caused by Mycobacterium infosus infection.
FIG. 6 is a graph showing necrotizing granuloma produced when immature dendritic cells are injected into TLR2 ^{- / -} mice upon infection with Mycobacterium abscessus.

Hereinafter, the present invention will be described in detail.

The present invention

(a) infecting an experimental animal with Mycobacterium abscessus; And

(b) injecting the immature dendritic cells into the experimental animal. The present invention also provides a method of producing an animal model of granuloma comprising the steps of:

The test animal of step (a) may be any animal except human, preferably a mammal, more preferably a rat, a mouse, a guinea pig, a hamster, a rabbit, a dog, Is a Toll-like receptor 2 (TRL 2) deficient mouse.

The amount of the M. tuberculosis infestation in step (a) is not limited as long as it causes infection with Mycobacterium infosus, and may vary depending on the weight and condition of the experimental animal. In an embodiment of the present invention, a mouse is used as an experimental animal, and 1 × 10 ⁵ to 1 × 10 ⁹ Mycobacterium infosus is preferably injected, but not limited thereto.

The immature dendritic cells of step (a) can be obtained through various methods known in the art. For example, dendritic cells can be obtained using monocytes, hematopoietic stem cells, or bone marrow cells.

The immature dendritic cells of step (b) are preferably injected within 12 to 48 hours after the infusion of M. tuberculosis, but the present invention is not limited thereto.

The amount of immature dendritic cells injected in step (b) may vary according to the weight and condition of the experimental animal, but is not limited thereto. In one embodiment of the present invention, a mouse is used as an experimental animal. In this case, 1 x 10 ⁴ to 1 x 10 ⁸ immature dendritic cells are preferably injected, but not limited thereto

The injection of the step (a) or step (b) may be performed through various routes, and preferably, it may be performed through intravenous, oral, rectal, muscular, subcutaneous, intrauterine or intracerebral injection, Preferably through intravenous injection.

The granuloma is preferably a necrotizing granuloma.

The present invention also provides an animal model of granuloma produced by the above method.

The animal may be any animal except human, preferably a mammal, more preferably a rat, a mouse, a guinea pig, a hamster, a rabbit, a dog and the like, more preferably TRL 2 (Toll- like receptor 2) deficient mice.

The granuloma animal model according to the present invention can be used for studying the pathological and immunological characteristics by using the granulomatous animal model because it can sustain the growth of M. occidentalisseus and form human necrotic granuloma, It can be useful for screening candidates for antimicrobial agents of granuloma treatment candidates and Mycobacterium infosus.

In addition,

(a) administering a candidate substance for treating granuloma to the animal model of granuloma; And

(b) confirming the therapeutic effect of the inflammation in the animal model.

In addition,

(a) administering to the animal model of granuloma an antimicrobial agent candidate of Mycobacterium infosus; And

(b) identifying an antimicrobial candidate substance of Mycobacterium infosus including the step of confirming the therapeutic effect of the infecting strain of Mycobacterium infosus in the animal model.

The method of administering the sample may be appropriately selected from the conventional methods of administering the sample, such as oral administration, intravenous injection, subcutaneous administration, intraperitoneal administration, etc. The dosage of the sample can be appropriately selected depending on the administration method,

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are provided for illustrating the present invention in detail and the scope of the present invention is not limited by these examples.

Example 1. Preparation of an animal model of granuloma

1-1. Isolation and induction of dendritic cells

Femur bone marrow was collected from C57BL / 6 mice using injection for bone marrow harvesting. After the collected bone marrow was washed, the erythrocytes were removed using ammonium chloride. Separated cells were cultured in 6-well plates in RPMI 1640 (10% FBS (Fetal bovine serum, calf serum), 2 mM L-glutamine, 100 U / ml penicillin / streptomycin, 50 μM mercaptoethanol, , 1 mM sodium pyruvate, 20 ng / ml GM-CSF, and 2.5 ng / ml IL-4). GM-CSF and IL-4 were used to induce differentiation into dendritic cells.

1-2. Cultivation of Mycobacterium abscessus

M. abscessus ATCC19977T) was distributed from the American Type Culture Collection (ATCC, Manassas, Va.). This was incubated for 7 days at 37 ° C in 7H9 broth (Difco Laboratories, Detroit, MI) containing 10% (v / v) oleic acid-albumin-dextrose-catalase (Becton Dickinson, Spark, Respectively. At this time, the M. tuberculosis shows smooth colonies. To transform it into a rough form, C57BL / 6J mice were infected and treated with a macrolide antibiotic Clarithromycin. Mucorcythelium pressisus switched to rough form from mouse lung was obtained and the genotype change was confirmed by VNTR method. The Mycobacterium infosus was cultured in a 7H9 broth medium containing OADC, centrifuged at 10,000 xg for 20 minutes, and washed three times with PBS. The obtained pellet was treated with a homogenizer for 1 minute and then passed through an 8 [mu] m filter to make a single cell. It was stored at -80 ° C.

1-3. Manufacture of animal models of granuloma

Six-week old female C57BL / 6 (H-2K ^b and IA ^b ) normal mice and C57BL / 6J TLR2 deficient mice (TLR2 ^{- / -} ; B6.129-Tlr2 ^tm1Kir / J) were used as experimental ^animals . Each mouse was infected with an intravenous injection of Mycobacterium infosus (1.5 x 10 ⁷ ) obtained in Example 1-2 above in the tail. 24 hours after infection, the immature dendritic cells (1 x 10 ⁶ ) obtained in Example 1-1 were intravenously injected into the tail to prepare an animal model of granuloma. This process is shown in Fig.

Experimental Example 1. Analysis of effect on mouse survival rate and number of bacteria

Mycobacterium infosus is naturally cleared by host immunity after a certain period of time when the mouse is infected. Therefore, it is important to establish an animal model of necrotizing granuloma so as to continuously increase the number of bacteria.

To confirm that the mouse prepared in Example 1 was suitable as an animal model of granuloma animal, mouse survival rate during the experimental period and inoculation of microbacterium Mycobacterium infiltration in vivo after 2, 7, 15 and 21 days of Mycobacterium infosus infection The number of bacteria was analyzed. The results are shown in Figs. 2 and 3, respectively.

As shown in Fig. 2, when the M. tumefaciensis was infected with a normal (WT) mouse, all mice survived until 30 days after the infection, whereas M. corbicillium abscessus was treated with TLR2 ^{- / -} mice , Most mice died after 20 days, and there was no significant difference in viability when immature dendritic cells were injected.

As shown in FIG. 3, when the M. tuberculosis isolate was infected with the normal (WT) mouse, the number of bacteria was increased in the lungs until the 7th day after infection, and the bacteria gradually disappeared after 14 days I could. On the other hand, when the M. tuberculosis isolate was infected with TLR2 ^{- / -} mice, a similar level of bacteria was detected up to 7 days after the infection, but the number of bacteria was continuously increased after 14 days, And it was confirmed that it continued to increase.

In addition, after 2, 7, 15 and 21 days after the infestation with M. tuberculosis, lung tissues were separated and subjected to acid-fast staining according to a conventionally known method. The results are shown in Fig.

As shown in FIG. 4, when M. canis infected with normal (WT) mice, the number of bacteria in the lung tissue was small and the bacteria were present in a form spread in a certain granuloma, Were infected with TLR2 ^{- / -} mice, the number of bacteria was observed more than that of normal mice, but it was confirmed that the bacteria existed in a form spread in granuloma. On the other hand, in the infusion of immature dendritic cells after infecting the TLR2 ^{- / -} mice with Mycobacterium abscessus, bacteria were concentrated in the central portion of the granulomas of the lung tissue and typical necrotic And it was confirmed that the distribution of bacteria according to the granuloma was shown.

From the above experimental results, it was confirmed that the mouse according to the present invention can be used as an animal model of granulomas because it is possible to continuously increase mycobacterium infiltrate and to form necrotizing granuloma.

Experimental Example 2. Analysis of Inflammation Level by Infection with Mycobacterium infestus

The effect of TLR2 deficiency or immature dendritic cell infusion on the degree of inflammation caused by Mycobacterium infosus infection was confirmed. The results are shown in Fig.

As shown in Fig. 5, when a normal (WT) mouse was infected with Mycobacterium abscessus, (Mild) in the range of 2 to 2.1, and that the infectivity of Mycobacterium infosus with TLR2 ^{- / -} mice was about 2.5. On the other hand, when infected with M. tuberculosis infected with TLR2 ^{- / -} mice, infiltration of immature dendritic cells showed a very strong inflammation level of about 3.5 to 4.

Experimental Example 3. Analysis of Influence on Inflammation of Granuloma by Mycobacterium infestus Infection

To confirm the effect of TLR2 deficiency or immature dendritic cell infusion on the granuloma formation due to Mycobacterium infosus infection, the mouse lung tissue obtained in Experimental Example 1 was subjected to haematoxin and eosin staining, And histopathologic changes were observed. The results are shown in Fig.

As shown in FIG. 6, when the M. tuberculosis was infected with normal (WT) mice, most of the granulomas in the pulmonary tissues were observed, and the M. tuberculosis was treated with TLR2 ^{- / -} Similar findings were seen when infected with mice. On the other hand, when immature dendritic cells were infected with TLR2 ^{- / -} mice after infecting M. tuberculosis isolates, necrotic debris was observed in the central part of the lung tissue, A typical necrotizing granuloma surrounding the cells was formed.

Claims (11)

(a) infecting a TLR2 (Toll-like receptor 2) deficient mouse with Mycobacterium abscessus; And
(b) injecting immature dendritic cells into said mouse. < Desc / Clms Page number 19 > delete delete 2. The method of claim 1, wherein the injection of step (a) or step (b) is performed by intravenous injection. The granuloma animal model according to claim 1, wherein the amount of the microbacterium infestus is 1 × 10 ⁵ to 1 × 10 ⁹ in the step (a). The method according to claim 1, wherein the immature dendritic cells are injected in step (b) within 12 to 48 hours after the infusion of M. tuberculosis. The method according to claim 1, wherein the amount of immature dendritic cells injected in step (b) is 1 × 10 ⁴ to 1 × 10 ⁸ . The method of claim 1, wherein the granuloma is a necrotizing granuloma. delete delete delete

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