CN112274535A

CN112274535A - Application of spermidine modified macrophage in development of immunotherapy drugs

Info

Publication number: CN112274535A
Application number: CN202011164840.3A
Authority: CN
Inventors: 时玉舫; 刘睿; 陈永井; 邵常顺
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2021-01-29
Anticipated expiration: 2040-10-27
Also published as: CN112274535B

Abstract

The invention discloses application of spermidine-modified macrophages in development of immunotherapeutic drugs, and belongs to the technical field of biomedicine. The invention can reprogram the macrophage glycometabolism mode from glycolysis to oxidative phosphorylation in vivo and in vitro through spermidine, and further can improve the anti-inflammatory capability of macrophages in vivo and in vitro, and spermidine can passivate the response of macrophages to LPS and maintain an anti-inflammatory phenotype, thereby providing the application of spermidine or spermidine in vitro modified macrophage immunotherapy reagent in treating inflammatory bowel diseases; the invention can improve the expression and secretion of macrophage liver growth factor (HGF) through spermidine, and can improve the expression of macrophages and Matrix Metalloproteinases (MMP) MMP8, MMP9, MMP12 and MMP13, thereby providing the application of spermidine or spermidine in-vitro modified macrophage immunotherapeutic agent in treating hepatic fibrosis.

Description

Application of spermidine modified macrophage in development of immunotherapy drugs

Technical Field

The invention relates to application of spermidine-modified macrophages in development of immunotherapeutic drugs, and belongs to the technical field of biomedicine.

Background

In today's society, autoimmune diseases and degenerative, chronic inflammatory diseases such as: crohn's disease, obesity, type II diabetes, liver and kidney fibrosis and the like, which are inflammatory bowel diseases, have become public health problems seriously endangering human health. Because of the lack of curative treatment, the treatment of patients often relies on the use of immunosuppressive drugs such as steroids, but many of these treatments are associated with a variety of side effects. In recent years, the preclinical and clinical studies of stem cell adoptive transplantation immunotherapy in inflammatory diseases have demonstrated an important role in the modulation of tissue microenvironment in the treatment of inflammatory diseases. The tissue microenvironment, as a complex ecosystem, is composed of parenchymal cells, stromal cells and various types of immune cells, and there are complex interactions between them, and its orderly dynamic regulation is an important guarantee for maintaining homeostasis. Among them, macrophages account for a large proportion of the tissue microenvironment, and excessive accumulation and inflammatory factors secreted from activated macrophages are important causes of inflammatory diseases. Although clinical therapeutic studies of anti-inflammatory factor antibodies such as TNF α and IL-6 have been conducted, the effects are still limited.

In fact, macrophages are very plastic and exhibit different phenotypes by executing different metabolic pathways in different microenvironments. At present, macrophages can be simply classified as classically activated macrophages (M1 macrophages, pro-inflammatory macrophage phenotype) and alternatively activated macrophages (M2 macrophages, anti-inflammatory macrophage phenotype). Macrophages initiate an energy-consuming, biosynthetic aerobic glycolysis under stimulation of LPS and inflammatory factors such as IFN gamma, present a proinflammatory M1 phenotype, secrete various proinflammatory cytokines and chemokines, express a large amount of MHCII and B7 molecules, improve antigen presentation, participate in Th1 type immune response, and kill infectious pathogens and tumor cells. In addition, macrophages, which perform oxidative catabolic-oxidative phosphorylation under the treatment of IL4, IL13, IL10 and the like, produce a large amount of ATP, mediate Th2 type immune responses, and are important participants in suppressing immune responses and performing tissue repair. Given that excessive accumulation of pro-inflammatory macrophages is often the leading cause of inflammatory disease, recent clinical data further demonstrate the safety of autologous macrophage reinfusion therapy, and therefore selective targeting of pro-inflammatory macrophages in the microenvironment, or reinfusion of patient autologous macrophages with anti-inflammatory phenotypes after external reprogramming, would be an important strategy for new inflammatory immunotherapy. There is a need in the art for the development of targeted macrophage based immunotherapy.

Disclosure of Invention

To solve the above problems, the present invention provides an agent for potentiating inflammatory disease treatment by achieving polarization of anti-inflammatory macrophages and improving their anti-inflammatory ability in vivo and in vitro through metabolic reprogramming, and use thereof.

The first object of the present invention is to provide the use of spermidine for the preparation of a medicament for the treatment of diseases based on metabolic reprogramming macrophage immunotherapy.

Further, the disease based on metabolic reprogramming macrophage immunotherapy is an autoimmune disease.

Further, the disease based on metabolic reprogramming macrophage immunotherapy is inflammatory bowel disease.

Further, the disease based on metabolic reprogramming macrophage immunotherapy is liver fibrosis.

Further, the metabolically reprogrammed macrophage is an in vitro reprogrammed macrophage or an in vivo reprogrammed macrophage.

Further, the medicament is a preparation containing spermidine or a preparation of macrophages treated with spermidine.

It is a second object of the present invention to provide a pharmaceutical preparation for the treatment of a disease based on metabolic reprogramming macrophage immunotherapy, said pharmaceutical preparation comprising spermidine-treated macrophages.

Further, the pharmaceutical preparation further comprises a carrier compatible with the macrophage.

Further, the carrier comprises one or more of saline, buffer, glucose, water, glycerol and ethanol.

Further, the carrier also comprises an auxiliary agent, wherein the auxiliary agent is one or more of a lubricant, a retention aid, a wetting agent, an emulsifier and a pH buffering agent.

The invention has the beneficial effects that:

the invention can reprogram the macrophage glycometabolism mode from glycolysis to oxidative phosphorylation in vivo and in vitro through spermidine, and further can improve the anti-inflammatory capability of macrophages in vivo and in vitro, and spermidine can passivate the response of macrophages to LPS and maintain an anti-inflammatory phenotype, thereby providing the application of spermidine or spermidine in vitro modified macrophage immunotherapy reagent in treating inflammatory bowel diseases; the invention can improve the expression and secretion of macrophage liver growth factor (HGF) through spermidine, and can improve the expression of macrophages and Matrix Metalloproteinases (MMP) MMP8, MMP9, MMP12 and MMP13, thereby providing the application of spermidine or spermidine in-vitro modified macrophage immunotherapeutic agent in treating hepatic fibrosis.

Drawings

Figure 1, spermidine was effective in alleviating dextran sulfate sodium salt (DSS) induced inflammatory bowel disease.

(A-B) oral administration of 4% DSS to wild-type C57 mice induced inflammatory bowel disease (5 in the control group, 6 in the spermidine-treated group). The mice were injected intraperitoneally daily with 50mg/kg spermidine in PBS as a control group and the weight changes were monitored and recorded daily. On day 7, mice were sacrificed, followed by isolation of colon tissue.

(C) Isolated mouse colon tissue was photographed and measured for statistical analysis

(D) Cleaning excrement in colon tissues by PBS, drying by absorbent paper, weighing, and counting the ratio of the weight to the length of the cecal tissues;

(E) mouse colon tissue was fixed with 4% PFA and paraffin embedded for paraffin sectioning, H & E tissue staining and imaging analysis. P <0.05, P <0.01, P < 0.001.

Figure 2, spermidine was able to reprogram macrophages towards a stable, anti-inflammatory phenotype in vitro and in vivo.

(A-D) isolation of mouse bone marrow-derived macrophages, flushing out a bone marrow cell suspension, culturing in DMEM/F12 medium containing 20% of the supernatant of L929 with 20. mu.M spermidine for 7 days, (A) extraction of total RNA from cells, and detection of the expression level of anti-inflammatory macrophage-associated genes using Q-PCR. (B) RIPA cell lysis, high-speed centrifugation to extract protein, and Western blot to detect the protein expression of anti-inflammatory macrophage Arg-1. (C) The expression of Arg-1 was observed by confocal microscopy and photographed. (D) The ratio of different phenotypic macrophages was measured using flow cytometry. (E) After bone marrow-derived macrophages were cultured in a spermidine (20. mu.M) -containing medium for 7 days, the supernatant was discarded, washed 3 times with PBS and replaced with a fresh spermidine-free complete medium for further culture for various periods of time, cellular proteins were extracted, and Western blot was used to detect protein expression of the anti-inflammatory macrophages Arg-1. (F-G) intraperitoneal injection of 1ml of starch broth, and daily intraperitoneal injection of 50mg/kg spermidine, DMSO as a control. On day three, mice were sacrificed and peritoneal macrophages were removed, (F) RNA was extracted and Q-PCR was used to detect gene expression of anti-inflammatory macrophages. (G) The phenotype of peritoneal macrophages was examined by flow cytometry. P <0.05, P <0.01, P < 0.001.

Figure 3, spermidine metabolism reprograms macrophage transitions from glycolysis to oxidative phosphorylation.

(A) Intraperitoneal injection of starch broth into wild type mice, intraperitoneal injection of 50mg/kg spermidine every day and PBS as a control group, sacrifice of mice on the third day, isolation of mouse peritoneal macrophages or washing out of bone marrow cell suspension of wild type mice in DMEM/F12 medium containing 20% L929 supernatant, addition of 20 μ M spermidine for culture for 7 days to obtain mouse bone marrow-derived macrophages, incubation with 2-NBDG probe for 1h, and flow cytometry to detect the glucose absorption amount of macrophages.

(B-C) separating and culturing to obtain mouse bone marrow-derived macrophages, labeling mitochondria in the macrophages by using a mito-traker probe, (B) detecting the generation amount of the mitochondria by flow cytometry, and (C) observing the mitochondrial condition of the macrophages by using a fluorescence microscope and taking pictures. (D) Flow cytometry detects macrophage mitochondrial membrane potential. (E) And (3) cracking cells, detecting the ATP content and the total protein content in cell lysate, and evaluating the ATP generation amount of macrophage unit. (F-G) extracting macrophage mitochondria and cracking, and detecting the expression of each compound of (F) mitochondrial carnitine transferase and (G) mitochondrial respiratory chain by Western blot. P <0.05, P <0.01, P <0.001, Scale bar 100 μm.

FIG. 4, inactivation of LPS sensitivity of spermidine-reprogrammed macrophages

(A-B) bone marrow-derived macrophages were cultured in a medium containing spermidine (20. mu.M) for 7 days, then washed out of spermidine with PBS, stimulated with LPS (100ng/ml) for 24 hours, and (A) RNA was extracted and the expression level of proinflammatory macrophage-associated genes was measured using Q-PCR. (B) The protein expression level of iNOS was detected using Western blot assay. And detecting the content of the nitric oxide in the culture medium by using a nitric oxide detection kit. P <0.05, P <0.01, P < 0.001.

FIG. 5 shows that spermidine-modified macrophages are effective in relieving inflammatory bowel disease

(A) A 4% DSS-induced model of inflammatory bowel disease in mice was administered orally daily, with spermidine-pretreated bone marrow-derived macrophages injected intraperitoneally on

days

1,3, and 5 of model induction, and mice were sacrificed on day 7 and the colon isolated for analysis (n-5/group). (B) The body weight of the mice was monitored and recorded daily for statistical analysis. (C-D) isolated Colon tissues 2 pictures (C) per group were taken, colon length was measured and statistical analysis (D) was performed. (E) After the feces in the colon tissue are cleaned, the colon tissue is weighed, and the ratio of the weight to the length of the colon tissue is counted. (F) The treated colon tissue of mice was fixed with 4% PFA and paraffin sections were made, stained with H & E tissue and photographed. P <0.05, p <0.01, p <0.001, and Scale bar 100 μm.

FIG. 6, the change of the secretion profile of spermidine-treated macrophages is beneficial for the treatment of liver fibrosis

(A-B) bone marrow-derived macrophages were cultured in a medium containing spermidine (20. mu.M) for 7 days, and after total cellular RNA was extracted, the expression levels of matrix metalloproteinase family members (A) and liver growth factor (B) were examined by Q-PCR. (C) Macrophage supernatant after spermidine stimulation is collected, and the content of HGF in the supernatant is detected by using an ELISA kit. (D) RIPA lysed cells, Western blot detection was used to detect the protein expression level of HGF. P <0.05, P <0.01, P < 0.001.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Intensive research by the present inventors revealed that spermidine can modify macrophages into a stable oxidative phosphorylation-dependent anti-inflammatory phenotype by metabolic reprogramming, thereby alleviating the progression of inflammatory diseases. Therefore, spermidine can be used as a drug for suppressing immune response. In addition, the spermidine can be used as a modifier for in vitro domestication of macrophages to realize immunotherapy of the macrophages. The present invention has been completed based on this finding.

Spermidine and uses thereof

Spermidine is an important metabolite of cellular amino acid (arginine) metabolism. The polyamine substances play an important role in maintaining the body steady state. The amount of spermidine in the human body is mostly reduced with age and is accompanied by a large number of age-related diseases. In recent years, a great deal of research shows that spermidine can initiate autophagy and inhibit cell senescence. The inventor of the invention reveals for the first time after intensive research: spermidine improves anti-inflammatory ability through metabolic reprogramming, further relieves the progress of inflammatory diseases, and meanwhile, the increased expression of forbidden metalloprotease is beneficial to the remodeling of extracellular matrix at the focus.

The inventor finds that spermidine can start a cell oxidative phosphorylation program depending on mitochondrial activity by reprogramming macrophage, and convert to anti-inflammatory macrophage phenotype, thereby improving the immunoregulation capability. First, spermidine significantly inhibits macrophage glucose uptake, while mitochondrial biosynthesis is significantly increased, oxidative phosphorylation activity is significantly enhanced and more ATP is produced, although mitochondrial membrane potential is not significantly increased, and active oxidative phosphorylation is required for anti-inflammatory macrophages. Furthermore, it has been found that spermidine-modified macrophages are able to maintain relatively stable anti-inflammatory phenotypes and blunt the response to M1-type macrophage stimulators such as LPS, which is more favorable for the modified macrophages to maintain anti-inflammatory phenotypes in complex microenvironments.

Application of spermidine in preparation of macrophage preparation

The present inventors next investigated whether spermidine-modified macrophages can treat inflammatory bowel disease, and it is noteworthy that spermidine-modified macrophages are still effective in alleviating the progression of inflammatory bowel disease. Based on the above new findings of the present inventors, the present invention provides that spermidine can metabolize the reprogrammed macrophage phenotype under in vivo or ex vivo conditions and enhance immunotherapy of inflammatory diseases by modulating immune microenvironment. One approach is, for example, to administer an effective dose of spermidine directly to a subject in need of treatment, thereby altering the immune microenvironment as a whole, increasing the reserve of peripheral anti-inflammatory macrophages, and achieving specific sites by recruiting migrations. Another way is for example to isolate macrophages from the patient in need of treatment or to isolate mononuclear cells (PBMCs) from peripheral blood, to treat and culture them ex vivo with spermidine, to modify and stably reprogram their anti-inflammatory phenotype, and to return them. It is to be understood that other modes of administration contemplated by those skilled in the art are also encompassed by the present invention, based on the novel discovery herein.

Based on the research results of the inventor, the spermidine also has the following characteristics: increasing expression of liver growth factor in macrophages; increasing the expression of macrophage matrix metalloproteinase family members MMP8, MMP9, MMP12 and MMP 13. Therefore, the invention also provides the application of spermidine in preparing macrophage preparations for liver fibrosis immunotherapy. The macrophage preparation with extracellular matrix reconstruction phenotype can be a cell fluid preparation of macrophages which are modified by spermidine. The spermidine treatment may be in vivo or ex vivo.

As used herein, the term "comprising" means that the various ingredients can be applied together in a mixture or composition of the invention.

As used herein, the term "effective amount" refers to an amount that is functional or active in a patient with an inflammatory disorder and is acceptable to the patient.

The composition with the pharmaceutically acceptable carrier may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as lubricants, retention aids, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

The compositions of the present invention comprise a safe and effective amount of the cells and a carrier compatible with the cells. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparations should generally be adapted to the mode of administration, and the compositions of the present invention may be prepared in the form of injections, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount.

In using the compositions of the invention, a safe and effective amount of the cells of the invention are administered to a mammal. Of course, the particular dose and frequency of administration will also take into account the weight, age, health, etc. of the patient to which it is administered, and are within the skill of those in the art.

The composition of the invention can be directly used for improving the proportion and the number of anti-inflammatory macrophages and regulating the microenvironment of inflammation. In addition, it may be used in combination with other therapeutic agents or adjuvants.

The invention will be further elucidated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to manufacturer's recommendations.

Example 1: spermidine is effective in relieving inflammatory bowel disease

C57BL/6 mice with inflammatory bowel disease were injected intraperitoneally with 50mg/kg spermidine daily, as shown in FIG. 1A. As a result, spermidine was found to significantly alleviate DSS-induced weight loss, colon shortening and colon wall thickening in mice, as shown in fig. 1B-D. Pathological analysis results also show that the structure of the colon tissue of the mice in the spermidine treatment group is basically complete, and inflammatory cell infiltration, colon goblet cells and crypt loss caused by DSS are also obviously improved. In conclusion, the administration of spermidine significantly inhibited inflammation due to colonic tissue damage.

Example 2: spermidine is capable of reprogramming macrophages to a stable, anti-inflammatory phenotype in vitro and in vivo

Macrophages of the pro-inflammatory phenotype play an important role in inflammatory bowel disease and are important players of the colonic inflammatory microenvironment. Therefore, in vitro using bone marrow-derived macrophages as the study object, to study whether spermidine has reprogramming effect on tumor-promoting macrophages and makes them play the role of inhibiting inflammation and promoting repair, we first added spermidine during macrophage culture process, and found that spermidine can promote macrophage to be polarized to anti-inflammatory phenotype, as shown in fig. 2A-D. Moreover, this anti-inflammatory phenotype was stable for at least 3 days, as shown in FIG. 2E. To determine the effect of spermidine on macrophages in vivo, mice were injected daily with 50mg/kg spermidine during induction of intraperitoneal macrophagy, resulting in almost all of the macrophages removed having the phenotype of anti-inflammatory macrophages, as shown in FIGS. 2F and 2G. Taken together, spermidine is capable of acclimatizing macrophages to (M2) macrophages of a relatively stable anti-inflammatory phenotype in vitro as well as in vivo.

Example 3: spermidine metabolism reprogramming the conversion of macrophages from glycolysis to oxidative phosphorylation

As mentioned above, spermidine is an important metabolite of spermidine metabolism, and maintains cellular function mainly by inducing autophagy. It was found that the spermidine was able to reprogram the carbohydrate metabolism of macrophages. In vitro results showed that both bone marrow-derived macrophages from spermidine-treated mice and freshly isolated macrophages from the abdominal cavity of mice injected daily with spermidine had significantly reduced glucose uptake capacity, as shown in figure 3A. At the same time, the number of mitochondria in these macrophages was significantly increased, as shown in fig. 3B and 3C. Although there was no significant change in mitochondrial membrane potential, as shown in fig. 3D, there was a significant increase in mitochondrial activity, as shown by more ATP production, more carnitine transferase expression, and an increase in oxidative phosphorylation complexes, as shown in fig. 3E-G. This result suggests that spermidine can decrease the glucose utilization by macrophages, which in turn translates into a mitochondrial dependent oxidative phosphorylation process.

Example 4: inactivation of LPS sensitivity of spermidine-reprogrammed macrophages

Due to the decreased barrier function of the damaged colon tissue, high levels of inflammatory factors as well as LPS are present in the microenvironment, and thus the educated anti-inflammatory macrophages are able to maintain the anti-inflammatory phenotype without being reprogrammed to pro-inflammatory macrophages to exacerbate the level of inflammation. Thus, spermidine-modified macrophages were incubated in LPS-containing medium for an additional 24h and the results showed that the polarization of macrophages with pro-inflammatory phenotypes was significantly inhibited in the absence of spermidine compared to macrophages without treatment, as shown in fig. 4A-B. This result indicates that spermidine confers an immunological memory to macrophages, blunting the response to LPS, making it more difficult to reprogram macrophages to a pro-inflammatory phenotype.

Example 5: spermidine-modified macrophages can effectively relieve inflammatory bowel disease

To verify the feasibility of spermidine-modified macrophage immunotherapy, mouse bone marrow-derived macrophages were treated with spermidine in vitro and reinfused 5 × 10 intraperitoneally on

days

1,3, and 5 established in an inflammatory bowel disease model⁶Cells, mice injected with normal macrophages were used as controls, as shown in fig. 5A. Spermidine-modified macrophages were found to significantly alleviate the weight loss and shortening and thickening of the colon caused by inflammatory bowel disease, as shown in figures 5B-E. Histopathological analysis also showed effective relief of spermidine-modified macrophagesInfiltration of inflammatory cells, disorganization of tissue structure, destruction of goblet cells, and loss of crypts due to colonic tissue injury, as shown in fig. 5F. Taken together, spermidine in vitro modified macrophages are able to exert anti-inflammatory effects in complex inflammatory microenvironments. The present inventors believe that this discovery provides a new approach to the clinical use of spermidine and to macrophage immunotherapy.

Example 6: modification of the expression secretion profile of spermidine-treated macrophages is beneficial for the treatment of liver fibrosis

Spermidine-modified macrophages not only play a role in suppressing inflammatory mediated immune responses, but also have great potential in reconstructing extracellular matrix. There was a clear increase in matrix metalloproteinase expression in spermidine-treated macrophages, as shown in figure 6A. In addition, the expression of Hepatic Growth Factor (HGF) was significantly increased after spermidine treatment, as shown in FIGS. 6B-D. In conclusion, spermidine can direct macrophages to modulate the immune microenvironment, and the potential role of spermidine and related macrophage preparations in liver fibrosis is suggested.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. Use of spermidine in the manufacture of a medicament for the treatment of a disease based on metabolic reprogramming macrophage immunotherapy.

2. The use of claim 1, wherein the disease based on metabolic reprogramming macrophage immunotherapy is an autoimmune disease.

3. The use of claim 1, wherein the disease based on metabolic reprogramming macrophage immunotherapy is inflammatory bowel disease.

4. The use of claim 1, wherein the disease based on metabolic reprogramming macrophage immunotherapy is liver fibrosis.

5. The use of claim 1, wherein the metabolically reprogrammed macrophage is an in vitro reprogrammed macrophage or an in vivo reprogrammed macrophage.

6. The use according to claim 1, wherein the medicament is a spermidine-containing preparation or a spermidine-treated macrophage preparation.

7. A pharmaceutical preparation for the treatment of a disease based on metabolic reprogramming macrophage immunotherapy, wherein the pharmaceutical preparation comprises spermidine-treated macrophages.

8. The pharmaceutical formulation of claim 7, further comprising a carrier compatible with said macrophage.

9. The pharmaceutical formulation of claim 8, wherein the carrier comprises one or more of saline, buffer, dextrose, water, glycerol, ethanol.

10. The pharmaceutical formulation of claim 8, wherein the carrier further comprises an adjuvant, wherein the adjuvant is one or more of a lubricant, a retention aid, a wetting agent, an emulsifier, and a pH buffer.