CN114732908A

CN114732908A - Application of HDAC6 inhibitor in severe infection

Info

Publication number: CN114732908A
Application number: CN202210209710.XA
Authority: CN
Inventors: 黄勇; 童德文; 王晓亚
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-07-12

Abstract

The invention discloses application of an HDAC6 inhibitor in severe infection. The inhibitor can obviously reduce the number and reactivity reduction degree of NK cells induced by severe infection, improve the clinical symptom score of the severe infection and relieve the histopathological injury of the severe infection to the lung, the liver and the kidney, can obviously enhance the capability of resisting the secondary infection of a severe infection organism by targeted inhibition of HDAC6 activity, and provides a new idea for effective prevention and treatment of the severe infection.

Description

Application of HDAC6 inhibitor in severe infection

Technical Field

The invention relates to the use of HDAC6 inhibitors for the prevention and treatment of severe infections.

Background

Severe infection (Severe infection) caused by pathogenic microorganisms such as bacteria and viruses can cause low immune function of the organism, secondary mixed infection of other various pathogens or chronic infection which cannot be cured, and seriously affect the life quality of patients. Immunosuppression caused by severe infection impairs the function of lymphocyte population, wherein the reduction of the number and reactivity of Natural Killer (NK) cells is closely related to the low immune function of body in later stage of infection, so that the improvement of the number and reactivity reduction of NK cells in the course of severe infection is one of the effective methods for improving survival rate of severe infection.

Studies have shown that the Expression of surface NKG2D is reduced and the killing function is greatly inhibited after NK cells are treated with valproic acid (a broad-Spectrum Inhibitor of histone deacetylase, propionic acid) (Ni L, Wang L, Yao C, et al. the histone deacetylase Inhibitor or a potent Inhibitor of Cancer NKG2D Expression in natural kit cells of 2015 3 and HDAC3.Rep,2017,7:45266.) while the broad-Spectrum Inhibitor of histone deacetylase Entinostat can enhance the killing of Cancer cells by NK cells by increasing NKG2D and its ligands (Cell, Zhu, center, et al. the Narrow-Spectrum HDAC Inhibitor of Cancer Cell NKG2D Expression, Cell of Cell. The histone deacetylase inhibitor can also play a role in protecting acute lung injury caused by sepsis, and the mechanism of the histone deacetylase inhibitor can be that the inflammatory response is reduced and the apoptosis is reduced (Chen Long et al. the histone deacetylase inhibitor, trichostatin A, has a protective effect on acute lung injury of sepsis mice. China journal of Emergency medicine, 2018,27(03): 275-282.). However, the toxicity and side effects of broad-spectrum deacetylation inhibitors such as trichostatin A limit the clinical application.

Research shows that Histone deacetylase 6 (HDAC 6) is an important regulatory molecule with deacetylase activity on non-Histone substrates and localized in cytoplasm, and is involved in the functional regulation of various immune cells in the inflammatory reaction process of organisms. For example, after the HDAC6 inhibitor CAY10603 is applied, the LPS-induced STAT1(Try701) activation and IRF-1 synthesis in mouse abdominal cavity macrophages can be inhibited, so that i NOS overexpression and NO overproduction can be inhibited (Wangbao HDAC6 promotes the mechanism research of sepsis development by regulating macrophage nitric oxide production. Jilin university 2020 DOI 10.27162/d. cnki. gjlin 2020.007259). However, reports that HDAC6 participates in the regulation of NK cell hypofunction induced by severe infection are not found at present. Meanwhile, the influence of Tubastatin A as a specific HDAC6 inhibitor on the NK cell function is not reported.

Disclosure of Invention

The present invention aims to address the deficiencies of the prior art by providing the use of HDAC6 inhibitors in severe infections.

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

the invention injects 1 XLD to abdominal cavity of mice₅₀Staphylococcus aureus (6.2X 10)⁸CFU) or E.coli (3.5X 10)⁷CFU), a C57BL/6J wild-type mouse severe infection model was constructed. 1h after bacterial infection, HDAC6 inhibitor is given, clinical symptoms of mice infected with different groups of bacteria are observed and recorded, the survival rate of the mice is counted, and the bacterial load in abdominal cavity is detected; taking lungs, livers and kidneys of mice infected with different groups of bacteria, making paraffin sections and observing histopathological changes; flow cytometry is used for detecting the number, the proportion and the reactivity change of NK cells in spleens of mice with different infectious bacteria. The results show that the degree of reduction of NK cell number and reactivity is significantly reduced in critically infected mice administered with HDAC6 inhibitor (e.g., a selective inhibitor such as Tubastatin a, targeted inhibition of HDAC6 activity) compared to critically infected mice administered with control solvent, and that survival of the mice is increased, clinical symptom scores are improved, and tissue damage is reduced.

The invention further carries out secondary infection on different groups of infectious microbe mice surviving at the later stage of severe infection, observes and records the clinical symptoms and the histopathological changes of the mice, and counts the survival rate of the mice after the secondary infection. The results show that critically infected mice that survived administration of HDAC6 inhibitor had significantly improved survival rates after secondary infection compared to critically infected mice that survived administration of control solvents, and that mice had significantly lower clinical symptom scores and less tissue damage.

The invention has the beneficial effects that:

the HDAC6 inhibitor is adopted to effectively intervene in the anti-infection effect of NK cells in severe pathogen (such as pathogenic bacteria) infection and the balance effect of regulating and controlling the inflammatory response of organisms, can reduce the death caused by severe infection and the like, and provides a new idea for the prevention and treatment of severe infection. The HDAC6 inhibitor adopted by the invention obviously improves the capability of the body with serious infection to resist secondary infection after survival.

Furthermore, the Tubastatin A (or its pharmaceutically acceptable salt, such as Tubastatin A Hydrochloride, Tubastatin A trifluoroacetate salt, etc.) used in the present patent can be used as a specific HDAC6 inhibitor, and can increase the number, proportion and reactivity of NK cells in a severely infected organism in the presence of inflammatory cytokine stimulation.

Drawings

FIG. 1 shows the results of the effect of the HDAC6 inhibitor Tubastatin A on the characteristics and anti-infective ability of a severely infected mouse; a, B shows survival rate of mice infected with Staphylococcus aureus and Escherichia coli at different time; C. d is the clinical symptom score of mice infected with staphylococcus aureus and escherichia coli at different time; E. f is the number of abdominal cavity bacteria 48 hours after the mice are infected with staphylococcus aureus and escherichia coli; indicates statistically significant differences relative to the "s.

FIG. 2 shows the effect of the HDAC6 inhibitor Tubastatin A on the pathological changes of lung tissues of the critically infected mice.

FIG. 3 shows the effect of the HDAC6 inhibitor Tubastatin A on pathological changes of liver tissue of mice infected with severe hepatitis.

FIG. 4 shows the effect of the HDAC6 inhibitor Tubastatin A on the pathological changes of kidney tissues of mice infected with severe symptoms.

FIG. 5 shows the result of flow cytometry to detect the effect of the HDAC6 inhibitor Tubastatin A on NK cells of mice infected with severe symptoms; wherein A is the change condition of the proportion and the number of the spleen NK cells after the mice are infected with staphylococcus aureus; b is the change condition of the proportion and the number of the spleen NK cells after the mice are infected with the escherichia coli; denotes p relative to control group (DMSO)<0.05, relative to control group (DMSO) p<0.01，^##Represents p relative to the infection group administered with DMSO<0.01。

FIG. 6 shows the result of flow cytometry to detect the effect of the HDAC6 inhibitor Tubastatin A on the reactivity of NK cells of severely infected mice; wherein A is the IFN-gamma positive rate of NK cells and the IFN-gamma expression level of the NK cells of mice spleen lymphocytes (separated 48h after staphylococcus aureus infection) under the condition of giving IL-2 and IL-12 in-vitro stimulation for 6 h; b is the positive rate of NK cell IFN-gamma and the expression level of NK cell IFN-gamma of mice spleen lymphocytes (separated 48h after escherichia coli infection) under the condition of giving IL-2 and IL-12 in-vitro stimulation for 6 h; denotes p versus control (DMSO)<0.01；^##Represents the low dose of infection group p relative to DMSO administration (Tubastatin A not administered)<0.01；^&Represents the high dose of infection group p relative to DMSO administration (Tubastatin A not administered)<0.05。

FIG. 7 shows the results of the physical effects of the intervention of Tubastatin A, an HDAC6 inhibitor, on the secondary infection of surviving mice; wherein A, B is the survival rate of mice after secondary infection (0 h is the starting time of secondary infection); C. d is the clinical symptom score after secondary infection of mice (starting time of primary infection as 0h), indicates p <0.01 relative to control group (DMSO).

FIG. 8 shows histopathological observation results of major organs of mice (infected with Staphylococcus aureus for the first time) after being infected with Escherichia coli for the second time.

FIG. 9 shows the histopathological observation results of the major organs of mice (infected with E.coli for the first time) infected with Staphylococcus aureus for the second time.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention.

The invention firstly injects 1 XLD to the abdominal cavity of mice₅₀Staphylococcus aureus (s. aureus) or escherichia coli (e. coli), a mouse severe infection model was constructed. 1h after bacterial infection, HDAC6 inhibitor (such as specific inhibitor Tubastatin A, Tub A for short) is given for intervention, the clinical symptoms of the mice are observed and recorded, the bacterial number in abdominal cavity of the mice 48h after infection is measured, and the histopathological changes of Lung (Lung), Liver (Liver) and Kidney (Kidney) are observed, and meanwhile, the number, proportion and reactivity change of NK cells in spleen of the mice are detected by flow cytometry. And then, carrying out secondary bacterial infection on the mice surviving in the late period of severe infection, and carrying out clinical symptom and histopathological observation on the mice after secondary infection. The above Staphylococcus aureus and Escherichia coli adopt isolate (16 s sequencing and gene evolutionary tree analysis show that the Staphylococcus aureus isolate and pathogenic Staphylococcus aureus with GenBank accession number CP049545.1 belong to kindred development branch, and the Escherichia coli isolateStrains belonging to a closely developing branch with pathogenic escherichia coli having GenBank accession number CP 027103.1). Establishing and grouping severe mouse infection model

C57BL/6 mouse control group (ctrl), each healthy mouse was intraperitoneally injected with 0.5mL of 0.9% sterile saline, and 1h after the injection of saline was intraperitoneally injected with an equal volume of DMSO (uninfected solvent control) or Tub A (uninfected inhibitor control); c57BL/6 mice were high dose infected with a priming group (S. aureus or E. coli + Tub A or DMSO), each healthy mouse at1 × LD₅₀Intraperitoneal injection of 6.2X 10⁸Staphylococcus aureus solution of CFU or 3.5 × 10⁷Injecting HDAC6 inhibitor Tub A (70mg/kg, solvent is DMSO) or equal volume of DMSO into abdominal cavity 1h after bacterial infection of CFU Escherichia coli liquid; c57BL/6 mice low dose infection intervention group, each healthy mice intraperitoneal injection 6.2X 10⁶Staphylococcus aureus solution of CFU or 3.5 × 10⁵Injecting HDAC6 inhibitor Tub A (70mg/kg, solvent is DMSO) or DMSO with the same volume into the abdominal cavity 1h after bacterial infection of Escherichia coli liquid of CFU; c57BL/6 mouse severe infection group, each healthy mouse intraperitoneal injection 6.2X 10⁸Staphylococcus aureus solution of CFU or 3.5 × 10⁷CFU escherichia coli solution.

(II) observation of clinical symptoms

Clinical symptoms of the test mice were recorded at 4h, 8h, 12h, 24h, 48h, 96h, 192h observation post bacterial infection, respectively, and semi-quantitative scores were performed according to the mouse clinical symptom scoring criteria (Okeke et al 2014): 0, no apparent clinical symptoms are shown; 1, the mouse is disorderly but lively by hair; 2, the mouse is disorderly in hair, depressed in spirit and sluggish in movement; 3, the mouse has bow waist, is difficult to move and has serious illness; 4, the canthus of the mouse has a large amount of secretion, constipation and dying; and 5, death. And drawing a clinical score mean curve, and counting the death condition of the mouse after the test is finished.

The results show that administration of the HDAC6 inhibitor Tub a after s.aureus or e.coli infection significantly improved survival of critically infected mice (fig. 1A, 1B) and reduced clinical symptoms in critically infected mice (fig. 1C, 1D).

(III) determination of bacterial load in abdominal cavity

Mice were killed 48h after bacterial infection and evaluated for their anti-infective ability by counting the number of bacteria in the abdominal cavity: injecting 4mL of sterile normal saline into the abdominal cavity of a mouse in a sterile super-clean bench, fully massaging the abdominal cavity of the mouse, sucking abdominal cavity liquid, performing gradient dilution on the abdominal cavity sucking liquid, uniformly coating the solution on an LB agar plate after the gradient dilution, culturing overnight at 37 ℃, and counting the plate bacteria.

The results show that critically infected mice administered the HDAC6 inhibitor Tub a had a significant reduction in the number of peritoneal bacteria at 48h post-infection (fig. 1E, fig. 1F).

(IV) histopathological Observation

At 48h post-bacterial infection, mice were dissected and HE stained paraffin tissue sections were prepared by the following steps:

material taking and fixing: the lung, liver and kidney of the test mouse were harvested, the harvested tissue was fixed in a wide-mouth bottle with 4% neutral formaldehyde for 48h to ensure a ratio of fixative to tissue of at least 10: 1.

Block repairing: trimming the fully fixed tissue block to a suitable size (size not exceeding 0.5 cm)³) The trimmed tissue blocks were then placed in tissue embedding frames and rinsed overnight with running water.

Dewatering, transparency and wax dipping: the embedding frame with the tissue block is sequentially subjected to 70%, 80%, 90%, 95% alcohol solutions (each for 1 hour), 100% alcohol I, II (each for 30min), xylene I, II (each for 2-5 min) and paraffin I, II (each for 1 hour), so that dehydration, transparency and wax dipping are completed.

Different tissues vary in time of transparency, liver: xylene I (5min), xylene II (5 min); lung: xylene I (3min), xylene II (2 min); kidney: xylene I (5min), xylene II (2 min).

Embedding: pouring molten wax into a mould, clamping a tissue block by using forceps, flatly placing a section at the center of the bottom surface of the mould, covering a disposable embedding box, injecting a little paraffin for preventing overflowing, slightly placing the disposable embedding box on a condensation table after a moment, and naturally solidifying.

Slicing, unfolding and baking: sampling and slicing, wherein the thickness of the slices is generally 5 mu m; placing the cut section selection part in warm water at 38 ℃, fishing out the section by using a glass slide after the section is fully unfolded, and adhering the section to the middle part of the glass slide as much as possible; and drying the adhered slices, placing the slices on a spreading plate, and drying the slices in a 37 ℃ thermostat overnight.

Dyeing: the dried slices were placed in sequence in xylene I, II (10 min each), 100%, 95%, 90%, 80%, 70% alcohol solutions (3min each), hematoxylin stain (15min), tap water rinse (5min), then 1% hydrochloric acid alcohol differentiation (10s), tap water rinse (5min), eosin stain (15s), 90% alcohol solution (2min), 95% alcohol solution (1.5min), 100% alcohol I, II (1.5min each), xylene I, II (10 min each).

Sealing: taking out the section from the dimethylbenzene, dripping a small amount of neutral gum in the center of the tissue slice, clamping a cover glass slide sealing sheet by using a forceps, placing the sealing sheet in a fume hood for natural drying, sticking a label, and finishing the preparation of the HE stained paraffin tissue section. The pathological changes of the tissue organs were observed and recorded under a microscope.

Observed under a microscope after HE staining. The results show that no significant tissue lesions appeared after healthy mice were injected with Tub a or the control solvent DMSO. After infection with staphylococcus aureus or escherichia coli, lung tissues of critically infected mice given DMSO are shown to be blood stasis, obvious alveolar wall thickening and massive inflammatory cell infiltration; disorder of hepatocyte cords, partial cell degeneration, necrosis, infiltration of a small amount of inflammatory cells; tubular epithelial cells swell and denature. Compared with the critically infected mice given DMSO, the Tub A intervention obviously reduces the tissue damage of the lung, the liver and the kidney of the critically infected mice (figure 2, figure 3 and figure 4).

(V) preparation of spleen lymphocyte suspension

Killing the mice by using a cervical dislocation method, disinfecting the whole skin surface of the mice by using alcohol, cutting the skin and peritoneum of the left ventral side of the mice in an ultraclean workbench, carefully picking up the spleen, washing the spleen twice by using PBS (phosphate buffer solution) with pre-cooled at 4 ℃ and containing 2% serum, grinding the spleen on a 200-mesh stainless steel screen, washing and filtering the ground spleen by using PBS containing 2% serum, collecting filtrate and transferring the filtrate to a 50mL centrifuge tube. Centrifuging at room temperature of 350 Xg for 7min, removing supernatant, adding 4mL of erythrocyte lysate, adding the lysate, immediately resuspending precipitate, slightly shaking every 1min to ensure more complete lysis, and adding 25mL of PBS containing 2% serum after 2-4 min of lysis at room temperature to stop lysis. Centrifuging at 350 Xg for 7min at room temperature, discarding the supernatant (if the lysis is incomplete, the lysis can be carried out again), washing twice with PBS containing 2% serum, and resuspending and precipitating with 4mL PBS containing 2% serum to obtain spleen lymphocyte suspension.

(VI) flow cytometry detection of NK cell proportion and IFN-gamma production level

After collecting the spleen lymphocyte suspension, counting the cells, and sucking 100. mu.L of cell suspension (2X 10)⁶Individual cells) were placed in a 1.5mL centrifuge tube, and antibodies (CD3-FITC, NK1.1-PE) against the labeled cell surface specific antigen were added, placed in the dark at 4 ℃ for 30min, washed 2 times with 4 ℃ pre-cooled PBS, resuspended in 500 μ L PBS, and the NK cell proportion was analyzed by flow cytometry after cell resuspension.

The results showed that Staphylococcus aureus induction (6.2X 10) compared to uninfected mice⁸CFU) while the number of splenic NK cells was significantly reduced in critically infected mice (DMSO administered), the number and proportion of NK cells in critically infected mice given Tub a was significantly higher than those in severely infected mice not given Tub a (DMSO administered) (fig. 5A); in the case of induction with E.coli (3.5X 10)⁷CFU) showed the same results in a severely infected mouse model (fig. 5B). The results indicate that the HDAC6 inhibitor Tub A can significantly reduce the proportion and the number of spleen NK cells of mice induced by severe infection.

Collecting spleen lymphocyte suspension, counting cells, sucking 100 mu L of cell suspension after counting, respectively inoculating the cell suspension into a 24-pore plate, respectively adding 1mL of RPMI-1640 cell culture medium, rmIL-2(100U/mL) and rmIL-12(5ng/mL) into each pore, gently mixing uniformly, then placing the mixture into a cell incubator to culture for 2h, then adding 1 mu L of monensin into each pore, continuing to culture for 4h, collecting the cells into a 1.5mL centrifuge tube, washing the cells twice by PBS, staining CD3 and NK1.1 according to the step of detecting cell surface markers by flow cytometry, washing the cells twice by PBS after the cell surface staining is finished, then adding 200 mu L of cell fixing solution into each tube, gently suspending the cells, and fixing the cells at 4 ℃ for more than 4 h. Washing twice with PBS after fixation, adding 200 μ L of penetrating fluid into each tube, resuspending the cells, permeabilizing the membrane for 7min, centrifuging at 450 Xg for 7min at room temperature, discarding the supernatant, and repeating the steps of permeabilizing the membrane once. Adding specific intracellular labeled antibody IFN-gamma antibody, and standing at normal temperature in dark place for 40 min. PBS washing 2 times, 500 u L PBS heavy suspension cells, by flow cytometry after heavy suspension of cells were detected and analyzed.

After detection analysis, the NK cell percentage of different groups of uninfected mice (Tub A inhibitor control group and DMSO solvent control group) and the expression level of NK cell IFN-gamma (positive) are not significantly different after IL-2 and IL-12 stimulation; 6.2X 10⁸CFU or 6.2X 10⁶Staphylococcus aureus infections with CFU all significantly increased IFN- γ + (positive) NK cell proportion and IFN- γ expression levels, but High dose (High dose) infection group (6.2X 10) given DMSO⁸CFU) mice had significantly lower IFN-gamma + (positive) NK cell ratios and IFN-gamma expression levels than the Low dose (Low dose) infection group given DMSO (6.2X 10)⁶CFU) mice (fig. 6A), suggesting that mild infection can significantly stimulate the production of IFN- γ by NK cells, while severe infection not only does not further enhance the ability of NK cells to produce IFN- γ, but also weakens the ability of NK cells to produce IFN- γ, inducing the formation of low reactivity of NK cells; similarly, similar results were obtained in the E.coli-infected group (FIG. 6B). At 6.2X 10⁶After infection with Staphylococcus aureus (CFU), the percentage of IFN-gamma + (positive) NK cells and the expression level of NK cells IFN-gamma after stimulation with IL-2 and IL-12 in the Tub A stem group were not significantly different from those in the Tub A non-stem (DMSO) group, but at 6.2X 10⁸After the infection of the CFU staphylococcus aureus, the percentage of IFN-gamma + (positive) NK cells and the expression level of the NK cells of the critically infected mice given Tub A intervention are obviously higher than those of critically infected mice not given Tub A (given DMSO) after the stimulation of IL-2 and IL-12 (figure 6A); similarly, similar results were obtained in the E.coli-infected group (FIG. 6B). The above results indicate that the HDAC6 inhibitor Tub a can be used to attenuate the reduction of NK cell reactivity caused by severe infections.

(VII) establishment of Severe infection mouse secondary infection model and clinical symptom and histopathological observation of grouping

Staphylococcus aureus infection control group of C57BL/6 mice, 6.2X 10 injection per mouse abdominal cavity⁸The bacterial liquid of Staphylococcus aureus of CFU was injected with DMSO of equal volume in the abdominal cavity 1h after bacterial infection, and then mice surviving after Staphylococcus aureus infection were infected with Escherichia coli (3.5X 10) with non-lethal dose⁵CFU), mice were observed after bacterial infection and recorded for clinical symptoms, survival and histopathological changes; a pretreatment group of C57BL/6 mice infected with staphylococcus aureus was injected into the abdominal cavity of each mouse at 6.2X 10⁸The bacterial infection of CFU Staphylococcus aureus was followed by intraperitoneal injection of Tub A (70mg/kg) 1h after bacterial infection, and then the surviving mice were secondarily infected with E.coli at the above dose.

C57BL/6 mice E.coli infection control group, each mouse intraperitoneal injection 3.5X 10⁷CFU E.coli solution was injected with equal volume of DMSO (1 h) in the abdominal cavity after bacterial infection, and then mice surviving E.coli infection were infected with Staphylococcus aureus (6.2X 10) in a secondary non-lethal dose⁶CFU), mice were observed after bacterial infection and recorded for clinical symptoms, survival and histopathological changes; c57BL/6 mice E.coli infection intervention group, each mouse intraperitoneal injection 3.5X 10⁷The Escherichia coli solution of CFU was subjected to intraperitoneal injection of Tub A (70mg/kg) 1h after bacterial infection, and then the surviving mice were subjected to secondary infection with the above-mentioned dose of Staphylococcus aureus.

The results show that the survival rate of mice given Tub a after the first (1st) infection after the second (2nd) infection is significantly higher than that of mice in the control group given DMSO after the first infection (fig. 7A, fig. 7B); likewise, the clinical score of mice given Tub a after the first (1st) infection after the second (2nd) infection was also significantly lower than that of control mice given DMSO after the first infection (fig. 7C, fig. 7D). The above results indicate that HDAC6 inhibitors can significantly improve the ability of surviving mice to resist secondary infection after severe infection.

The results also show that mice given Tub a after the first (1st) infection had significantly less lung, liver and kidney damage after the second (2nd) infection than control mice given DMSO after the first infection (fig. 8, fig. 9). The results suggest that pathological damage of lung, liver and kidney of mice after secondary infection is further aggravated, and the pathological damage of lung, liver and kidney of mice after secondary infection can be relieved by the HDAC6 inhibitor Tub A.

In a word, the results obtained by experiments show that the application of the HDAC6 inhibitor can remarkably recover the number and the reactivity of spleen NK cells of a severely infected mouse, reduce the clinical symptom score and the histopathological injury of lung, liver and kidney of the severely infected mouse, and remarkably enhance the capability of the severely infected mouse in resisting secondary infection.

Claims

The application of the HDAC6 inhibitor in preparing a medicine for preventing and treating severe infection.
2. Use according to claim 1, characterized in that: the pathogens of severe infections are of bacterial origin.
3. Use according to claim 1, characterized in that: the pathogen of severe infection is Staphylococcus aureus or Escherichia coli.
4. Use according to claim 1, characterized in that: the HDAC6 inhibitor is Tubastatin A or one of pharmaceutically acceptable salts thereof.
5. Use according to claim 1, characterized in that: the HDAC6 inhibitor can increase the number and proportion of NK cells of a severe infection organism.
6. Use according to claim 1, characterized in that: the HDAC6 inhibitor increases NK cell reactivity in critically infected organisms.
7. Use according to claim 1, characterized in that: the HDAC6 inhibitors increase severe infection survival.
8. Use according to claim 1, characterized in that: the HDAC6 inhibitor enhances the anti-infective ability of critically infected organisms.
Use of an HDAC6 inhibitor for the preparation of an immunomodulatory formulation.
10. Use according to claim 9, characterized in that: the HDAC6 inhibitors attenuate decreases in NK cell number, proportion and reactivity.