Methods of treating cytokine storm associated with acinetobacter baumannii infection

Abstract

The present application relates to methods of treating cytokine storms associated with Acinetobacter baumannii infection. Provided is a method for treating cytokine storm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a therapeutic agent selected from the group consisting of (+)-(S)-2-(6-methoxynaphthalen-2-yl) propionic acid, acetylsalicylic acid, dexamethasone, azathiopurine, and pharmaceutically acceptable salts thereof, wherein the cytokine storm is associated with an Acinetobacter baumannii infection in the subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 551,622, filed on February 9, 2024, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing

[0004] The sequence listing identified as Sequence_Listing_P25718US00.xml is incorporated herein by reference in its entirety, has a size of 30.5 kilobytes, and was created on January 24, 2024. Technical Field

[0005] The present disclosure provides methods of treating a cytokine storm associated with an Acinetobacter baumannii infection in a subject in need thereof. Background Art

[0006] Acinetobacter baumannii (Ab) is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals. In recent years, nosocomial infections caused by Ab have become a global health problem due to the emergence of multidrug-resistant (MDR) strains. 1,2 This pathogen can cause infections in multiple body sites, such as the skin, soft tissues, and urinary tract. More severe infections, including ventilator-associated pneumonia and sepsis, are associated with an unacceptably high mortality rate. 3,4 The challenge of treating clinical Ab infections is further exacerbated by the continuous evolution of drug-resistant phenotypes in these organisms. The lack of active antimicrobial drugs has prompted the development of new therapies to control these infections. Furthermore, the nature of the interaction between Abs and the human host is currently only superficially understood. Therefore, a comprehensive study of Ab-induced immune responses in humans will help identify potential targets for the development of new treatments for Ab infections.

[0007] Current data suggest that sepsis caused by Ab infection is due to dysregulation of the innate immune system, the function of which is mediated by pattern recognition receptors (PRRs). Therefore, it is necessary to maintain a delicate balance between inflammatory and anti-inflammatory responses. 5,6 Several studies have shown that pro-inflammatory cytokines are beneficial for the clearance of Abs and infection control. 7,8 However, excessive inflammatory responses can lead to systemic inflammatory response syndrome, organ damage, and even death. 9 Toll-like receptors (TLRs) and other PRRs are crucial for initiating immune responses but are also responsible for regulating the magnitude of inflammatory responses by recognizing specific pathogen-associated molecular patterns (PAMPs) by Abs. 10,11Therefore, a thorough understanding of Ab-host interactions and the mechanisms regulating cytokine production and inflammatory responses during Ab infection is essential. Likewise, improved methods for treating Ab-associated cytokine storms in subjects in need thereof are therefore needed. Summary of the Invention

[0008] As disclosed herein, alveolar and interstitial macrophages have been found to be key components of the host immune system that regulate changes in the Ab-induced inflammatory microenvironment. These macrophages can polarize to the M1 phenotype and cause a cytokine storm, which often leads to host death. In addition, we found that the M1 polarization events triggered by Ab are mainly mediated by the TLR2 / MyD88 / NF-κB signaling pathway, which can even lead to excessive production of cytokines. Importantly, studies have shown that the drugs naproxen (NPXS), acetylsalicylic acid (ASA), dexamethasone (DXMS) and azathioprine (AzA) can effectively inhibit the excessive inflammatory response of infected mice by inhibiting the TLR2 / MyD88 / NF-κB signaling pathway, thereby protecting infected mice from death. These findings suggest that the use of immunosuppressive drugs (NPXS) is a promising therapeutic strategy for managing acute Ab infection.

[0009] In a first aspect, provided herein is a method of treating a cytokine storm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a therapeutic agent selected from the group consisting of (+)-(S)-2-(6-methoxynaphthalen-2-yl)propionic acid (naproxen), acetylsalicylic acid (ASA), dexamethasone (DXMS), azathioprine (AzA), and pharmaceutically acceptable salts thereof, wherein the cytokine storm is associated with an Acinetobacter baumannii infection in the subject.

[0010] In certain embodiments, the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof.

[0011] In certain embodiments, the Acinetobacter baumannii infection activates the TLR2 / MyD88 / NF-κB signaling pathway in the subject.

[0012] In certain embodiments, the subject overexpresses one or more proinflammatory factors selected from the group consisting of IL-1β, IL-6, IL-10, IL-12, IL17a, IL23, IL-27, IFN-γ, and TNF-α.

[0013] In certain embodiments, the infection with Acinetobacter baumannii activates the TLR2 / MyD88 / NF-κB signaling pathway in the subject; the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof; and administration of naproxen results in a decrease in the relative expression of genes associated with the TLR2 / MyD88 / NF-κB signaling pathway.

[0014] In certain embodiments, the TLR2 / MyD88 / NF-κB signaling pathway-related genes are selected from the group consisting of toll-like receptor 2 (TLR2), primary response to myeloid differentiation 88 (Myd88), nuclear factor κB subunit 1 (Nfkb1), nuclear factor κB subunit 2 (Nfkb2), interleukin 1β (Il1b), interleukin 6 (Il6) and tumor necrosis factor (Tnf).

[0015] In certain embodiments, the A. baumannii infection is caused by antibiotic-resistant A. baumannii.

[0016] In certain embodiments, the antibiotic-resistant Acinetobacter baumannii is resistant to one or more antibacterial agents selected from the group consisting of aminoglycosides, fluoroquinolones, and carbapenems.

[0017] In certain embodiments, the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof, and the A. baumannii infection is caused by antibiotic-resistant A. baumannii.

[0018] In certain embodiments, the Acinetobacter baumannii infection is caused by an Acinetobacter baumannii strain selected from the group consisting of ATCC 17978, ATCC 19606, AB5075, ATCC 9955, ATCC 17904, R 477, and R 0211019.

[0019] In certain embodiments, the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof; and the Acinetobacter baumannii infection is caused by an Acinetobacter baumannii strain selected from the group consisting of ATCC 17978, ATCC 19606, AB5075, ATCC 9955, ATCC 17904, R 477, and R0211019.

[0020] In certain embodiments, the A. baumannii infection is present in one or more of a wound, a surgical site, a catheter site, the blood, the urinary tract, the skin, the lungs, or the respiratory tract.

[0021] In certain embodiments, the method further comprises diagnosing the subject with an Acinetobacter baumannii infection prior to administering the therapeutically effective amount of naproxen.

[0022] In certain embodiments, the method further comprises co-administering to the subject a therapeutically effective amount of an antibacterial agent.

[0023] In certain embodiments, the antibacterial agent is selected from the group consisting of meropenem, colistin, polymyxin B, sulbactam, piperacillin / tazobactam, minocycline, tigecycline, and aminoglycosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other objects and features of the present disclosure will become apparent from the following description of the present disclosure when taken in conjunction with the accompanying drawings.

[0025] Figure 1 Evaluation of Ab virulence levels in a mouse sepsis model. (a) Kaplan-Meier survival curves of mice infected with Ab strains (n = 5 per group). (b) Relative weight loss of Ab-infected mice at 12 hpi. (c) Percentage of spleen weight and representative images of spleens in mice after Ab inoculation. (d) Bacterial burden in lung, spleen, kidney, liver, and blood samples from mice at 12 hpi. (e) Survival of intracellular bacteria obtained from lung and spleen samples of infected mice at 12 hpi. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, significant.

[0026] Figure 2 Ab-induced cytokine storm in the host. (a) Percentage of F4 / 80+ macrophages and Ly6G+ neutrophils in lung cells gated on total CD45+ cells, analyzed by flow cytometry. (b) Quantification of total F4 / 80+ and Ly6G+ cells, as well as expression of CD86 and CD206 on the surface of F4 / 80+ macrophages, expressed as mean fluorescence intensity (MFI) values in lung cells. (c) Percentage of CD3+ T cells, CD19+ B cells, and NK1.1+ NK cells gated on total CD45+ cells. (d) Quantification of total CD3+, CD19+, NK1.1+ cells, and lymphocytes. (e) KEGG enrichment analysis delineates pathways most affected by Ab infection. (f and g) Heatmaps showing differential expression patterns underlying M1 polarization (f) and chemokine production (g) in healthy and Ab-infected mice. (h) Transcription levels of proinflammatory cytokines (top) and chemokines (bottom) in different mouse groups. (i) Expression levels of IL-1b, IL-6, and TNF-α in the serum of Ab-infected and uninfected mice. **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0027] Figure 3Molecular cell atlas and intercellular signaling networks of lung cells depicted based on single-cell RNA sequencing data. (a) UMAP projection of cells from the lungs of healthy mice and ATCC 17978-infected mice showing major clusters and their respective cell type assignments. Each dot corresponds to an individual cell. Different colors represent different cell clusters. (b) Cells of origin with the same inset as in (a). Different colors depict infected and uninfected lung cells. (c) Total RNA expression of Plet1 and Csf1r in the same inset as in (a). (d) Detection frequency of the different cell types shown in (a). (e) Differences in overall information flow in the interaction network between healthy and Ab-infected mice. Signaling pathways labeled in red are more enriched in infected individuals, while those labeled in black are equally enriched in both groups; signaling pathways labeled in blue are more enriched in healthy individuals. (f and g) Circular plots show the inferred intercellular communication networks for the TNF signaling pathway (f) and the TGFb signaling pathway (g) in healthy mice (left) and ATCC 17978-infected mice (right). Circle size is proportional to the number of cells in each group, edge width represents the probability of communication, and edge color corresponds to the source of signaling.

[0028] Figure 4 Ab mediates the onset of the cytokine storm by inducing M1 polarization. (a) UMAP of AM and corresponding group assignments. (b and c) Pseudo-time trajectories of total AM cells stained by pseudo-time (b) and different samples (c). (d) Cxcl2 expression in healthy and infected mice in the same inset as (a). (e) Percentages of Cxcl2+ and CD36+ cells in AM. (f) UMAP of IM with cell type assignments. (g and h) Pseudo-time trajectories of total IM cells stained by pseudo-time (g) and different samples (h). (i) Il1b expression in healthy and infected mice in the same inset as (f). (j) Percentages of Tnf+ and Gadd45g+ cells are shown in IM. (k and l) KEGG enrichment analysis showing the most affected pathways in AM (k) and IM (l) after Ab infection.

[0029] Figure 5Blocking the TLR2 / Myd88 / NF-κB pathway attenuates M1 polarization and cytokine storm induced by Ab infection. (a) Heatmap depicting the differential expression profiles of toll-like receptors (TLRs) and the TLR2 / Myd88 / NF-κB pathway in the AM and IM of healthy and Ab-infected mice. (b) Relative transcript levels of genes involved in the TLR2 / Myd88 / NF-κB pathway in different mouse groups. (c) TLR2 MFI on the surface of F4 / 80+ macrophages in the lungs of infected mice. (d) Pseudo-time traces of total cells colored by expression levels of Tlr2, Myd88, Nfkb1, Nfkb2, Il1b, Il6, and Tnf in the IM and AM. (e and f) Surface CD86 (e) and CD206 (f) MFI on RAW264.7 cells pretreated with medium, C29, TJ-M2010-5, or JSH-23 and then infected with ATCC 17978. (g and h) TNF-α (g) and IL-6 (h) levels in supernatants of Ab-infected RAW264.7 cells pretreated with medium, C29, TJ-M2010-5, or JSH-23. (i) Kaplan-Meier survival curves of WT and TLR2- / - mice infected with ATCC 17978. (j) Percentage of F4 / 80+ macrophages gated on total CD45+ cells determined by flow cytometry. (k) Quantification of total F4 / 80+ cells analyzed in (j). (l) Surface CD86 expression on F4 / 80+ macrophages in the lung. (m and n) Relative transcript levels of proinflammatory cytokines (m) and chemokines (n) in different mouse groups. (o) Serum expression levels of IL-1b, IL-6, and TNF-α in healthy mice, infected WT mice, and infected TLR2- / - mice. (p) Tlr2 mRNA expression in RAW264.7 cells incubated with strain ATCC17978 and ΔOmpA (MOI 5) for 3 hours. (q, r, and s) Adhesion (q), invasion (r), and intracellular survival (s) of strains ATCC17978 and ΔOmpA in macrophages. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

[0030] Figures 6a-6j NPXS inhibits the cytokine storm caused by Ab infection. ( Figure 6a ) Kaplan-Meier survival curves of Ab-infected mice treated with immunosuppressive drugs. ( Figure 6b ) Relative weight loss at 12 hpi in healthy mice, Ab-infected mice, and Ab-infected mice treated with NPXS. Figure 6c) Spleen weight percentage and representative images of healthy mice, mice infected with ATCC 17978, and mice infected with ATCC 17978 + NPXS treatment. ( Figure 6d ) Percentage of F4 / 80+ macrophages gated on total CD45+ cells as determined by flow cytometry. ( Figure 6e )( Figure 6d ) and quantification of total F4 / 80+ cells analyzed in the lungs and surface CD86 expression on F4 / 80+ macrophages. Figure 6f ) Heat map showing the differential expression profiles of pro-inflammatory marker genes in control mice, mice infected with ATCC 17978, and mice infected with ATCC 17978 + NPXS. ( Figure 6g ) mRNA expression of M1 markers and proinflammatory cytokines in the lungs of mice receiving different treatments. ( Figure 6h ) Quantification of TNF-α, IL-1b and IL-6 production in serum analyzed by ELISA. ( Figure 6i ) Heat map of the TLR2 / Myd88 / NF-κB signaling pathway in the three groups of mice. ( Figure 6j ) Relative transcription levels of genes related to the TLR2 / Myd88 / NF-κB signaling pathway. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

[0031] Figure 7 Differential gene expression analysis of lung cells from healthy and infected mice by RNA-seq. (a) Volcano plot of differentially expressed genes. Heat maps of DEGs for interleukins (b), TNF family clusters (c), and neutrophil activation markers (d).

[0032] Figure 8 Differential gene expression analysis of Ly6G+ cells from healthy and Ab-infected mice. Heatmap depicts relative gene expression levels of TLR (a), TLR2 / Myd88 / NF-κB pathway genes (b), and neutrophil activation markers (c). (d) KEGG enrichment analysis shows the pathways most affected after Ab infection.

[0033] Figure 9 Deletion of the ompA gene in ATCC 17978. (A) Colony PCR results show that only the scar sequence remains at the locus in the mutant strain. Lane CK represents the PCR band of the wild-type ATCC 17978 strain as a control, while lanes 1, 2, 4, 5, 6, and 7 represent mutants in which the ompA gene has been successfully deleted. (B) Sequencing results of the mutation sites in ATCC 17978 and ΔompA.

[0034] Figure 10 . A table showing the strains and plasmids used in this study is depicted.

[0035] Figure 11 . A table showing the primers and ssDNA used in this study is depicted. DETAILED DESCRIPTION

[0036] definition

[0037] Throughout this disclosure, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of the specified integer or group of integers but not the exclusion of any other integer or group of integers. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprises", "comprises", "comprising", etc. may have the meanings ascribed to them in U.S. patent law; for example, they may mean "includes", "included", "including", etc.; and terms such as "consisting essentially of" have the meanings ascribed to them in U.S. patent law, for example, they allow for the presence of elements not expressly recited, but exclude elements present in the prior art or elements that affect the basic or novel characteristics of the invention.

[0038] Furthermore, throughout this disclosure and claims, unless the context requires otherwise, the word "include" or variations such as "includes" or "including", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0039] Unless otherwise expressly stated, the singular as used herein includes the plural (and vice versa). In addition, if the term "about" is used before a quantitative value, the present teachings also include the specific quantitative value itself, unless otherwise specifically stated. As used herein, unless otherwise stated or inferred, the term "about" refers to a variation of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% of the nominal value.

[0040] As used herein, the term "therapeutically effective amount" refers to that amount of a compound or therapeutic agent that elicits the biological and / or pharmaceutical response that is being sought by the researcher, veterinarian, clinician, or physician, including alleviation of the symptoms of the disease, condition, or disorder being treated, in a cell culture, tissue system, subject, animal, or human.

[0041] As used herein, the terms "treat" and "treat" and the like refer to the alleviation or improvement of a condition / disease and / or symptoms associated therewith. It should be understood that, although it cannot be excluded, the treatment of a disorder or condition does not require the complete elimination of the disorder, condition or symptoms associated therewith. In certain embodiments, treatment includes the prevention of a disorder or condition and / or symptoms associated therewith. The terms "prevention" or "prevent" as used herein refer to any action that inhibits or at least delays the development of a condition, disorder or symptoms associated therewith. Prevention can include primary, secondary and tertiary prevention, wherein: a) primary prevention avoids the development of the disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing the chances of interventions to prevent the progression of the disease and the onset of symptoms; and c) tertiary prevention reduces the negative effects of existing diseases by restoring function and reducing complications associated with the disease.

[0042] The terms "co-administration" and "co-administration" refer to both simultaneous administration (two or more therapeutic agents administered at the same time) and non-simultaneous administration (one or more additional therapeutic agents administered at a different time than one or more therapeutic agents), so long as the therapeutic agents are present in the patient's body at some point at the same time.

[0043] As used herein, the term "subject" refers to any animal (eg, mammal), including but not limited to humans, non-human primates, canines, felines, and rodents.

[0044] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, Berg et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1-4alkyl) 4 salts. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions (e.g., halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates) where appropriate. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In certain embodiments, pharmaceutically acceptable base addition salts are selected from ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts.

[0045] The present disclosure provides a method for treating cytokine storm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a therapeutic agent selected from the group consisting of naproxen, ASA, DXMS, AzA, and pharmaceutically acceptable salts thereof, wherein the cytokine storm is associated with an Acinetobacter baumannii infection in the subject. In certain embodiments, the therapeutic agent is naproxen.

[0046] In certain embodiments, infection with Acinetobacter baumannii activates the TLR2 / MyD88 / NF-κB signaling pathway in the subject, which can increase TLR2, MyD88, and NF-κB mRNA and / or protein in the subject.

[0047] In certain embodiments, infection with Acinetobacter baumannii results in overexpression of one or more proinflammatory factors selected from the group consisting of IL-1β, IL-6, IL-10, IL-12, IL17a, IL23, IL-27, IFN-γ, and TNF-α.

[0048] Administration of a therapeutically effective amount of naproxen to a subject can result in a decrease in the relative expression of genes related to the TLR2 / MyD88 / NF-κB signaling pathway, such as toll-like receptor 2 (TLR2), primary response to myeloid differentiation 88 (Myd88), nuclear factor κB subunit 1 (Nfkb1), nuclear factor κB subunit 2 (Nfkb2), interleukin 1β (Il1b), interleukin 6 (Il6), and tumor necrosis factor (Tnf), which may lead to a decrease in TLR2, Myd88, Nfkb1, Nfkb2, Il1b, Il6, and Tnf mRNA and protein.

[0049] Acinetobacter baumannii infections can be skin infections (including but not limited to skin ulcers, bedsores, diabetic foot ulcers, etc.), infections in and around wounds, postoperative infections, infections associated with catheters, surgical drains, other medical / diagnostic tools and medical procedures, bloodstream infections, respiratory tract infections, cerebrospinal fluid infections, peritoneal fluid infections and urinary tract infections.

[0050] In certain embodiments, the Acinetobacter baumannii infection is caused by a strain selected from the group consisting of AB001, AB002, AB0057, AB967, AB2828, AB3340, AB3560, AB3638, AB3785, AB3806, AB3917, AB3927, AB4025, AB4026, AB4027, AB4052, AB4269, AB4448, AB4456, AB4490, AB4498, AB4795, AB4857, AB4878, AB4932, AB4957, AB4991, AB5001, AB5075, AB5197, AB5256, AB5674, AB5711, ATCC 9955, ATCC 17904, ATCC 17978, ATCC 19606, R In some embodiments, the infection is caused by an Acinetobacter baumannii strain selected from the group consisting of ATCC 17978, ATCC 19606, and AB5075.

[0051] In certain embodiments, the Acinetobacter baumannii infection is caused by antibiotic-resistant Acinetobacter baumannii. In certain embodiments, the antibiotic-resistant Acinetobacter baumannii is resistant to one or more antibiotics selected from the group consisting of colistin, erythromycin, ampicillin, vancomycin, linezolid, methicillin, oxacillin, cefotaxime, rifampicin, amikacin, gentamicin, kanamycin, tobramycin, neomycin, ertapenem, doripenem, imipenem, meropenem, ceftazidime, cefepime, ceftaroline, ceftobiprole, aztreonam, piperacillin, polymyxin B, ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, piperacillin, minocycline, tigecycline, cotrimoxa, and derivatives thereof. In certain embodiments, the Acinetobacter baumannii is a multidrug-resistant Acinetobacter baumannii that is resistant to aminoglycosides, fluoroquinolones, and carbapenems.

[0052] In certain embodiments, methods described herein further include testing the Acinetobacter baumannii infection of a subject or diagnosing the Acinetobacter baumannii infection of a subject. In certain embodiments, the step of diagnosing a subject suffering from Acinetobacter baumannii infection includes providing a sample from the subject, carrying out bacterial culture to the sample, identifying the bacterium in the bacterial culture, and diagnosing the subject suffering from Acinetobacter baumannii infection based on the bacterial diagnosis of the identification. Sample can include or can be derived from blood, feces, urine, mucus, spinal fluid or other body fluid samples obtained from the subject.

[0053] The methods described herein can be used in combination with standard of care for treating Acinetobacter baumannii infections. Thus, in certain embodiments, the methods described herein further comprise co-administering to the subject a therapeutically effective amount of an antibacterial agent.

[0054] Any antibacterial agent that can be used to treat Acinetobacter baumannii infection can be used. The selection of a suitable antibacterial agent is well within the skill of those of ordinary skill in the art. Exemplary antibacterial agents include, but are not limited to, carbapenems, such as meropenem, colistin (colistin), polymyxin B, sulbactam, piperacillin / tazobactam, minocycline, tigecycline, and aminoglycosides.

[0055] result

[0056] Establishment of mouse Ab infection sepsis model

[0057] To investigate the lethality and pathogenesis of Ab, a mouse sepsis infection model was established. Briefly, 7.5 × 10 7 After infection with CFU ATCC 17978 strain, all mice died within 36 hours ( Figure 1 , a). At 12 hpi, poor health, weight loss, and spleen enlargement were observed in each mouse ( Figure 1 , b and c). At 12 hpi, lung, spleen, liver, kidney and blood samples were collected from infected mice; bacterial loads in these samples were counted and found to be as high as 10 7 -10 9 CFU / g( Figure 1 , d). To assess the intracellular survival of Ab, cells obtained from lung and spleen samples were incubated with 300 μg / mL gentamicin to eliminate extracellular bacteria. A large number of ATCC 17978 strains were found to remain viable in phagocytes at 12 hpi; the total bacterial load was found to be higher than the intracellular population, indicating that this strain can survive both intracellular and extracellular environments ( Figure 1 , e), and the ATCC 17978 strain in a mouse sepsis model could not be effectively cleared by the immune system.

[0058] Ab infection-induced immune response fluctuations and the occurrence of cytokine storm in mice

[0059] Given the ability of ATCC 17978 to persist both intracellularly and extracellularly, we hypothesized that the symptoms of infection were induced by an overactive immune response triggered by persistent stimulation of the host immune system by Ab. To evaluate the systemic immune response induced by Ab, we analyzed the changes in the number of immune cells in the lungs and spleens of Ab-infected mice by flow cytometry. We observed that macrophages (CD11b + f / 4 / 80 + ) and neutrophils (CD11b + Ly6G + ) infiltration was enhanced, but lymphocyte populations (including T cells (CD19 - CD3 + ), B cells (CD3 - CD19 + ) and natural killer (NK) cells (CD3 - NK1.1 + ))'s number is reduced ( Figure 2 , a to d). Rapid recruitment and activation of neutrophils and macrophages is observed in the early stages of Ab infection. Macrophages play a crucial role in phagocytosis of Ab and release of cytokines that recruit neutrophils to control infection. 12 Previous studies have shown that neutrophils are essential for Ab infection control, and therefore neutrophil-depleted mice have a dramatic increase in mortality and infection severity. 13-15 Furthermore, lymphopenia is a predictor of systemic inflammatory response to bacterial infection, characterized by a decrease in T cells, B cells, and NK cells in the lungs of Ab-infected mice, where decreased CD206 MFI levels and CD11b + f / 4 / 80 + Increased CD86 MFI levels on the cell surface indicate that these cells are polarized to a pro-inflammatory M1 phenotype ( Figure 2 , b).

[0060] We then used RNA-seq to investigate the differences in gene transcription levels between cells collected from healthy and infected mice. We found that a total of 3,212 genes were significantly upregulated and 3,514 genes were downregulated after Ab infection ( Figure 7 , a). KEGG enrichment analysis showed that metabolic pathways that were highly responsive to infection were those involved in the expression of cytokines and chemokines in the innate immune system (marked in red) ( Figure 2 , e). In addition, pro-inflammatory (M1 polarization) markers ( Figure 2 , f), chemokines ( Figure 2, g), interleukins, TNF family and neutrophil activation markers ( Figure 7 , b to d) were depicted by constructing a heat map. Several differentially expressed pro-inflammatory marker genes were selected and their expression levels were verified by RT-qPCR ( Figure 2 , h). It was found that the levels of cytokines (TNF-α, IL-1b, and IL-6) in mouse serum increased more than 100-fold at 12 hpi ( Figure 2 , i). These findings are consistent with observations of pro-inflammatory M1 macrophage polarization. In summary, our data indicate that Ab infection systemically triggers significant macrophage and neutrophil recruitment, lymphopenia, and dysregulated cytokine production and an exaggerated inflammatory response.

[0061] scRNA-seq reveals the functional role of macrophages in mediating excessive inflammatory responses during Ab infection

[0062] Given the changes in the structure of immune cell populations and the initiation of strong inflammatory responses during Ab infection, a comprehensive analysis of the excessive immune response induced by Ab was performed by scRNA-seq. By using a unified single-cell analysis pipeline diagram, a cell atlas was generated, and the gene expression profiles of 20,774 cells were obtained, of which 11,046 were from healthy mice and 9,728 were from ATCC 17978-infected mice. The data were presented using a dimensionality reduction method involving uniform manifold approximation and projection (UMAP). Data for 14 major cell clusters are shown ( Figure 3 , a to c). ATCC 17978 infection was found to result in an increase in the total number of macrophages, accompanied by a decrease in the frequencies of detection of various lymphocyte compartments (T cells, B cells, and NK cells); thus, these findings are consistent with the flow cytometry data ( Figure 3 , d). The lower-than-expected number of neutrophils may be due to their susceptibility to degradation and lower RNA expression levels compared to other cells, which may lead to underestimation of neutrophil numbers in 10× Genomics scRNA-seq. 16-18 To complement the scRNA-seq data, Ly6G + Neutrophil differential expression analysis was performed ( Figure 8 , a to d).

[0063] To further understand the nature of the interactions between different cell clusters, Cellchat 19Intercellular communication analysis was performed. A global communication atlas of lung cells collected from infected and uninfected mice found that after Ab infection, the inferred number and intensity of interactions between multiple immune cells and between immune cells and non-immune cells increased overall. The information flow of each signaling pathway was determined to describe the global immune response spectrum. After Ab infection, we observed a large enrichment of multiple signaling pathways, most of which were associated with inflammatory responses. It is worth noting that the information flow of signaling pathways associated with pro-inflammatory responses was turned on (IFN-II, CD37) or enhanced (TNF, VISFATIN), while the information flow of anti-pro-inflammatory signaling pathways was reduced (TGFb)( Figure 3 ,e). These data suggest that the inflammatory microenvironment may have an important impact on the progression of Ab-induced sepsis. Based on this finding, we investigated specific cellular components that are critical for the relevant pathways. In the TNF signaling pathway, the overall signal intensity of cell interactions was increased in Ab-infected individuals compared with healthy individuals, particularly in alveolar macrophages (AM), interstitial macrophages (IM), and neutrophils, where significantly enhanced paracrine and autocrine signaling was observed ( Figure 3 ,f). In the TGFβ signaling pathway, the interaction of both macrophages with other immune cells was strongly weakened, and autocrine signaling was completely lost in IM after Ab infection, while the interaction of natural killer cells and neutrophils with other cells was enhanced ( Figure 3 ,g). When interacting with other cells, neutrophils, IM, and AM showed significant changes in signal intensity in inflammatory signaling pathways; in particular, the signal intensity of IM and AM showed a consistent pro-inflammatory trend after Ab infection. 16-18 Although the number of neutrophils that can be detected in the lungs is limited, alveolar and interstitial macrophages are considered to be the key cellular components mediating Ab-induced hyperinflammatory responses.

[0064] Ab infection induces a cytokine storm in mice by regulating macrophage polarization

[0065] Since IM and AM are likely to be the main targets of Ab during the infection process, we further re-clustered these two types of macrophages and subsequently constructed a cell lineage of differentiation trajectories to determine whether the M1 polarization pattern and timing could be observed after Ab infection. + M2 macrophages and Ear2 in IM + As time progresses, a gradual evolution of M2 macrophages towards Cxcl2- or Tnf-labeled M1 macrophages can be observed in both populations. This evolutionary process leads to an increase in M1-like macrophages and a decrease in M2-like macrophages in the AM of Ab-infected mice compared to healthy mice ( Figure 4 ,a, b, c and e). After Ab infection, the number of M1 and M2 macrophages in the IM subcluster also increased to a similar extent ( Figure 4 ,f,g,h, and j). Therefore, the results of pseudo-time trajectory analysis indicate that there is a transition from the M2 polarization state to the M1 polarization state in IM and AM, and this transition becomes more obvious in Ab-infected animals. This conclusion is also supported by the increase in CD86 MFI and the decrease in CD206 MFI on the surface of macrophages after Ab infection as shown by flow cytometry analysis ( Figure 2 ,b).

[0066] Consistently, AM and IM from Ab-infected individuals often exhibited M1 polarization and expression of pro-inflammatory marker genes (Cxcl2, Cxcl10, Tnf, Il1b, Ptgs2, Csf3, Figure 4 , d and i and Figure 8 , b and c) expression levels were enhanced. Enrichment analysis showed that the vast majority of the top 20 significantly activated KEGG pathways in AM and IM were involved in cytokine production and robust inflammatory response ( Figure 4 , k and l). In summary, single-cell sequencing data further confirmed that Ab infection promotes the polarization of macrophages to the M1 phenotype and leads to the formation of a cytokine storm.

[0067] Activation of the TLR2 / MyD88 / NF-κB signaling pathway promotes Ab-induced M1 macrophage polarization and triggers the occurrence of cytokine storm

[0068] M1 polarization occurs in an inflammatory environment dominated by TLRs, which are usually associated with activating immune responses to fight bacterial infections. 20 Analysis of macrophage enhancer activity indicated that nuclear factor-κB (NF-κB) appears to be a key transcription factor involved in M1 polarization. 21 Therefore, RNA-seq and qPCR were performed to analyze the differential transcriptional levels of TLRs shared by humans and mice; the results showed that Tlr2 expression was significantly upregulated in response to Ab infection. In addition, increased transcript abundance of genes related to the TLR2 / Myd88 / NF-κB signaling pathway and expression of downstream proinflammatory cytokines were found in the lungs of Ab-infected mice ( Figure 5 , a and b). When measured by flow cytometry, Tlr2 was found to be highly expressed on the surface of ATCC 17978-infected macrophages ( Figure 5 ,c). In addition, the transcription levels of the above genes and pro-inflammatory marker genes were significantly enhanced, leading to the polarization of M2 macrophages toward the M1 phenotype, indicating that the expression of these genes was also elevated in IM and AM during Ab infection ( Figure 5 ,d and Figure 6c and6d These findings led us to hypothesize that the TLR2 / Myd88 / NF-κB signaling pathway is involved in Ab-mediated M1 polarization, which in turn leads to the development of cytokine storm. To test this hypothesis, we used a TLR2 inhibitor (C29) 22 , Myd88 inhibitor (TJ-M2010-5) 23 or NF-κB inhibitor (JSH-23) 24 After blocking the TLR2 / Myd88 / NF-κB pathway, RAW264.7 cells were infected with ATCC 17978. Compared with cells not treated with the inhibitor, we observed no difference in CD206 MFI levels, but a decrease in CD86 MFI levels ( Figure 5 , e and f), indicating that the TLR2 / Myd88 / NF-κB pathway is involved in Ab-induced M1 polarization. IL-6 and TNF-α levels were also significantly reduced, with a more than 5-fold decrease observed in the supernatant of RAW264.7 ( Figure 5 , g and h). To confirm that the cytokine storm of Ab in vivo is mediated by TLR2, TLR2 knockout mice (KO) and WT mice were infected with ATCC17978. The results showed that when infected with Ab, TLR2 - / - The mortality rate of mice was reduced by 40% ( Figure 5 ,i). In addition, we observed that TLR2 - / - Decreased macrophage infiltration in mice ( Figure 5 , j and k), M1 polarization weakens ( Figure 5 ,l), decreased expression of cytokines and chemokines ( Figure 5 , m to o), indicating that Ab-induced cytokine storm is TLR2-dependent. - / - The surviving and dying groups of mice followed two distinct trajectories that correlated with the outcome of the infection: infected mice with significantly reduced cytokine production remained relatively healthy, but significant levels of cytokine production were observed in diseased mice. Taken together, these findings further support the theory that Ab infection can activate the TLR2 / MyD88 / NF-κB signaling pathway, which is a key mediator of Ab-induced M1 macrophage polarization and, therefore, a strong trigger of cytokine production.

[0069] Previous studies have identified outer membrane protein A (OmpA) as a potential pathogen-associated molecular pattern (PAMP) recognized by TLR2 in Abs 25 To investigate the contribution of OmpA in the development of Ab-induced immune responses, we examined its effect on Tlr2 mRNA expression and found that the transcript abundance of Tlr2 was reduced in strain ΔOmpA ( Figure 5,j). In the presence of C29, we observed decreased adhesion and invasiveness, leading to reduced survival of ATCC 17978 in macrophages. Notably, these effects were more pronounced when TLR2-inhibited macrophages were infected with strain ΔOmpA ( Figure 5 , k to m). These observations suggest that OmpA activates TLR2 on the surface of macrophages, promoting cell adhesion and invasion of Ab.

[0070] NPXS rescues Ab-infected mice by attenuating cytokine storm

[0071] In order to confirm that there is a high correlation between the occurrence of cytokine storm and the mortality rate of mice after Ab infection, immunosuppressive drugs were administered intraperitoneally to mice to weaken the cytokine production of infected mice and determine whether the mortality rate could be reduced. Another purpose of this experiment was to test whether a new method for treating Ab infection could be developed by inhibiting the strong inflammatory response caused by Ab. The therapeutic drugs used in this study included naproxen (NPXS), acetylsalicylic acid (aspirin, ASA), dexamethasone (DXMS), azathioprine (AzA) and cyclosporine A (CsA). Except for CsA treatment, which did not improve the survival of ATCC17978-infected mice, the administration of other immunosuppressants prolonged their survival. The survival rates of mice infected with ATCC17978 after treatment with ASA, AzA and DXMS were 60%, 40% and 40% at 24 hours, 36 hours and 48 hours, respectively, while NPXS protected 100% of the infected mice from death within 120 hours, indicating that it has superior effectiveness compared with other treatment methods ( Figure 6a In addition, after NPXS treatment, it was found that the degree of weight loss decreased from 6.7% to 3.6% on average, and the degree of splenomegaly decreased from 0.48% to 0.38% on average ( Figure 6b and 6c Although NPXS treatment did not lead to the + f / 4 / 80 + Macrophage infiltration was reduced, but it effectively attenuated the CD86 MFI level, thereby inhibiting the polarization of macrophages to the M1 phenotype ( Figure 6d and 6e Consistently, the transcript levels of most pro-inflammatory genes were significantly decreased in Ab-infected mice after NPXS treatment ( Figure 6f and 6g ). It was also found that NPXS could reduce the production of serum cytokines (TNF-α, IL-6, and IL-1b) in Ab-infected mice by more than 50-fold ( Figure 6h ).

[0072] Nonsteroidal anti-inflammatory drugs (NSAIDs) such as NPXS and ASA inhibit the cyclooxygenase (COX) isoforms COX-1 and COX-2. 26 It plays an effective anti-inflammatory and immunosuppressive role. It is worth noting that the most effective drug NPXS is a non-selective inhibitor of COX-1 and COX-2, which converts arachidonic acid into prostaglandins (PGs), such as PGD 2, PGE 2, etc. 27 Both COX-2 / PGE2 and TLR / MyD88 signaling pathways are involved in inflammatory responses, and the interaction between these two different pathways is crucial for regulating the inflammatory microenvironment. 28 Furthermore, deletion of the genes encoding COX-1 / COX-2 alters the response to TLR activation in a TLR-specific manner. 29 RNA-seq analysis showed that after NPXS treatment, the expression of COX1 (Log2FC: 0.4194) and COX2 (Log2FC: 0.2429) genes was suppressed in ATCC 17978-infected mice, which led to a further decrease in the relative expression of genes related to the TLR2 / Myd88 / NF-κB signaling pathway, ultimately attenuating the cytokine storm induced by Ab infection ( Figure 6i and 6j These findings also suggest a potential role for the TLR2 / Myd88 / NF-κB signaling pathway in regulating the cytokine storm induced by M1 polarization. Taken together, these data suggest that Ab-induced mortality is attributed to excessive cytokine production during infection, and that NPXS could serve as a novel therapeutic agent to rescue mice from lethal Ab infection by attenuating the cytokine storm.

[0073] Abs pose a serious threat to public health. Existing approaches to treating Ab infections rely primarily on the use of antimicrobial drugs. However, the increasing prevalence of MDR infections has made antibiotic therapy increasingly challenging. In this study, we provide new insights into the treatment of Ab infections by employing an immunosuppressive approach. We focused on the innate immune response triggered by Abs. In a mouse sepsis model, we found that Ab infection led to polarization of AMs and IMs to the M1 phenotype, triggering a cytokine storm and ultimately leading to host death. The polarization of both types of macrophages toward the M1 phenotype was primarily mediated by the TLR2 / MyD88 / NF-κB signaling pathway, which then triggered excessive cytokine production. This excessive inflammatory response could be attenuated by NPXS, an immunosuppressant, which was found to effectively protect mice from death after Ab infection.

[0074] The immune response triggered by Ab is a highly complex process involving a range of antimicrobial activities mediated by the host's macrophages and other immune cells. In the early stages of Ab infection, macrophages are recruited in large numbers due to the activation of the innate immune response. Although the activity of macrophages has a bactericidal effect, our results show that a considerable number of Abs remain active at 12 hpi. Previous studies have shown that ATCC 17978 exhibits a higher level of virulence by evading macrophage-mediated killing. 30 Therefore, this organism can show resistance to macrophages in the extracellular environment and secrete OMVs to deliver virulence factors into host cells, inducing apoptosis. 31 Within the cell, Ab can replicate in vacuoles and escape from the macrophage 32 The presence of live Abs continuously stimulates the membrane-bound pattern recognition receptor TLR2, upregulating the TLR2 / MyD88 pathway, activating the transcription factor NF-κB, and promoting the polarization of M1 macrophages, thereby inducing excessive production of inflammatory factors (IL-1β, IL-6, and TNF-α). The result is a severe cytokine storm, resulting in a systemic inflammatory response syndrome, which is often associated with systemic organ failure and even host death.

[0075] Different inflammatory responses induced by M1 macrophages have different effects on host innate immunity, leading to different clinical outcomes. Appropriate polarization of M1 macrophages can protect the host during acute infectious diseases. 33 Listeria monocytogenes has been shown to trigger the M1 program, which prevents bacterial phagosome escape and promotes intracellular bacterial killing. 34 . Prolonged or excessive activation of the M1 program can trigger a cytokine storm, which is harmful to the host. Uncontrolled activated M1 macrophages may produce high levels of type 1 cytokines and chemokines, leading to inflammation in multiple organs and potentially fatal outcomes. Our results are the first to show that Ab infection induces this uncontrolled M1 polarization and cytokine storm, which is associated with severe complications, multiple organ failure and host death. Cytokine storms are also thought to be triggered by a variety of viral, bacterial or fungal infections. Many Gram-negative bacteria have been reported to cause excessive inflammatory responses in the host, including Yersinia pestis. 35 , Francisella tularensis 36 , Pseudomonas aeruginosa 37 and Klebsiella pneumoniae 38Therefore, inhibiting M1 macrophage hyperpolarization or cytokine overexpression may represent a novel approach to treat infections caused by Ab and other pathogens, regardless of their antibiotic susceptibility properties.

[0076] We found that immunosuppressive drugs can be used to suppress the inflammatory response induced during Ab infection. Our results show that NSAIDs are highly effective as therapeutic agents, acting by suppressing the cytokine storm triggered by Ab, with naproxen being the most effective. A previous study also demonstrated that naproxen is effective in controlling the cytokine storm in chronic and severe cases of novel coronavirus disease. 39 Naproxen is a nonselective inhibitor of COX-1 and COX-2, inhibiting constitutive expression of COX-1 in most cells and COX-2 expression induced in response to inflammatory stimuli. 40 In this study, we found that the anti-inflammatory efficacy of aspirin was inferior to that of naproxen during Ab infection. COX-2 is considered the most suitable target for anti-inflammatory drugs. In addition, the study also showed that the therapeutic anti-inflammatory effects of NSAIDs are achieved by inhibiting COX-2, while the adverse side effects are mainly related to COX-1 inhibition. 41 Compared with aspirin, naproxen showed a stronger inhibitory effect on COX-2, demonstrating a clear difference in the anti-inflammatory effects and potency of the two drugs. Furthermore, naproxen's effects were longer-lasting, requiring a lower dose for treatment.

[0077] Our study has several limitations. First, while interstitial and alveolar macrophages are the primary targets of the cytokine storm induced by Ab infection, the involvement of other immune populations in this process, such as T cells, B cells, neutrophils, and natural killer cells, requires further exploration. Furthermore, our studies demonstrate that AbOmpA can trigger TLR2 activation on the macrophage surface, enhancing cell adhesion and invasion. However, future studies will require additional supporting evidence to determine whether AbOmpA can robustly induce M1 polarization and the associated inflammatory response by upregulating the TLR2 / MyD88 / NF-κB pathway. Finally, our studies used C7BL / 6 mice, which may not perfectly mimic Ab infection in humans. However, insights gained from animal experiments are valuable for understanding the immune responses triggered by Ab in humans and pave the way for the introduction of naproxen into clinical use through further clinical trials. Given that anti-inflammatory drugs such as naproxen can effectively protect mice from potentially lethal Ab infection, this drug could be considered a promising therapeutic agent for the future treatment of human infections.

[0078] Materials and Methods

[0079] Bacterial strains and culture conditions

[0080] Acinetobacter baumannii strain ATCC 17978 was purchased from the American Type Culture Collection (VA, USA) and cultured in LB liquid medium and agar plates at 37°C unless otherwise stated. When necessary, 50 μg / mL cloxacillin was added for selection purposes. The bacterial strains and plasmids used in this study are shown in Figure 10 middle.

[0081] mice

[0082] C57BL / 6 and TLR2 - / - Mice (5-7 weeks old) were used in animal experiments. C57BL / 6 mice were provided by the Laboratory Animal Research Unit of the City University. - / - Mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and bred in-house at the Hong Kong Polytechnic University Shenzhen Research Institute. - / Mice were randomly placed into cages at a standard density and housed at 21°C under a 12:12 dark / light cycle with adequate water and food. Figure 11 The genotypes of the KO mice were identified by PCR using the primers listed in All animal experiments were approved by the City University of Hong Kong and the Shenzhen Research Institute of the Hong Kong Polytechnic University.

[0083] Mouse sepsis infection model

[0084] The immune response induced by Ab was tested in vivo using a mouse infection model. Mice of the same sex were randomly divided into several groups (5 per group) and then intraperitoneally administered with 7.5×10 7 The mice were infected with the test strain containing 100 CFU of the Ab. The body weight of all mice was recorded before and 12 hours after infection. The survival of the test animals was determined by recording the mortality rate during the 120-hour experimental period. At 12 hpi, the mice were sacrificed, and the organs were collected and homogenized for bacterial load measurement and analyzed by flow cytometry, RNAseq, scRNAseq, and qRT-PCR assays. Mouse serum was collected for ELISA testing. At least two animal experiments were performed to assess the consistency of the data.

[0085] Collection of spleen and lung single cells

[0086] To collect lung cells, the whole lung sample was minced into a homogenous paste with a scalpel and digested in HBSS containing 1x HEPES and 0.4 mg / ml collagenase I at 37°C for 1 hour. The digested tissue was minced and filtered through a 70 μm filter to obtain a lung cell suspension. Cells from the spleen were pushed through a 70 μm filter and allowed to fall into RPMI 1640 medium containing 5% fetal bovine serum (FBS). ACK lysis buffer was used to lyse red blood cells. 42 .

[0087] Determination of bacterial load in vivo

[0088] To measure the total bacterial load in different organs (including lung, spleen, liver, kidney homogenates and blood samples), serial dilutions were prepared and plated on agar plates containing 50 μg / mL cloxacillin. To further assess the number of intracellular bacteria in vivo, single-cell suspensions of lung and spleen were washed twice with PBS and suspended in RPMI 1640 medium containing 5% FBS and 300 μg / mL gentamicin and incubated at 37°C for 2 hours to eliminate extracellular bacteria. The cells were then lysed with 0.2% Triton X-100, and the cell lysates were plated on agar plates to count the number of phagocytosed bacteria.

[0089] Flow cytometric analysis

[0090] Single lung and spleen cells isolated from test mice were incubated with different combinations of fluorochrome-conjugated antibodies against mouse: CD45, CD11b, Ly6G, F4 / 80, CD206, CD86, CD3, CD19, CD4, CD8, NK1.1; appropriate isotype controls were obtained from Biolegend. TM Dead cells were excluded by using Purple 510 (Tonbo Biosciences, CA, USA) or propidium iodide (Sigma-Aldrich, MI, USA). TM Flow cytometric analysis was performed using a flow cytometer (BD Biosciences, CA, USA); data analysis was performed using FlowJo software (version 10.8.1, Treestar, CA, USA).

[0091] RNA extraction and real-time quantitative PCR assay

[0092] After Ab infection, TRIzol TMMethods Total RNA was extracted from homogenized lung samples or RAW264.7 cells (ATCC, CA, USA) after Ab infection (Thermo Fisher Scientific, MA, USA). TM Kit (Invitrogen, CA, USA) was used to remove DNA contaminants. TM 1 μg of purified mRNA was reverse transcribed into cDNA using the PCR amplification kit (Invitrogen) and diluted 1:10 for subsequent qRT-PCR analysis. qRT-PCR was then performed using the PowerUP SYBR Green master mix (Applied Biosystems) on an ABI QuantStudio 7Flex real-time PCR system (Applied Biosystems, CA, USA). The primers used are shown in Table 1. Figure 11 The target gene transcript levels were quantified by the comparative Ct method and normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GADPH).

[0093] RNA sequencing

[0094] Total RNA extracted from lung samples was sequenced by Novogene Technology (Hong Kong SAR, China). RNA integrity was measured using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA); cDNA libraries for Illumina sequencing were constructed using the NEB Next Ultra RNA Library Preparation Kit (NEB, CA, USA). Index-encoded sample clustering was performed using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia, CA, Illumia) on the cBot Cluster Generation System. Libraries were sequenced using paired-end 150 bp reads on the Illumina Novaseq platform. HISAT2 was used for sequencing read alignment. Fragments of each gene were counted based on exonic regions using FeatureCounts v.1.6.2. 43 Differential mRNA level analysis was performed using the DESeq2 R package. Unigenes with a P-value < 0.05 and a |Log2 fold change| > 1 were characterized as differentially expressed genes. Gene enrichment analysis was performed for gene ontology enrichment analysis. Up-regulated / down-regulated DEGs were visualized using volcano plots.

[0095] Serum collection

[0096] Blood was collected from test mice by orbital bleeding and allowed to clot at room temperature for 1 hour. The blood was then centrifuged at 10,000 g for 25 minutes at 4°C. Serum was aliquoted into tubes and immediately stored at –80°C until cytokine analysis.

[0097] Cytokine analysis

[0098] Cytokines in mouse serum and cell supernatant were analyzed using enzyme-linked immunosorbent assay (ELISA). Mouse serum was extracted as described above. Pretreated macrophage supernatant was harvested and centrifuged to remove impurities. According to the manufacturer's protocol, IL-1β, IL-6, and TNFα mice were not coated with ELISA kits (Thermo Fisher Scientific) to measure the levels of IL1β, IL6, and TNF-α.

[0099] Single-cell RNA sequencing and data processing

[0100] Single cell suspensions extracted from mouse lung samples were sent to BGI Genomics (Hong Kong SAR, China) for 10X genome single cell sequencing. Chromium TM Pre-made libraries were constructed using the System Single Cell 3' Library Construction Kit v3 and the Next Generation GEM Single Cell 3' Library Construction Kit v3.1. After quality analysis of the pre-made libraries, BGI's proprietary DNBseq TM NGS technology was used to convert these cells into DNA nanoballs (DNBs) for sequencing. Demultiplexing, barcoding, and single-cell 5' unique molecular identifier (UMI) counting were performed using the Cell Ranger software suite (v.3.1.0). Valid cells were filtered based on gene number, UMI count, and mitochondrial gene percentage.

[0101] Dimensionality reduction and clustering

[0102] The filtered gene barcode matrix for all samples was analyzed using Seurat v.4.3 using default parameters. 44Integration and normalization were performed. Highly variable genes were validated using the “vst” method in the Seurat FindVariableFeatures function. The variables “percent.mito” and “nCount_RNA” were regressed in the scaling step. PCA was performed with highly variable genes. To visualize the cells, UMAP was performed on the frontiers (principal components). In addition, the resolution was set to 0.2 to perform graph-based clustering analysis on the PCA-reduced data. All clusters were subjected to differential gene expression analysis using FindAllMarkers to validate the marker genes of interest. Marker genes for each cluster were selected to annotate fine clusters based on normalized RNA expression values (RNA expression values were >0.25 log-fold higher than the mean expression value and had detectable expression in >25% of all cells). Alveolar macrophages and intestinal macrophages were also reintegrated and re-clustered in the same manner.

[0103] Intercellular communication analysis

[0104] CellChat (v1.6.1) package 19 Used to infer, analyze, and visualize intercellular communication between various immune cells, using the CellChat database (github.com / sqjin / CellChat) as a reference. For cell interaction analysis, expression levels related to the total number of reads were calculated and mapped to the same set of coding genes in all transcriptomes. The expression values in each cell sample or single cell cluster were averaged. By calculating and comparing the information flow of each signaling pathway, differential analysis of intercellular communication between infected and non-infected groups was performed, defined as the probability of all communication between all pairs of cell populations in the inferred network.

[0105] Pseudo-time trajectory analysis

[0106] After designating the corresponding cells as root nodes, trajectory analysis was performed by Monocle version 3 to investigate the relationship between trajectories and macrophage subsets. 45 We used the “plot_cells” function to visualize the pseudo-time trajectories and arranged all cells as well as cells from healthy donors or Ab-infected donors onto the trajectories.

[0107] Differential gene expression and KEGG pathway enrichment analysis

[0108] Differential gene expression analysis was performed using FindAllMarkers in Seurat. One-tailed Wilcoxon rank sum tests were performed, and the p-values were adjusted for multiple testing using Bonferroni correction. KEGG pathway enrichment analysis was performed using the R package clusterProfiler using differentially expressed genes (DEGs) with adjusted p-values < 0.01. 24 .

[0109] Mouse Ly6g+ neutrophil isolation

[0110] Using MojoSort TM Mouse Ly-6G selection kit (Biolegend, CA, USA) was used in MojoSort TM Lung single cell suspensions were sorted on a magnet to select for Ly6G + Neutrophils. Briefly, Ly6G was labeled by mixing the sample with a biotin-antibody cocktail and then adding magnetic streptavidin nanobeads. + These magnetically labeled cells are retained by a magnetic separator. The purity of cells enriched from single-cell populations is consistently >90%, which meets the requirements for flow cytometry.

[0111] Inhibitor treatment in RAW264.7

[0112] RAW264.7 cells were treated with culture medium, C29 (100 μM) 22 、TJ-M2010-5(20μM) 23 and JSH-23 (30 μM) 24 (MCE, NJ, USA) were pretreated for 1 hour and then stimulated with Ab (MOI = 5) for 6 hours in the presence of culture medium, C29, TJ-M2010-5, or JSH-23. Cell supernatants were collected for subsequent cytokine measurements. RAW264.7 cells were incubated with fluorescently labeled antibodies against mouse F4 / 80, CD206, and CD86 for flow cytometric analysis, then washed three times with PBS and resuspended in PBS containing 2.5% FBS.

[0113] OmpA knockout based on CRISPR-Cas9 genome editing platform

[0114] This study used the dual-plasmid genome editing system pSGAb-pCasAb, which combines the CRISPR-Cas9 genome cleavage system and the RecAb recombination system to efficiently perform gene deletion in strain ATCC 17978. 46,47 The specific 20 bp spacer sequence (sgRNA) upstream of the PAM site of OmpA was generated by sgRNAcas9 software.48 The competent cells of ATCC 17978 were selected and cloned into pSGAb-spe plasmid (Addgene, MA, USA). The competent cells were prepared by subculturing in LB liquid medium overnight and then shaking cultured at 37°C until OD 600 The value reached 0.4 to 0.7. The fresh exponentially growing culture was then washed with ddH2O and then washed with 10% glycerol cooled to 4°C. The pCasAb-apr plasmid (Addgene, MA, USA) was then transformed into ATCC 17978 competent cells by electroporation. After recovering in LB liquid medium for 1 hour, the mixture was plated on a selective LB agar plate containing 100 μg / mL apramycin. Transformants containing the pCasAb-apr plasmid were selected and enriched by incubating at 37°C until the OD600 reached 0.1-0.15, and then IPTG was added to a final concentration of 1 mM to induce the expression of the Cas9 nuclease and the RecAb recombination system; the mixture was then incubated for a further 2 hours, then washed with ddH2O, and then washed with 10% glycerol at 4°C. Next, ssDNA (ATCC17978 genome repair template by homologous recombination) and the pSGAb-spe plasmid introducing sgRNA were simultaneously transformed into ATCC 17978 carrying the pCasAb-apr plasmid by electroporation. For recovery and genome editing, the culture was incubated for another 1.5 hours and then spread on LB agar plates containing 100 μg / mL apramycin and 50 μg / mL spectinomycin. After incubation overnight, colonies were collected and confirmed by PCR and nucleotide sequencing whether the ompA gene had been deleted. The PCR verification results of ATCC17978 and ΔOmpA are shown as follows. Figure 9 The primers and ssDNA used in this study are listed in Figure 11 middle.

[0115] Bacterial adhesion, invasion, and survival in RAW264.7 cell culture

[0116] Mouse macrophage-like RAW264.7 cells were plated at 2 × 10 5 Cells were seeded into 24-well culture plates at a density of 100 μM C29 or 400 μM C29. 22Treat for 1 hour and then infect with Acinetobacter baumannii strain (MOI=10). For cell adhesion assays, cells were washed 3 times with PBS 30 minutes after infection and then lysed in 1 ml of 0.2% Triton X-100. To analyze the invasion rate, DMEM culture medium containing 300 μg / mL gentamicin and supplemented with 10% FBS was added 1 hour after infection and cultured for another 2 hours to eradicate all extracellular bacteria. Next, the cells were washed 3 times with PBS and lysed in 1 ml of 0.2% Triton X-100 to release invading bacteria in infected cells. For RAW264.7 intracellular survival assays, cells were incubated in culture medium containing 300 μg / mL gentamicin for 2 hours; after washing with PBS, culture medium containing 15 μg / mL gentamicin was added to control the growth of intracellular bacteria released from lysed macrophages. After extended incubation for 4 hours, the cells were washed and lysed in 1 ml of 0.2% trionX-100. The lysate was serially diluted and plated on LB agar plates.

[0117] In vivo drug treatment

[0118] To test the efficacy of NSAID treatment, mice were intraperitoneally injected with 50 mg / kg NPXS and 100 mg / kg ASA 1 hour before infection with ATCC 17978. A control group of mice received an equal amount of PBS. The survival rate, health status, and body weight of the test animals were recorded over the next 120 hours.

[0119] Statistical analysis

[0120] All experiments were performed at least twice, with at least three biological replicates per experiment. Statistical data were plotted using GraphPad Prism 8; analysis of variance was performed with Tukey's correction for multiple comparisons. The log-rank (Mantel-Cox) test was used to compare survival rates in the animal experiments.

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Claims

1. Use of a therapeutic agent in the preparation of a medicament for treating cytokine storm in a subject in need thereof, wherein the therapeutic agent is selected from the group consisting of (+)-(S)-2-(6-methoxynaphthalen-2-yl)propionic acid (naproxen), acetylsalicylic acid (ASA), dexamethasone (DXMS), azathioprine (AzA) and pharmaceutically acceptable salts thereof, and wherein the cytokine storm is associated with an Acinetobacter baumannii infection in the subject.

2. The method of claim 1, wherein the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, wherein the infection with Acinetobacter baumannii activates the TLR2 / MyD88 / NF-κB signaling pathway in the subject.

4. The use according to claim 1, wherein the subject overexpresses one or more proinflammatory factors selected from the group consisting of IL-1β, IL-6, IL-10, IL-12, IL17a, IL23, IL-27, IFN-γ and TNF-α.

5. The use according to claim 1, wherein the infection with Acinetobacter baumannii activates the TLR2 / MyD88 / NF-κB signaling pathway in the subject; the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof; and administration of naproxen results in a decrease in the relative expression of genes related to the TLR2 / MyD88 / NF-κB signaling pathway.

6. The use according to claim 5, wherein the TLR2 / MyD88 / NF-κB signaling pathway-related genes are selected from the group consisting of toll-like receptor 2 (TLR2), primary response to myeloid differentiation 88 (Myd88), nuclear factor κB subunit 1 (Nfkb1), nuclear factor κB subunit 2 (Nfkb2), interleukin 1β (Il1b), interleukin 6 (Il6) and tumor necrosis factor (Tnf).

7. The method of claim 1, wherein the Acinetobacter baumannii infection is caused by antibiotic-resistant Acinetobacter baumannii.

8. The use according to claim 7, wherein the antibiotic-resistant Acinetobacter baumannii is resistant to one or more antibacterial agents selected from the group consisting of aminoglycosides, fluoroquinolones and carbapenems.

9. The use according to claim 7, wherein the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof.

10. The use according to claim 1, wherein the Acinetobacter baumannii infection is caused by an Acinetobacter baumannii strain selected from the group consisting of ATCC 17978, ATCC 19606, AB5075, ATCC 9955, ATCC 17904, R 477 and R0211019.

11. The use according to claim 10, wherein the therapeutic agent is naproxen or a pharmaceutically acceptable salt thereof.

12. The method of claim 1, wherein the Acinetobacter baumannii infection is present in one or more of a wound, a surgical site, a catheter site, blood, urinary tract, skin, lung, or respiratory tract.

13. The use of claim 1, wherein the subject was diagnosed with Acinetobacter baumannii infection before being administered the medicament.

14. The use of claim 1, wherein the medicament is co-administered with a therapeutically effective amount of an antibacterial agent.

15. The use according to claim 14, wherein the antibacterial agent is selected from the group consisting of meropenem, colistin, polymyxin B, sulbactam, piperacillin / tazobactam, minocycline, tigecycline and aminoglycosides.

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