Regulation and control of IER3-CSF2 axis in sepsis immune hyporeaction of high-toxicity klebsiella pneumoniae and application of IER3-CSF2 axis

Abstract

The invention relates to a technical scheme in the field of immune regulation and control in high-toxicity klebsiella pneumoniae infection, and particularly discloses regulation and control of an IER3-CSF2 axis in high-toxicity klebsiella pneumoniae sepsis immune low reaction and application of the IER3-CSF2 axis in high-toxicity klebsiella pneumoniae sepsis immune low reaction. Researches find that after the Ier3 gene is knocked down, tissue damage in high-toxicity klebsiella pneumoniae infected sepsis is aggravated, and the survival rate is reduced. With the decline of CSF2 expression and the migration of macrophages to M2 type, the secretion of proinflammatory factors is insufficient, and the immune response is impaired. Further in-vitro cell experiments show that supplement of CSF2 can reverse reduction of expression of inflammatory factors. Based on the discovery, the invention provides an immune state evaluation method taking an IER3-CSF2 signal axis as a target spot and a potential immune regulation strategy, and the immune state evaluation method and the potential immune regulation strategy are suitable for development of an intervention scheme of bacterial infection, especially sepsis, and have important clinical transformation application value.

Description

Technical Field

[0001] This invention relates to the field of infection immunity and immune regulation research, specifically to a technical solution for the regulation of bacterial sepsis immune status based on the IER3 gene and its downstream CSF2 signaling axis. Background Technology

[0002] Klebsiella pneumoniae is an important Gram-negative opportunistic pathogen that causes a variety of infectious diseases, including urinary tract infections, sepsis, pneumonia, and liver abscesses. Highly virulent Klebsiella pneumoniae (hvKP) is an evolving strain, more virulent than classic Klebsiella pneumoniae (cKP), characterized by its ability to evade the host's immune system and cause infection in immunocompetent hosts. hvKP infections often occur at multiple sites or subsequently spread metastatically, usually requiring source control. Due to management deficiencies, antimicrobial resistance determinants of cKP and virulence factors of hvKP fuse on the same or coexisting plasmids, ultimately leading to the emergence of multidrug-resistant (MDR) hvKP. These strains are resistant to a variety of commonly used antibiotics, including β-lactams (such as penicillins and cephalosporins), aminoglycosides, and even carbapenems (such as imipenem and meropenem). Carbapenems are often considered a last resort for treating MDR infections, making their resistance particularly concerning. Therefore, this poses a significant challenge to the clinical treatment of sepsis caused by highly virulent Klebsiella pneumoniae. We urgently need to develop new therapeutic targets or interventions.

[0003] Sepsis is defined as "life-threatening organ dysfunction caused by an abnormal host response to infection." Sepsis is not merely a systemic inflammatory response or immune disorder; it involves changes in the function of multiple organs. At the cellular and molecular levels, the pathogenesis of sepsis is extremely complex, including inflammatory imbalances, immune dysfunction, mitochondrial damage, coagulopathy, neuroendocrine-immune network abnormalities, endoplasmic reticulum stress, autophagy, and other pathophysiological processes, ultimately leading to organ dysfunction. With a deeper understanding of the pathogenesis of sepsis, new paradigms for the mechanisms and solutions of sepsis, as well as the consequences for sepsis survivors, are emerging. However, current research largely focuses on suppressing early excessive inflammatory responses. For the immunodeficiency state that occurs during sepsis progression, especially the molecular regulatory mechanisms of weakened macrophage function and insufficient secretion of inflammatory factors, there is still a lack of clear targets and effective intervention strategies. Macrophages are important innate immune cells, and numerous studies have demonstrated that deep activation of macrophages plays a crucial role in the immunopathogenesis and regulatory development of sepsis. The secretion of inflammatory factors is a double-edged sword. Components of the outer membrane of Klebsiella pneumoniae (such as LPS) stimulate macrophages to secrete large amounts of pro-inflammatory cytokines, leading to a "cytokine storm." Conversely, insufficient secretion of inflammatory factors can result in decreased pathogen clearance, worsened infection, delayed tissue repair, and immune imbalance, all of which can exacerbate the disease. Therefore, regulating genes that release inflammatory factors may become an important target for the treatment of Klebsiella pneumoniae sepsis. Summary of the Invention

[0004] The lack of effective and modifiable molecular targets for addressing the immunodeficiency that occurs during sepsis progression makes precise intervention in host immune function difficult. While the IER3 gene has been reported to be associated with inflammation regulation, its regulatory mechanism in bacterial infections, particularly sepsis caused by highly virulent Klebsiella pneumoniae, remains unclear. Whether IER3 regulates macrophage immune status and participates in the formation of immunodeficiency in sepsis through specific downstream signaling pathways still lacks systematic research and definitive technical solutions.

[0005] The purpose of this invention is to provide a technical solution for regulating macrophage immune response based on the IER3–CSF2 signaling axis, revealing that IER3 affects macrophage polarization state by regulating CSF2 expression, thereby regulating the secretion of inflammatory factors and the host's antibacterial immune capacity, and applying this mechanism of action to the regulation and intervention of sepsis-induced hyporesponsiveness caused by highly virulent Klebsiella pneumoniae infection.

[0006] To achieve the above objectives, the technical solution provided by this invention includes the following:

[0007] By regulating the expression level of the IER3 gene, the expression or secretion state of its downstream CSF2 is affected, thereby modulating the polarization of macrophages from pro-inflammatory to immunosuppressive or vice versa, thus altering the secretion level of inflammatory factors and the strength of the host's immune response to highly virulent Klebsiella pneumoniae infection. The immunomodulatory effect described in this invention depends on the presence and functional state of macrophages.

[0008] In one embodiment of the present invention, downregulation of IER3 expression leads to a decrease in CSF2 expression, which causes macrophages to shift towards the M2 immunophenotype, reduces the secretion of inflammatory factors, and decreases the ability to clear bacteria, thereby aggravating infection-related tissue damage and reducing host survival.

[0009] In another embodiment of the present invention, by regulating CSF2 levels, the ability of macrophages to secrete pro-inflammatory factors can be partially restored, thereby improving the host's immune response to bacterial infection.

[0010] Furthermore, this invention provides a technical approach for studying and regulating the low immune response state of highly virulent Klebsiella pneumoniae infection. Specifically, it targets the IER3–CSF2 signaling axis and its mediated macrophage immune function regulation, and modulates macrophage immune function by intervening in key nodes of this signaling axis. This technical approach is applicable to the study of bacterial infection-related immune dysfunction and can be used for the design and development of immunomodulatory strategies for sepsis.

[0011] The beneficial effects of this invention are as follows: it clarifies the regulatory role of the IER3–CSF2 signaling axis in the hyporesponsiveness of sepsis caused by highly virulent Klebsiella pneumoniae infection, and confirms that this role depends on macrophage mediation. It reveals the mechanism by which IER3 affects macrophage polarization and inflammatory factor secretion by regulating CSF2, providing a new molecular target and technical solution for the immune regulation of bacterial sepsis, which has important research value and potential application prospects. Attached Figure Description

[0012] Figure 1 This is a schematic diagram showing the changes in Ier3 expression in mouse peritoneal macrophages under conditions of infection with highly virulent Klebsiella pneumoniae. Figure 1 (A) The transcription level of the Ier3 gene in peritoneal macrophages of mice with highly virulent Klebsiella pneumoniae sepsis; Figure 1 (B) The transcription level of the Ier3 gene in mouse peritoneal macrophages at different time points after stimulation with highly virulent Klebsiella pneumoniae in vitro. Figure 1 (C) represents the expression level of Ier3 protein in mouse peritoneal macrophages at different time points after stimulation with highly virulent Klebsiella pneumoniae.

[0013] Figure 2A schematic diagram illustrating the effect of knocking down the Ier3 gene on the immune response to highly virulent Klebsiella pneumoniae sepsis. Figure 2 (A) The transcriptional level of inflammatory factors 12 hours after knocking down mouse peritoneal macrophages Ier3 and stimulating them with highly virulent Klebsiella pneumoniae. Figure 2 (B) Bacterial load in tissues of mice with highly virulent Klebsiella pneumoniae sepsis model after knocking down the mouse Ier3 gene; Figure 2 (C) HE staining results of tissues from a mouse model of highly virulent Klebsiella pneumoniae sepsis after knocking down the mouse Ier3 gene; Figure 2 (D) shows the survival rate and weight changes of mice with highly virulent Klebsiella pneumoniae sepsis after knocking down the Ier3 gene.

[0014] Figure 3 This is a schematic diagram illustrating the IER3 function dependence under macrophage depletion conditions. Figure 3 (A) The bacterial load in tissues of mice with high-virulence Klebsiella pneumoniae sepsis model after depletion of mouse peritoneal macrophages; Figure 3 (B) HE staining results of tissues from a highly virulent Klebsiella pneumoniae sepsis model mouse with knocked-down IER3 gene after exhaustion of mouse peritoneal macrophages.

[0015] Figure 4 This is a schematic diagram illustrating the regulation of CSF2 expression and macrophage polarization by IER3. Figure 4 (A) The transcriptional and protein secretion levels of the Csf2 gene 12 hours after knocking down mouse peritoneal macrophages Ier3 and stimulating them with highly virulent Klebsiella pneumoniae. Figure 4 (B) After knocking down mouse peritoneal macrophages Ier3, the expression levels of iNOS and Arg1 genes were detected by qPCR. Figure 4 (C) Flow cytometry was used to detect the expression of CD86, iNOS and CD206 after knocking down mouse bone marrow-derived macrophages Ier3; Figure 4 (D) The expression of inflammatory factors after knocking down mouse peritoneal macrophages Ier3 and supplementing with exogenous CSF2 protein.

[0016] Figure 5 This diagram illustrates the regulatory mechanism of the IER3-CSF2 signaling axis in the low immune response of bacterial sepsis. It is used to explain the overall technical approach that changes in IER3 expression affect macrophage polarization by regulating CSF2 levels under bacterial infection conditions, thereby regulating the secretion of inflammatory factors and the host's anti-infection immune response. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0018] Example 1: Candidate gene screening and its validation in a highly virulent pulmonary sepsis model

[0019] (I) Gene Screening Methods

[0020] This embodiment first utilizes a publicly available transcriptome database related to bacterial sepsis and employs a standard differential gene analysis procedure to screen for genes with significantly different expression levels between the infection group and the control group. Through cross-referencing across multiple datasets, candidate genes that are stably upregulated under bacterial infection conditions and participate in the inflammatory response were selected, ultimately identifying Ier3 as the core target of this invention.

[0021] (II) Establishment of a mouse model of infection with highly virulent Klebsiella pneumoniae

[0022] Six- to eight-week-old C57BL / 6 mice were selected and an infection model was established by intraperitoneal injection of a suspension of highly virulent Klebsiella pneumoniae (hvKP) according to standard methods. Liver, spleen, lung, and kidney tissues, heart blood, and peritoneal lavage fluid were collected 24 hours after infection for histopathological analysis and bacterial load detection.

[0023] (III) Validation of Ier3 expression

[0024] The expression level of the Ier3 gene in peritoneal macrophages after infection was detected by qPCR, and the results showed that Ier3 was significantly increased in the infected group. In vitro cultured macrophages stimulated with hvKP also showed upregulation of Ier3 expression, as detected by qPCR and Western blot, suggesting that Ier3 is closely related to the immune response of macrophages in bacterial infection.

[0025] Example 2: Knocking down IER3 reduces macrophage inflammatory cytokine secretion and exacerbates highly virulent Klebsiella pneumoniae sepsis (I) Experimental method for knocking down IER3 in macrophages in vitro

[0026] Peritoneal macrophages from 6-8 week old C57BL / 6 mice were isolated and cultured using standard methods. Small interfering RNA (siRNA) was used to transfect the cells to reduce Ier3 expression levels; a negative control group was included. Twenty-four hours after transfection, the cells were stimulated with inactivated HvKP (highly virulent strain) solution, and cellular RNA was collected after stimulation. The expression levels of inflammatory factors in the cellular RNA were then detected. (II) In vivo mouse infection experiment after Ier3 interference.

[0027] Six- to eight-week-old C57BL / 6 mice were randomly divided into groups. Ier3 expression in mice was reduced via tail vein injection of an interfering RNA complex, with a negative interference control group included. A sepsis model was established 24 hours post-interference by intraperitoneal injection of highly virulent Klebsiella pneumoniae. Liver, spleen, lung, and kidney tissues, heart blood, and peritoneal lavage fluid were collected 24 hours after infection for bacterial load analysis. Liver, spleen, lung, and kidney tissues were stained with hematoxylin and eosin (HE). Mouse weight and survival status were continuously recorded post-infection.

[0028] (III) In vitro macrophage knockdown of IER3 results showed that macrophages with Ier3 knockdown expressed lower levels of various inflammatory factors than the negative control group after stimulation, indicating that the pro-inflammatory response capacity of macrophages was weakened after the Ier3 level decreased. In vivo mouse infection experiments after Ier3 interference showed that mice in the Ier3 interference group had a higher bacterial load, more obvious tissue inflammatory infiltration, more severe structural damage, faster weight loss, and lower survival rate than the control group.

[0029] Example 3: The effect of IER3 disappears after macrophage depletion.

[0030] Six- to eight-week-old C57BL / 6 mice were randomly divided into groups. Macrophages were depleted using Clodronate Liposomes, while Control Liposomes (PBS) and PBS served as control groups. Ier3 expression in mice was reduced via tail vein injection of an interfering RNA complex. In the hvKP sepsis model, bacterial load no longer increased significantly, and there was no significant difference in tissue HE staining. These results indicate that the regulatory role of IER3 in the immune response to bacterial sepsis is dependent on the presence of macrophages.

[0031] Example 4: IER3 downregulation affects CSF2 expression, macrophage polarization direction, and the salvage effect of CSF2.

[0032] (I) Transcriptome sequencing analysis and validation

[0033] Mouse peritoneal macrophages were transfected with IER3 siRNA. After 24 hours of transfection, they were stimulated with hvKP inactivation solution for 12 hours. A negative interference control group was included. Total RNA was extracted from the cells and sent to a professional sequencing platform for transcriptome sequencing analysis. Differential gene analysis showed that multiple inflammatory and immune regulation pathways were affected, with colony-stimulating factor 2 (CSF2) significantly downregulated. Further bioinformatics enrichment analysis indicated that IER3 downregulation led to a shift in macrophage phenotype from pro-inflammatory to immunosuppressive, with increased expression levels of genes associated with the M2 phenotype and decreased expression of genes associated with the M1 phenotype.

[0034] (II) Result Verification

[0035] Mouse peritoneal macrophages were transfected with IER3 siRNA. After 24 hours of transfection, they were stimulated with hvKP inactivation solution for 12 hours. A negative interference control group was included. Total RNA was extracted from the cells, and the transcriptional levels of CSF2, iNOS (nos2), and Arg1 were detected by qPCR. Cell supernatant was collected, and CSF2 protein secretion levels were detected by ELISA; CSF2 secretion was decreased. BMDM cells were used, and the experimental procedure and grouping were consistent with the previous methods. After stimulation, cells were collected and stained with CD11b, F4 / 80, CD86, CD206, and iNOS antigens. Cell polarization was analyzed by flow cytometry. The results showed that after knocking down the Ier3 gene, CD86 and iNOS expression decreased (M1 marker), while CD206 expression increased (M2 marker).

[0036] (III) CSF2 replenishment

[0037] Mouse peritoneal macrophages were transfected with IER3 siRNA, and two groups of IER3 siRNA were set up. 24 h after transfection, the negative interference control group was stimulated with hvKP inactivation solution for 12 h. One of the two IER3 siRNA groups was supplemented with exogenous CSF2 protein and simultaneously stimulated with hvKP inactivation solution for 12 h. Results showed that after CSF2 supplementation, the expression levels of TNF-α, IL-6, and IL-1β were significantly restored.

Claims

1. A method for assessing and regulating the immune response to infection with highly virulent Klebsiella pneumoniae, characterized in that, include: S1. In vitro knockdown of the Ier3 gene in mouse peritoneal macrophages; S2. Expose the macrophages to highly virulent Klebsiella pneumoniae; S3. Detect changes in the expression of inflammatory factors in the macrophages; S4. Knock down the Ier3 gene in mice in vivo and establish a highly virulent Klebsiella pneumoniae infection model. S5. The impact of Ier3 on infection was assessed through bacterial load, histopathology, and survival observation.

2. A method for sepsis immunomodulation based on IER3 regulation of the CSF2 signaling pathway to affect macrophage polarization, characterized in that, include: (1) Knock down the Ier3 gene in mice or mouse-derived macrophages; (2) Detect changes in the expression or secretion of CSF2; (3) Assess the polarization changes of macrophages toward the M1 / M2 phenotype.

3. A sepsis immune regulation intervention method based on the IER3–CSF2 signaling axis, characterized in that, This includes supplementing exogenous CSF2 into Ier3 knockdown mice or macrophages to assess its regulatory effect on the production of inflammatory factors.

4. An experimental method for verifying the role of macrophages in IER3-mediated immune regulation of sepsis, characterized in that, This includes using macrophage depletion techniques to observe changes in infection-related indicators in mice with Ier3 gene knockdown.

5. An experimental method for screening novel immunomodulatory factors in highly virulent Klebsiella pneumoniae sepsis, characterized in that, include: S1. Screen differentially expressed genes based on infection model data; S2. Knock down candidate genes and detect changes in inflammatory factors; S3. Perform transcriptome sequencing to screen downstream regulatory molecules; S4. Perform reverse verification by supplementing downstream molecules; S5. Detect changes in macrophage M1 / M2 polarization; S6. Assess the criticality of candidate genes in infection models through macrophage exhaustion.

6. The method according to claim 5, characterized in that, include: S1. The expression of IER3 was detected by qPCR or Western blot. S2. The expression or secretion of CSF2 is detected by ELISA or mRNA quantification. S3. The M1 / M2 polarization is detected by mRNA quantification or flow cytometry for CD86, CD206, iNOS, and Arg1.

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