Application of interleukin-40 as a biomarker in the diagnosis of sepsis

Abstract

The present invention discloses the application of interleukin 40 as a biomarker in the diagnosis of sepsis, and belongs to the technical field of sepsis diagnosis; the present invention explores that the expression of the novel immunomodulator IL-40 in patients with sepsis is significantly increased; a sepsis mouse model is induced by cecal ligation and puncture (CLP), and it is found that the expression level of IL-40 in the blood, peritoneal lavage fluid (PLF), liver and kidney of sepsis mice is significantly increased. A sepsis model is constructed by CLP using IL-40 knockout mice, and it is proved that inhibition of IL-40 can significantly reduce the bacterial load in sepsis mice and reduce their multi-organ damage, thereby significantly improving the survival rate of sepsis mice. Therefore, the present invention explores a new biological marker IL-40, which can not only be used for the diagnosis and prognosis of sepsis, but also provides a new technical means for the treatment of sepsis.

Description

Technical Field

[0001] The present invention belongs to the technical field of sepsis diagnosis, and specifically relates to the application of interleukin 40 as a biomarker in sepsis diagnosis. Background Art

[0002] The onset of sepsis involves complex inflammatory imbalance and immune dysfunction. In the early stage of sepsis, bacteria and their components bind to pattern recognition receptors such as TLRs and NOD of host cells, activate the intracellular signaling system, synthesize and release a variety of inflammatory mediators, form a cascade-like inflammatory response, and then the body produces a compensatory anti-inflammatory response, causing immune dysfunction and triggering organ failure in multiple systems such as cardiovascular, respiratory, urinary, nervous, blood and liver. Therefore, immune dysfunction with inflammatory disorders as the main manifestation plays a key role in the development of sepsis. Finding new targets for rapid diagnosis and treatment from the perspective of host immune regulatory factors is of great significance to improving the survival and prognosis of patients.

[0003] At present, the diagnosis of sepsis is mainly based on the diagnostic criteria of Sepsis 3.0 jointly released by the American Society of Critical Care Medicine and the European Society of Intensive Care Medicine in 2016: infection combined with Sequential Organ Failure Assessment (SOFA) score ≥ 2. Among them, the early diagnosis and initial screening of sepsis infection in clinical practice rely on procalcitonin (PCT), C-reactive protein (CRP) and pathogen detection, but there are the following disadvantages: 1) PCT can be used to guide the use of antibiotics and has high specificity for systemic bacterial infection, but the increase in local infection, viral infection and intracellular bacterial infection such as Mycoplasma pneumoniae infection is small; it has no diagnostic value for skin and soft tissue infection; and PCT increases to varying degrees in pathological conditions such as postoperative surgery, cardiogenic shock, and acute graft-versus-host disease, with low specificity and no independent diagnostic ability. 2) CRP is a common diagnostic indicator of inflammation with poor specificity. 3) Clinical microbiological examination (staining microscopy + culture) is still the gold standard for diagnosing pathogenic infection, but it has the disadvantages of being time-consuming, highly subjective, and having many interfering substances. 4) SOFA score involves the comprehensive evaluation of multiple systems of the body, which also brings great challenges to its early diagnosis. Therefore, it is urgent to find new biomarkers for the early diagnosis and treatment of sepsis.

[0004] Interleukin IL-40 (IL-40) is a 27kDa secretory protein discovered by Albert Zlotnik et al. in 2017, encoded and synthesized by C17orf99. As a new cytokine, the coding gene C17orf99 of IL-40 is mainly expressed in the bone marrow, fetal liver, and B cells activated by IL-4 and TGF-β. It has been confirmed that IL-40 is a key effector protein in humoral immune response. IL-40 is upregulated in rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and primary Sjögren's syndrome, and is positively correlated with disease activity and autoantibody levels. Biological studies have further confirmed that IL-40 plays a powerful pro-inflammatory role. It can not only promote the release of IFN-γ by B cells, TNF-α and IL-17A by CD4 / CD8+, but also promote the release of metal matrix proteases MMPs by synovial fibroblasts. These studies have shown that IL-40 is closely related to immune regulation and is a novel immune regulatory factor. However, there have been no reports on the study of IL-40 in sepsis. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide an application of interleukin 40 as a biomarker in the diagnosis of sepsis, thereby solving the problems in the prior art.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] Application of interleukin 40 as a biomarker in the preparation of a kit for diagnosing or screening sepsis.

[0008] Application of interleukin 40 detection reagent in preparing a kit for diagnosing or screening sepsis.

[0009] Application of interleukin 40 detection reagent in the preparation of a kit for monitoring the prognosis of sepsis.

[0010] A kit for diagnosing or screening sepsis, comprising a detection reagent for interleukin 40.

[0011] Use of interleukin 40 inhibitors in preparing drugs for treating sepsis.

[0012] A drug for treating sepsis, comprising an interleukin 40 inhibitor and a carrier thereof.

[0013] Furthermore, the carrier is a virus or liposome capable of carrying an IL-40 inhibitor.

[0014] Beneficial effects of the present invention:

[0015] 1) The present invention provides that IL-40 can be used as a diagnostic indicator for sepsis.

[0016] 2) The present invention provides that IL-40 can be used as an indicator for evaluating the severity of sepsis, and also for the first time finds that the prognosis of sepsis can be predicted based on the expression level of IL-40.

[0017] 3) The present invention provides the use of IL-40 inhibitors and carriers in the preparation of drugs for improving and / or treating sepsis, providing a new idea and option for the treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 a in Figure 1 b in Figure 1 c in the figure respectively show the IL-40 expression level detection result graphs in the peripheral blood of clinical adult sepsis patients and healthy subjects, the IL-40 expression level detection result graphs in the peripheral blood of adult septic shock patients and non-septic shock patients, and the IL-40 expression level detection result graphs in the peripheral blood of deceased adult sepsis patients and surviving adult sepsis patients in the embodiment of the present invention;

[0020] Figure 1 In d, Figure 1 e in Figure 1 f in the figure respectively show the IL-40 expression level detection result graph in the peripheral blood of clinical pediatric sepsis patients and healthy subjects in the embodiment of the present invention, the IL-40 expression level detection result graph in the peripheral blood of pediatric septic shock patients and non-septic shock patients, and the IL-40 expression level detection result graph in the peripheral blood of deceased pediatric sepsis patients and surviving pediatric sepsis patients;

[0021] Figure 1 In the g, Figure 1 h in the figure respectively show the results of correlation analysis between IL-40 expression and SOFA in the peripheral blood of adult and pediatric sepsis patients in the embodiment of the present invention;

[0022] Figure 2 a in Figure 2 b in the figure respectively shows the schematic diagram of the predictive effect of the expression of IL-40, PCT and CRP in adults and children on the 28-day mortality of sepsis patients by comparing the ROC curve in the embodiment of the present invention;

[0023] Figure 3 Shown is a diagram of the process of establishing a sepsis mouse model by cecal ligation and puncture (CLP) in an embodiment of the present invention;

[0024] Figure 4 This is a graph showing the results of an experiment to detect the expression of IL-40 in blood, peritoneal lavage fluid, liver and kidney in an animal sepsis model in an embodiment of the present invention;

[0025] Figure 5 a in the figure is a statistical graph of the survival rates of septic mice in the IL-40 protein treatment group and the control group in the embodiment of the present invention;

[0026] Figure 5 b is a statistical graph of the survival rate of septic mice in the IL-40 gene knockout mouse group and the wild control group;

[0027] Figure 6 The bacterial load in the heart blood, spleen, and peritoneal lavage fluid (PLF) of the IL-40 gene knockout septic mouse group and the wild septic mouse group in the embodiment of the present invention is shown.

[0028] Figure 7 Shown is a graph of protein content detection in PLF of the IL-40 gene knockout septic mouse group and the wild septic mouse group in the embodiment of the present invention;

[0029] Figure 8 Shown are graphs showing the results of liver / kidney function damage detection in the IL-40 gene knockout septic mouse group and the wild septic mouse group in the examples of the present invention, as well as organ pathological sections of the liver, kidney, spleen and lung. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] Detection of IL-40 in the blood of patients with sepsis and its correlation with SOFA score

[0033] Blood from patients with sepsis treated in Zhongda Hospital affiliated to Southeast University from August 2021 to December 2023 was collected as the experimental group, and healthy subjects who underwent physical examination during the same period were the healthy control group (their basic information is shown in Tables 1 and 2). The expression of IL-40 was detected using an ELISA kit (purchased from MyBiosource). The specific operation steps strictly refer to the instructions of the kit; the sequence of IL-40 has been published in "Identification of IL-40, a Novel B Cell-Associated Cytokine" published in "Journal of Immunology" in 2017. SPSS27.0 software was used for statistical analysis, and GraphPadPrism 10.0 software was used for drawing. The specific experimental details are as follows:

[0034] 1) Grouping of research subjects and establishment of inclusion / exclusion criteria: divided into sepsis group, septic shock group and healthy control group. Establishment of inclusion criteria: ① Sepsis patients: ICU hospitalized patients; aged 18 years or older; clinically diagnosed with sepsis, and the diagnostic criteria are in accordance with the "2021 International Guidelines for the Management of Sepsis and Septic Shock". ② Septic shock patients: ICU hospitalized patients; aged 18 years or older; clinically diagnosed with septic shock, and the diagnostic criteria are in accordance with the "2021 International Guidelines for the Management of Sepsis and Septic Shock", specifically manifested as refractory hypotension on the basis of sepsis, the need for continuous use of vasopressors to maintain a mean arterial pressure of more than 65 mmHg and blood lactate> 2 mmol / L. ③ Healthy controls: healthy physical examination subjects matched with the disease group in age and gender. Exclusion criteria: patients with malignant tumors; patients with severe blood system diseases, including but not limited to lymphoma, acute / chronic leukemia, aplastic anemia, etc.; patients with active autoimmune system diseases, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, etc.; patients with chronic liver damage (score greater than 3 points), chronic renal failure (stage 3 and above); pregnant women; patients with continuous hormone therapy (0.5 mg / kg) for >30 days.

[0035] 2) Sample data collection: record the patient's personal information, medication status and vital signs; calculate the patient's acute physiology and chronic health evaluation score (APACHEⅡ) and sequential organ failure assessment (SOFA); and record the patient's 28-day survival status (as shown in Tables 1 and 2).

[0036] 3) Sample collection and processing: For patients in the experimental group who met the inclusion criteria, 2 mL of peripheral venous blood was collected on the first day after admission, and 2 mL of peripheral venous blood was collected on the day of physical examination in the control group. The blood was centrifuged within 1 hour (3500 rpm, 10 minutes) and the serum was separated and packaged in 200 μL / tubes and stored at -80°C.

[0037] 4) Sample testing: ELISA was used to test the expression level of IL-40 protein in serum. At the same time, the samples were sent to the laboratory for testing of laboratory indicators, including PCT, CRP, blood routine, liver and kidney function, and coagulation function.

[0038] 5) Data analysis: Nonparametric paired test was used to compare the expression differences of IL-40 between sepsis patients and healthy subjects, septic shock patients and non-septic shock patients, and deceased sepsis patients and surviving sepsis patients; correlation analysis was used to determine the correlation between IL-40 and SOFA score; ROC curve was used to calculate the area under the curve (AUC), sensitivity (Sensitivity), specificity (Specificity), IL-40 cut-off value for predicting 28-day survival of sepsis and the corresponding Youden Index.

[0039] Table 1 Basic information statistics of adult sepsis patients and healthy subjects

[0040]

[0041]

[0042] Table 2 Basic information statistics of children with sepsis and healthy subjects

[0043]

[0044] The experimental results of this embodiment are as follows Figure 1 As shown in Figure 2, IL-40 expression is significantly increased in adult patients with sepsis ( Figure 1 In a), the expression level of IL-40 in the peripheral blood of patients with septic shock was significantly higher than that of patients with sepsis without shock ( Figure 1 b), and the IL-40 content in the peripheral blood of patients who died of sepsis was significantly higher than that of survivors ( Figure 1 c), with statistically significant differences. The IL-40 expression in children with sepsis was also significantly higher than that in normal children ( Figure 1 d), the expression of IL-40 in peripheral blood of children with septic shock was significantly higher than that of children with sepsis without shock ( Figure 1 e), and the IL-40 level in children who died of sepsis was also significantly higher than that in those who survived ( Figure 1 f). Further correlation analysis revealed that the expression of IL-40 in the peripheral blood of adult and pediatric sepsis patients was positively correlated with their SOFA scores. Figure 1 gh), indicating that IL-40 expression is elevated in sepsis and may become a novel diagnostic indicator for sepsis, and the expression level of IL-40 is positively correlated with the severity of sepsis.

[0045] Example 2

[0046] ROC curve analysis was used to compare the prognostic evaluation effects of serum IL-40, PCT and CRP on sepsis;

[0047] Then, adult / children sepsis patients were divided into survival group and death group according to their 28-day survival status. The expression of IL-40 in their peripheral blood was detected by ELISA, and the laboratory indicators PCT and CRP levels of sepsis patients were collected through the LIS system of the laboratory department. Then, ROC curve was drawn using SPSS27.0 software, and the statistical differences of indicators such as the area under the ROC curve were analyzed and compared.

[0048] Table 3 ROC curve analysis of the prognostic efficacy of IL-40 / CRP / PCT in adults with sepsis

[0049]

[0050] The results are as follows Figure 2 Figure a and Table 3 show that in adult sepsis, the areas under the ROC curve of serum IL-40, PCT, and CRP for predicting 28-day survival in sepsis were 0.801, 0.445, and 0.489, respectively. The area under the curve of IL-40 was significantly greater than that of PCT and CRP, which was statistically significant, indicating that IL-40 can be used to predict the prognosis of patients with sepsis.

[0051] Table 4 ROC curve analysis of the prognostic efficacy of IL-40 / CRP / PCT in children with sepsis

[0052]

[0053] The results are as follows Figure 2 Figure b and Table 4 show that in children with sepsis, the areas under the ROC curves of serum IL-40, PCT, and CRP for predicting 28-day survival in sepsis were 0.876, 0.570, and 0.519, respectively. The area under the curve of IL-40 was significantly greater than that of PCT and CRP, and was statistically significant, further indicating that IL-40 can be used to predict the prognosis of patients with sepsis.

[0054] Example 3

[0055] Cecal ligation and puncture (CLP) was used to establish the animal model of sepsis.

[0056] Experimental animals: The mice used in the experiment were male, weighing 18-22g, and about 6-8 weeks old. The mice used in the experiment were all SPF (Specific Pathogen Free) grade experimental animals and were raised in the SPF grade laboratory of the Experimental Animal Center of Southeast University. Wild type C57BL / 6 mice (Wild type, WT) were purchased from Beijing Huafukang Biotechnology Co., Ltd. IL-40-deficient mice were purchased from Guangzhou Saiye Biotechnology Co., Ltd.

[0057] Methods: Mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital, fixed on an operating board, and the abdomen was shaved with a pet electric clipper. The skin was disinfected, and a 1-cm long incision was made in the middle of the abdomen. The cecum was ligated and punctured with a 26-gauge syringe needle. Figure 3 As shown. Finally, the wound is sutured and the skin is disinfected. (Specific reference: Daniel Rittirsch, Peter A Ward, et al. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc. 2009; 4(1): 31–36). This modeling method is a classic modeling method for sepsis animal models, and this model is currently the gold standard animal model for studying sepsis.

[0058] Example 4

[0059] Detection of IL-40 content in sepsis mouse model;

[0060] The mouse sepsis model was established by CLP (as shown in Example 3), and the mouse heart blood, peritoneal lavage fluid (PLF), liver and kidney samples were collected at 6 hours and 24 hours, respectively, and the expression of IL-40 was detected by ELISA kit (purchased from MyBiosource). The specific operation steps were strictly referred to the kit instructions. GraphPad Prism 10.0 software was used for statistical analysis and drawing.

[0061] like Figure 4 a in Figure 4 b in Figure 4 c and Figure 4 As shown in (d), compared with wild-type control mice, the expression of IL-40 in peripheral blood, PLF, liver and kidney of septic mice was significantly increased, with statistical significance.

[0062] Example 5

[0063] Survival rate experiment:

[0064] A mouse sepsis model was established (as shown in Example 3) and divided into two groups (control group and recombinant IL-40 protein treatment group). The control group was given an intraperitoneal injection of 100 μL sterile PBS buffer after modeling, and the protein treatment group was given 1 μg of recombinant IL-40 protein (purchased from MyBiosource) in a volume of 100 μL and intraperitoneally injected into the mice after modeling. The survival of the mice was observed and recorded twice a day until the mice stopped dying, for about 2 weeks;

[0065] The results are as follows Figure 5 As shown in a, treatment with exogenous recombinant IL-40 (1 μg / 100 μL, 1 μg IL-40 dissolved in 100 μL sterile PBS buffer, the same below) can reduce the survival rate of septic mice from 50% to 0%, suggesting that IL-40 is not conducive to the survival of septic mice.

[0066] To further verify this conclusion, in this example, IL-40- / - mice were constructed and a mouse sepsis model was established by CLP (as shown in Example 3), and the mice were divided into a wild-type group (WT) and an IL-40- / - group. After modeling, 100 μL of sterile PBS was intraperitoneally injected, and the survival of the mice was observed for 2 consecutive weeks, twice a day.

[0067] The results are as follows Figure 5 As shown in b, compared with the wild-type septic mouse group, the survival rate of septic mice in the IL-40- / - group was significantly increased, increasing the survival rate of septic mice from 20% to 60%, which was statistically significant, further demonstrating that IL-40 plays a damaging role in sepsis.

[0068] Example 6

[0069] Determination of bacterial load;

[0070] A mouse sepsis model was established by CLP (as shown in Example 3). After 24 hours, the mice were anesthetized and fixed, and peritoneal lavage fluid, heart blood and spleen were taken respectively. The blood was diluted 10-fold serially and 100 μl was plated. The peritoneal lavage fluid was diluted 100-fold and 10,000-fold serially and 100 μl was plated. The spleen was first homogenized and then diluted 10-fold serially and 100 μl was plated. After 18-24 hours, the number of colonies on the blood agar plate was counted.

[0071] The results are as follows Figure 6 a in Figure 6 b in Figure 6 As shown in (c), compared with wild-type septic mice, the bacterial load in the blood, PLF, and spleen of septic mice in the IL-40- / - group was significantly reduced, suggesting that IL-40 can impair the body's ability to clear bacteria.

[0072] Example 7

[0073] Determination of total protein content of peritoneal lavage fluid;

[0074] A mouse sepsis model was established by CLP (as shown in Example 3). After 24 hours, the mice were anesthetized and fixed, and the peritoneal lavage fluid was collected and the total protein content was detected using a protein detection kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0075] The results are as follows Figure 7 As shown, compared with wild-type septic mice, the protein content in PLF of septic mice in the IL-40- / - group was significantly reduced, and the difference was statistically significant, further indicating that IL-40 can aggravate vascular damage in septic mice.

[0076] Example 8

[0077] Biochemical and pathological examination of various organ damage

[0078] A mouse sepsis model was established by CLP (as shown in Example 3). After modeling, 100 μL of sterile PBS buffer was intraperitoneally injected. After 24 hours, the mice were anesthetized and killed, and the heart blood was collected. The supernatant was collected after centrifugation to detect liver / kidney injury-related indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and urea (Urea); at the same time, the lungs, livers, spleens and kidneys of the septic mice were removed, fixed in 4% paraformaldehyde for 24 to 48 hours, dehydrated, waxed, embedded, and sliced, and examined under a microscope after H&E staining.

[0079] The results are as follows Figure 8 As shown in ad, compared with wild-type septic mice, the levels of AST, ALT, Urea and LDH, biochemical markers of liver and kidney injury, in the serum of IL-40- / - septic mice were significantly reduced, indicating that IL-40 aggravated the multi-organ damage in septic mice ( Figure 8 ad in the figure); pathological sections showed that the inflammation of the liver, kidney, lung and spleen of IL-40- / - group septic mice was significantly alleviated, and the manifestations of inflammatory cell infiltration, protein inflammatory exudation, tissue congestion and edema, and cell necrosis were significantly improved ( Figure 8 e); This suggests that IL-40 can reflect the multi-organ inflammatory damage in sepsis.

[0080] In summary, the present invention found that the expression of IL-40 in adults and children with sepsis was significantly upregulated compared with healthy subjects, and was positively correlated with the SOFA score of sepsis patients, which can be used as a new early diagnostic biomarker for sepsis; and ROC curve analysis found that IL-40 can effectively predict the 28-day survival rate of sepsis compared with PCT and CRP. Further in vivo animal experiments found that septic mice can cause increased expression levels of IL-40, and IL-40 is positively correlated with the severity of the disease; after knocking out IL-40, the bacterial load and liver and kidney function damage in septic mice can be significantly reduced, and the survival rate of septic mice can be effectively improved. Therefore, the present invention provides IL-40 as a diagnostic, prognostic and monitoring indicator for sepsis, and provides IL-40 inhibitors and carriers as new drugs for improving and / or treating sepsis.

[0081] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0082] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Application of interleukin 40 as a biomarker in the preparation of a kit for diagnosing or screening sepsis.

2. Use of interleukin-40 detection reagent in the preparation of a kit for diagnosing or screening sepsis.

3. Application of interleukin-40 detection reagent in the preparation of a kit for monitoring the prognosis of sepsis.

4. Use of an interleukin 40 inhibitor in the preparation of a drug for treating sepsis, wherein the interleukin 40 inhibitor is a reagent for knocking out interleukin 40.

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