INTRODUCTION
A cytokine storm (CS) is an uncontrolled systemic inflammatory response involving elevated circulating cytokine levels and immune cell over-activation, which can be triggered by treatment, pathogenic infections, cancer, and autoimmune diseases [
1,
2]. Although the factors driving CS may differ, late-stage clinical manifestations converge and often overlap [
3].Under normal stress, the inflammatory response mediated by increased cytokines helps control infection, which is beneficial for the body to maintain the stability of the internal environment [
4]. However, uncontrolled inflammatory responses can mediate CS that can cause severe damage to the host, including acute respiratory distress syndrome, kidney failure, acute liver injury, multiple organ failure (MOF), and even death [
2]. Therefore, accurately identifying inflammatory responses and CS is vital for treating patients with associated diseases using selective anti-inflammatory therapies.
With the continuous outbreak of the Ebola virus, avian influenza, SARS coronavirus, and novel Coronavirus in recent years, the role of CS in patients with sepsis, MOF, and severe pneumonia has become a significant concern [
5,
6]. Defining the normal inflammatory response and CS have attracted considerable attention for preventing and treating severe patients. The tumor necrosis factor-α (TNF-α), interleukin-18 (IL-18), interleukin-6 (IL-6), interferon-γ (IFN-γ), interleukin-4 (IL-4), and interleukin-10 (IL-10) cytokines represent the key mediators driving CS. Therefore, clinical attempts have been made to distinguish CS by detecting cytokine levels [
7]. Due to the short half-life of cytokines, the changes in the cytokine content are not completely consistent with the severity of the disease. It is difficult to distinguish a CS from an inflammatory response by detecting cytokine levels in the systemic circulation. Furthermore, it cannot accurately reflect the severity of the disease or confirm the diagnostic threshold of a CS [
5]. In recent years, some studies have shown that the content changes in non-specific inflammatory markers, such as C-reactive protein (CRP), are related to the severity of the disease and can be used for the auxiliary detection of CS [
8,
9]. However, the sensitivity and specificity of these markers still do not meet the needs for the clinical diagnosis and treatment of CS.
TNF-α is divided into transmembrane TNF-α (tmTNF-α) and soluble TNF-α (sTNF-α). tmTNF-α is a precursor of sTNF-α, expressed as 26kd on the cell membrane and cleaved to sTNF-α (blood TNF-α) via the TACE enzyme [
10]. However, the role of tmTNF-α in disease development differs from sTNF-α. Moreover, tmTNF-α is often difficult to detect or can only be determined at low levels, even upon stimulation, due to being cleaved chiefly by the TNF-α converting enzyme [
11]. Several studies have shown abnormally high tmTNF-α expression in the neutrophils during acute inflammation [
12], non-small cell lung cancer [
13], and primary breast cancer [
14]. Therefore, the role of tmTNF-α in diseases has attracted increasing attention. This study aims to elucidate the diagnostic value of tmTNF-α in CS involving MOF.
MATERIALS AND METHODS
Experimental Animals
The animal study was approved by the Animal Care and Use Committee of Guizhou University of Traditional Chinese Medicine. Six- to 7-week-old male C57BL/6 mice were purchased from Sibefu Biotechnology (BeiJin, China). The animals were housed in pathogen-free conditions and allowed free access to water and food.
Animal Model Preparation
Liver failure was induced via intraperitoneal injection with 10 μg/kg LPS (Sigma-Aldrich; Escherichia coli 0111: B) and 500 mg/kg D-gal (Sigma-Aldrich). Liver tissue and blood samples were then collected before (0 h) and after (2 h, 4 h, and 6 h) injection.
Flow Cytometry (FCM) Analysis
Here, 50 ML of blood was incubated at room temperature for 40 min with 5μL of a PE-conjugated anti-mouse TNF-α antibody, after which 300μL stromatolyser-4DL FFD-201A was added for 5 min, followed by 500μL PBS. The mixture was centrifuged at 2500 g for 5 min, after which the supernatant was discarded, and 300μL PBS was added for resuspension. The stained cells were analyzed using an LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA). The cytometric data were analyzed using FlowJo software. The expression levels of tmTNF-α in the neutrophils and peripheral blood mononuclear cells (PBMCs) were analyzed after removing the adherent cells and a small number of dead cells. All the reagents involved in FCM were purchased from BD Biosciences.
Enzyme-linked Immunosorbent Assay (ELISA)
The TNF-α, IL-18, INF-γ, IL-6, IL-4, and CRP levels were measured using commercial ELISA kits (HePeng Biological) according to the protocols of the manufacturer. Briefly, the plasma and standard were transferred to an ELISA plate and mixed for 1 h at 37 °C. After washing, enzyme-labeled antibody working liquid was added to each well for 30 min at 37 °C. Subsequently, the reaction was terminated using a termination solution, and the absorbance of each well was measured at a wave length of 450 nm. The sample content was determined via a standard curve, which consisted of the concentration gradient of the standard substance on the X-axis plotted against the optical density value on the Y-axis.
Histology
The liver and kidney tissues were fixed with 10% formalin for 24 h and then embedded in paraffin wax. The tissue Sects. (4 μm) were routinely stained with hematoxylin–eosin (H&E) for morphological evaluation.
The Detection of Liver and Renal Function
The ALT and AST levels of the creatinine (Cr) and cystatin C (CysC) were measured using a Mindray ExC8100 system in the clinical laboratory of the Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine.
Statistical Analysis
The data were shown as the median with standard deviation. The differences between the two groups were assessed via Mann–Whitney tests(unpaired tests). A value of P < 0.05 was considered statistically significant. An AUC was used to sensitively and specifically determine the predictive values of the tested biomarkers to discriminate between the mice with liver failure (6 h after injection) and the HC group and between the mice displaying an inflammatory reaction and CS. All statistical analyses were performed using GraphPad Prism V9 software.
DISCUSSION
No single definition of CS is commonly accepted, while there is disagreement about how these disorders differ from an appropriate inflammatory response [
3]. However, studies have shown that CS, in addition to high serum cytokine levels, is often accompanied by organ dysfunction and histopathological damage, which are closely related to the occurrence of acute liver failure, MOF, and sepsis [
15‐
17]. In recent years, research has indicated that severe Ebola, avian influenza, SARS coronavirus, and novel coronavirus infections are also associated with the occurrence of CS [
18,
19]. However, a reliable method for diagnosing CS is still lacking.
CS is closely associated with the exacerbation of acute liver failure [
20,
21]. Liver injury induced by LPS/D-gal is a common liver failure model, presenting similar clinical symptoms to patients with liver failure. In addition to liver tissue damage and abnormal liver function, it is often accompanied by hepatic encephalopathy, lung injury, and renal injury [
22,
23]. This study indicated that the blood TNF-α at 2 h after LPS/D-gal injection was significantly higher than the normal control but decreased to normal levels at 6 h. However, the tmTNF-α expression differed from the blood TNF-α, which was relatively low at 2 h and 4 h after injection but was substantially higher at 6 h, increasing with the deterioration of the damaged liver and kidney tissues. Common clinical markers for CS diagnosis include IL-6, IFN-γ, IL-4, and IL-18 [
24‐
28]. Here, changes in the blood levels of these cytokines were different from those in tmTNF-α, which, despite their high levels, were not consistent with the severity of liver and kidney tissue injury. The expression levels of these cytokines did not reach a maximum in the most severe liver tissue damage cases. Except for IL-18, no significant differences were evident in the serum cytokines between 6 and 4 h after LPS/D-gal injection or between the live and dead mice. Significant differences were evident between the tmTNF-α expression levels at 6 h and 4 h after injection, as well as between the survival and death rates of the mice, suggesting that tmTNF-α displayed potential for diagnosing CS.
ROC curve analysis is a commonly used statistical method in clinical diagnostic tests [
29,
30]. The diagnostic ability of test indexes can be directly judged according to the AUC value. To clarify the diagnostic ability of tmTNF-α, this study calculated the AUC value of tmTNF-α for CSwhileusingIL-18 as a control. The liver and renal tissue damage were mild 4 h after LPS/D-gal injection, with slightly abnormal liver and renal function, which was used as the normal inflammatory reaction stage. However, 6 h after this treatment, the liver and renal tissue damage were severe, while the functionality of these organs was highly abnormal, representing the CS stage. ROC curve analysis showed that the tmTNF-α AUC value was significantly higher than IL-18, suggesting that tmTNF-α could effectively distinguish between the inflammatory response and CS. Furthermore, this study evaluated the AUC value of tmTNF-α in distinguishing between the dead and surviving mice to assess its role in mitigating the disease severity caused by CS, indicating that the AUC value was significantly higher than IL-18. This suggests that the tmTNF-α expression level can effectively diagnose the disease severity resulting from CS.
Although CRP is a non-specific marker for diagnosing CS [
31‐
33], its specificity and sensitivity levels do not meet clinical needs [
34]. Combined with CRP, the tmTNF-α increased significantly compared to the value of CRP alone in differentiating inflammatory responses from CS, as well as in distinguishing between live and dead mice. The ROC curve analysis showed that CRP differentiated CS from inflammatory responses with an AUC value of 0.84 (95%CI, 0.73–0.94), while the tmTNF-α and CRP combination significantly increased the diagnostic value with an AUC value of 0.98 (95% CI, 0.95–1.00). Similarly, the AUC value of CRP was only 0.82 (95% CI, 0.70–0.94) in differentiating between the surviving and dead mice, while the combined CRP displayed a significantly higher AUC value of 0.98(95%CI, 0.94–1.00). These results imply that tmTNF-α can improve the diagnostic efficiency of CRP.
This study indicates that the tmTNF-α expression in the peripheral blood neutrophils can be used to diagnose CS and assess its severity. However, at present, it is only limited to animal experiments. It is unclear whether the expression of neutrophils in the peripheral blood of patients with MOF involving CS is increased and whether the expression level is related to disease severity. Moreover, whether the tmTNF-α expression in the peripheral blood neutrophils of patients with different degrees of CS display similar results requires further investigation.
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