Exogenous administration of appropriate dose of IL-17A helps to improve multiple organ dysfunction and improve survival in septic mice

doi:10.21203/rs.3.rs-2483805/v1

Download PDF

Research Article

Exogenous administration of appropriate dose of IL-17A helps to improve multiple organ dysfunction and improve survival in septic mice

https://doi.org/10.21203/rs.3.rs-2483805/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Purpose

To explore the effect of exogenous interleukin-17A on multi-organ function and prognosis in septic mice.

Methods

Mice model of sepsis was established by cecal ligation and puncture (CLP) method, and the mice were randomly divided into 8 groups according to different intervention measures: Sham + PBS, CLP + PBS, CLP + 0.25µg IL-17A, CLP + 0.5µg IL-17A, CLP + 1µg IL-17A, CLP + 2µg IL-17A, CLP + 4µg IL-17A and CLP + Anti-IL-17A. Survival rates of each group were monitored and recorded at 12-hour intervals. The blood of each group of mice was collected at each observation node under aseptic condition, and the expression changes of blood routine, related inflammatory cytokines, liver and kidney function indexes. Pathological injuries of lung, liver and kidney of mice in each group were detected by H&E staining, and the evaluation of bacterial translocation in vitro was performed by inoculation medium.

Results

Except for the sham-operated group, the 7-day survival rate of the mice in the CLP + 1µg IL-17A group was the highest (75%) and exogenous administration of appropriate dose of IL-17A was beneficial to reduce alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cre) in septic mice. The pathological damage of lung, liver and kidney tissue also can be alleviated, and the colony count of peripheral blood and spleen tissue of mice were also significantly decreased.

Conclusion

Exogenous administration of appropriate dose of IL-17A can improve the bacterial clearance ability of septic mice, and improve the multiple organ dysfunction and 7-day survival rate of the septic mice.

sepsis

cecal ligation and puncture

cytokines

liver and kidney function

colony count

Sepsis is a major public health concern and Sepsis-3, released in 2016, is the third consensus definition of sepsis and septic shock. Here, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection[1]. Around the world, 30 million people suffer from sepsis every year, and with a mortality rate of 30–40%, it is one of the leading causes of death in critically ill patients worldwide. Sepsis can occur in anyone who is infected, and it occurs in up to 1–2% of all hospitalized patients. As a result, sepsis is also the most expensive of all diseases to treat [2, 3]. Despite recent advances in therapeutic measures such as anti-infective therapy and organ function support, the incidence of sepsis continues to rise.

Interleukin-17A (IL-17A) is the earliest known member of the IL-17 family, first identified in 1993 and originally known as cytotoxic lymphocyte antigen 8, in previous studies[4], the inflammatory response mediated by IL-17A has been shown to be required for host protection and survival against infection[5, 6]. At the same time, many studies have confirmed that IL-17A has important effects on host defense, cell transport, immune regulation and tissue repair, and plays a vital role in inducing congenital immunity defense [7]. IL-17A, as an important part of the host immune response, plays an indispensable role in sepsis, and elevated levels of IL-17A have been observed in both laboratory and human sepsis[6, 8]. However, IL-17A does not blindly play a protective role. Excessive elevation of serum IL-17A levels may be concerned with a higher risk of sepsis, and severe sepsis is often accompanied by multiple organ dysfunction, some studies have also documented that excessive production of IL-17A exaggerates the immune response and may therefore cause impaired organ function[9]. In the associated with multiple organ damage during sepsis, an increased level of IL-17A can also be found in the tissue and plasma[10–16].In conclusion, IL-17A is not only a predictor of sepsis progression, but also a therapeutic target worthy of further exploration[17].

Previous studies have demonstrated that IL-17A-deficient mice were more likely to develop sepsis than wild-type controls [10], and blocking IL-17A will hurt peritoneal clearance of E. coli. This suggests that IL-17A may be able to prevent sepsis in some circumstances [18], and some studies suggest that administration of IL-17A can restore the impaired immunity of chemokine (C-X-C motif) ligand 1(CXCL1) knockout mice[19]. Likewise, administration of IL-17A improved host antibacterial immunity after mice were challenged with Beta streptococcus and Streptococcus pneumoniae[17]. However, studies have also shown that neutralizing IL-17A protects the integrity of the intestinal barrier, reduces systemic inflammation and bacterial dissemination, and reduces mortality in mice with sepsis [20]. Zhang, Y, et al.[21] found that knockout of IL-17A in mice attenuated cardiac disorders, myocardial ischemia-reperfusion injury and remodeling. In conclusion, IL-17A plays a key role in regulating host defense against invading pathogens, for one thing, it can interact with various mediators (such as tumor necrosis factor-α(TNF-α), IL-1, IL-6) to attract tissue infiltration of neutrophils to clear invading pathogens; for another thing, it can interact with other pro-inflammatory cytokines to drive an excessive immune response, which in turn leads to the progression of inflammation and autoimmune diseases. In other words, while IL-17A helps protect the host from a variety of infectious organisms during sepsis, it also induces hyperinflammation that is detrimental to the host under certain conditions. Therefore, specific circumstances must be considered when measuring the role of IL-17A in sepsis, whether it is protective or pathogenic under specific conditions, and whether there is a does limit, which requires further research and exploration. In this experiment, different does of IL-17A were administered exogenously to septic mice. By detecting the levels of multiple inflammatory factors, changes in blood routine indexes, liver and kidney function, multi-organ pathological damage and 7-day survival rate of sepsis mice at three time points of 12h, 24h and 7 days to evaluate the effect of different doses of exogenous IL-17A on multiple organ function and prognosis in septic mice.

Animals

A total of 144 specified pathogen-free (SPF) male C57BL/6 mice (aged 6–8 weeks, weighing 20 ± 2g) were purchased from Jinan Pengyue Laboratory Animal Co., Ltd. The mice were placed in controlled temperature and humidity conditions, on a 12-hour light/dark cycle, and provided with food and water at will. All mouse experiments were conducted in strict accordance with the Regulations on the Administration of Laboratory Animals of the Ministry of Science and Technology. Meanwhile, the experiments related to animals in this study were approved by Ethics Committee of Qingdao Municipal Hospital.

Group of mice

96 mice were randomly divided into 8 groups (n = 12) according to whether received CLP and intraperitoneal injection of equal volume of PBS/IL-17A were performed, namely: Sham + PBS, CLP + PBS, CLP + 0.25µg IL-17A, CLP + 0.5µg IL-17A, CLP + 1µg IL-17A, CLP + 2µg IL-17A, CLP + 4µg IL-17A and CLP + Anti-IL-17A. Among them, the dose of Anti-IL-17A in mice of CLP + Anti-IL-17A group depends on the preliminary experimental results of other groups. Besides, 48 mice were divided into 8 groups according to the above grouping method, and were further divided into 12 h and 24 h subgroups according to the time of sacrifice after CLP (n = 3).

Cecal ligation and puncture (CLP) procedure and management

Cecal ligation and puncture (CLP) procedure was used to establish a mouse septicemia model. Say simply, preoperative mice celiac injection of 4% chloral hydrate anesthesia (100 mg/kg). Then, during sterile conditions, an incision of about 2 cm in length was made in the left lower abdomen to expose the cecum, which was ligated 1/2 with 4 − 0 silk thread. An 18-gauge needle is used to make a penetrating puncture into the ligation of the cecum, and the stool is gently squeezed out, minimizing damage to the blood vessels. Finally, after suturing the abdominal wall and skin using 4 − 0 silk, 1ml of saline was injected subcutaneously for fluid resuscitation. Then, depending on the group, mice were intraperitoneally injected with 100µl PBS or 100µl IL-17A at different does.

Sample Collection and Treatment

Survival observation experiment

All treated mice were fed and watered ad libitum on a daily basis, and the survival rate of mice was measured every 12 hours for a total of 7 days.

Complete blood count, kidney and liver function index test

After observing the survival rate of mice in every group for 7 days, the surviving mice were anesthetized with 4% chloral hydrate, and peripheral blood was collected under aseptic conditions. A part of the blood was directly measured by a veterinary blood cell analyzer for complete blood count; the other part of the blood was centrifuged at 2500r/min for 20min, and the supernatant was stored at -80℃ until measurement. Indicators include: alanine aminotransferase enzyme (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cre). Mice in each subgroup were treated in the same way after 12h and 24h respectively and the above indicators were determined.

Enzyme linked immunosorbent assay (ELISA) detection

The levels of TNF-α, IL-6, IL-10 and IL-17A in serum of septic mice in each group were detected by corresponding ELISA method. The kit (JINGMEI, Jiangsu, China) conformed to the standard protocol provided by the supplier.

Histological Processing

Lung, liver, kidney and other important tissues and organs of mice in each group were taken under sterile conditions, fixed in 4% paraformaldehyde for 24–48 hours, and then subjected to a series of operations such as paraffin embedding, tissue sectioning, and H&E staining. Microscopy for histological analysis. Lung, liver and kidney histopathological scoring was performed blindly by pathologists, and scoring indicators included hemorrhage, edema, degree and extent of inflammatory cell infiltration, degree of necrosis, and so on[22–24].

Colony load detection

Peripheral blood supernatant and spleen were collected under sterile conditions. After the supernatant of each group was diluted by the same multiple, 100µl of the dilution was evenly spread on the blood agar medium; after the spleen tissue was weighed, 50 mg of tissue was minced, separated and filtered using 1 ml of normal saline to homogenize, and the diluted tissue suspension of 100µl was inoculated in blood AGAR medium. All blood AGAR media were cultured in an incubator at 37°C for 24 h. Afterwards, count bacterial colonies and count bacterial colony formation units (CFU) per gram of tissue culture, CFU/g = plate colony number*dilution factor/tissue mass (g).

Statistical analysis

Tests of normality were performed on all measurement data. R 4.0.5 software (http://www.R-project.org) and GraphPad Prism 9.0 (GraphPad Software, USA) were used for the statistical analysis. Kaplan-Meier curve and log rank test were used for survival analysis. Values are presented as the means ± standard deviation (SD). Data were analyzed using Student’s t-test and one-way ANOVA. P < 0.05 was considered statistically significant.

survival of mice

We constructed a mice model of sepsis by CLP method, and divided them into different groups according to the aforementioned method, and observed their 7-day survival rate under conditions suitable for mice survival. The survival rate results are as follows: Sham + PBS (100%), CLP + PBS group (25%), CLP + 0.25µg IL-17A (50%), CLP + 0.5µg IL-17A (58.33%), CLP + 1µg IL-17A (75%), CLP + 2µg IL-17A (58.33%) and CLP + 4µg IL-17A (0%). It can be seen that the 7-day survival rate of CLP + 1µg IL-17A group was the highest. Therefore, we selected CLP + 1µg IL-17A group mice as the main study object, to further verify whether exogenous administration of 1µg IL-17A was the most beneficial to the survival of septic mice and whether exogenous administration of the same does of IL-17A antibody would reduce this survival advantage, we investigated the molecular weight of IL-17A and anti-IL-17A and converted it to dose. In other words, mice in CLP + Anti-IL-17A group were intraperitoneally injected with 100µl IL-17A antibody, the dose of which was equal to 1µg IL-17A. The results showed that under the same conditions, the 7-day survival rate was 33.33%. Through statistical analysis, we found that the 7-day survival rate of CLP + 1µg IL-17A group was significantly higher than that of CLP + PBS group (P < 0.05) and CLP + 4µg IL-17A group (P < 0.01), while the 7-day survival rate of CLP + 4µg IL-17A group was significantly lower than that of CLP + PBS group (P < 0.05). Meanwhile, the 7-day survival rate of CLP + 1µg IL-17A group was higher than that of CLP + Anti-IL-17A group, but there was no statistically significant difference (P > 0.05). (Fig. 1)

Behavioral performance and abdominal condition of mice

Compared with sham-operated mice, mice undergoing CLP surgery had different behavioral performances at different time periods. At 6h after CLP, mice showed symptoms such as decreased activity, decreased food and water intake, erected hair and unsteady gait. At 12h after CLP, mice tended to curl up, and showed symptoms such as increased heart rate, rapid breathing, and tremors. At 24h after CLP, white pus exudation from eyes and abdominal swelling could be observed in mice. The abdominal cavity of the mice was observed by opening the abdomen. After CLP, cloudy and foul-smelling ascites could be seen in the abdominal cavity of the mice, and some were even bloody. In addition, obvious distention of the small intestine, blackening and necrosis of the distal end of the cecal ligation, and liver edema can be observed, while upward exploration of the thoracic cavity can see scattered bleeding spots on the surface of the lungs. None of the mice in the Sham + PBS group had the above abnormalities. Compared with the other groups, no matter at 12h, 24h or 7 days after CLP, the peritoneal lesions in the CLP + 1µg IL-17A group were lighter than those in the CLP + PBS group, mainly manifested as clearer ascites and lower intestinal distention, while the general conditions of the other groups were no significant difference with the naked eye.

Blood Cell Count

As shown in Table 1, the platelet count, the neutrophil-to-lymphocyte ratio (NLR), the platelet-to-lymphocyte ratio (PLR) of mice in the Sham + PBS group and the CLP + 1µg IL-17A group, whether at 12h, 24h or 7 days after CLP group were lower than in the CLP + PBS group (all P < 0.05). At the same time, the blood cell distribution width (RDW) of the mice in the Sham + PBS group and the CLP + 1µg IL-17A group were lower than in the CLP + PBS group at 12h, 24h, and 7 days after CLP, except at 24 hours after CLP (P > 0.05), other differences were statistically significant (all P < 0.05). Moreover, at 12h and 24h after CLP, the white blood cell (WBC) counts of mice in the Sham + PBS group and the CLP + 1µg IL-17A group were higher than that in CLP + PBS group (all P < 0.05), in contrast, the WBC count of mice in the Sham + PBS group and the CLP + 1µg IL-17A group were lower than in the CLP + PBS group at 7 days after CLP (all P < 0.05). However, the neutrophil count of mice in the Sham + PBS group and CLP + 1µg IL-17A group were higher than in the CLP + PBS group only at 12h after CLP (all P < 0.05), and at 24h and 7 days after CLP were lower than the CLP + PBS group (all P < 0.05). For hemoglobin level, the hemoglobin of mice in Sham + PBS group and CLP + 1µg IL-17A group at each time point was higher than in CLP + PBS group, but only 7 days after CLP had statistical significance (P < 0.05). (Fig. 2)

Compared with the CLP + Anti-IL-17A group, the CLP + 1µg IL-17A group mice had lower neutrophil count, platelet count, NLR, PLR and RDW at 12h, 24h and 7 days after CLP (all P < 0.05), however, the neutrophil count was only statistically significant at 7 days after CLP (P < 0.05), and the other items were statistically significant at three time points (all P < 0.05); while the WBC count of mice in the CLP + 1µg IL-17A group at 12h and 24h after CLP were higher (all P < 0.05),and WBC count was only lower at 7 days after CLP (P < 0.05). In addition, at 12h, 24h, and 7 days after CLP, although the hemoglobin of mice in the CLP + 1µg IL-17A group was higher than that in CLP + PBS group, only the 7th day after CLP had statistical significance (P < 0.05). (Fig. 2)

Table 1

White blood cell count, neutrophil count, red blood cell distribution width (RDW), platelet count, hemoglobin, the neutrophil-to-lymphocyte ratio (NLR), and the platelet-to-lymphocyte ratio (PLR) of mice(x ± s) (*P < 0.05 and ** P < 0.01)
Group	White blood cell count(10^9/L)			Neutrophil count(%)			Red blood cell distribution width(RDW)༈%༉			Platelet count(10^9/L)			Hemoglobin(g/L)			Neutrophil-to-lymphocyte ratio (NLR)			Platelet-to-lymphocyte ratio (PLR)
	12h	24h	7d	12h	24h	7d	12h	24h	7d	12h	24h	7d	12h	24h	7d	12h	24h	7d	12h	24h	7d
Sham + PBS	6.17 ± 0.30 **	7.47 ± 0.04 **	7.21 ± 1.44 **	1.68± 0.02 **	2.28± 0.01 *	1.96 ± 0.15 **	15.33 ± 0.33 *	16.1 ± 0.03	16.03 ± 1.80 *	543.67 ± 2082.33 **	667.00 ± 2308.00 **	599.00 ± 484.91 **	168.33 ± 342.33	184.67 ± 50.33	174.83 ± 79.61 **	0.42 ± 0.00*	0.46 ± 0.00 **	0.39 ± 0.00 **	134.15 ± 32.62 **	134.77 ± 89.06 **	122.15 ± 309.45 **
CLP + PBS	2.37 ± 0.00	5.13 ± 0.01	13.80 ± 0.07	1.07 ± 0.01	2.70± 0.03	6.31 ± 0.12	17.03 ± 0.02	16.77 ± 0.16	18.23 ± 0.76	1282.00 ± 16176.00	1662.67 ± 3646.33	1960.67 ± 3070.33	153.67 ± 74.33	174.33± 74.33	141.33 ± 9.33	0.96 ± 0.03	1.64 ± 0.01	0.91 ± 0.01	1131.02 ± 12088.31	1012.05 ± 63.63	281.16± 25.49
CLP + 1µg IL-17A	3.93 ± 0.09 **	5.77 ± 0.01*	9.86 ± 0.06 **	1.31 ± 0.01 *	2.01± 0.00 **	3.13 ± 0.02 **	15.30 ± 0.37 *	16.20 ± 0.12	16.26 ± 0.72 *	827.67 ± 15772.33*	924.67 ± 82.33 **	790.89 ± 3099.36 **	168.33 ± 357.33	177.67± 22.33	166.89 ± 45.11 **	0.61 ± 0.00*	0.57 ± 0.00 **	0.50 ± 0.00 **	385.23 ± 69.30 **	319.81 ± 121.90 **	125.59 ± 75.19 **
CLP + Anti-IL-17A	3.37 ± 0.02 *	5.17 ± 0.02 *	10.75 ± 0.22 **	1.33 ± 0.01	2.20 ± 0.06	3.59± 0.02 **	17.87 ± 0.10 **	17.50 ± 0.25 *	18.20 ± 0.13 **	1418.67 ± 1192.33 **	1784.33 ± 7089.33 **	1082.50 ± 17481.00 **	140.67 ± 56.33 **	172.00 ± 31.00	114.50 ± 187.00 **	0.67 ± 0.00 *	0.81 ± 0.02 *	0.53 ± 0.00 *	742.42 ± 391.53 **	654.08 ± 778.05 **	160.98 ± 198.03 **

Multiple organ function effects

To investigate the effects of IL-17A on liver and kidney function in septic mice, the liver and kidney function indexes of mice in each group were measured at different time points. According to the results, ALT, AST, BUN and Cre in CLP + PBS group were higher than those in the Sham + PBS group and the CLP + 1µg IL-17A group, with statistical significance (all P < 0.05). Similarly, the ALT, AST, BUN, and Cre of mice in the CLP + Anti-IL-17A group were also higher than those in the CLP + 1µg IL-17A group at every point in time (all P < 0.05). (Fig. 3)

H&E staining of pathological sections showed that the lungs, livers and kidneys of mice in the Sham + PBS group were not significantly different from those of normal mice, while the other groups showed different degrees of inflammatory infiltration, tissue edema and necrosis, and gradually increased with time. According to the pathological manifestations, the lung is the first and most severely damaged organ, while the liver and kidney are relatively less affected. Among them, the pathological scores of various organs of mice in the CLP + PBS group were higher than that in other groups at 12h, 24h and 7 days after CLP, the differences were statistically significant (all P < 0.05). The degree of involvement of various organs in the CLP + Anti-IL-17A group was also more severe than that in the CLP + 1µg IL-17A group, and the pathological scores were higher (all P < 0.05). (Fig. 4)

Changes in the levels of sepsis-associated inflammatory cytokines

To evaluate the effect of IL-17A on systemic inflammatory responses in septic mice, the levels of relevant inflammatory cytokines in peripheral blood supernatants were measured at 12 h, 24 h and 7 d after CLP. The levels of plasma inflammatory cytokines (IL-6, IL-17A, TNF-α, IL-10) in septic mice were increased at 12 and 24 h after CLP, among which IL-6, IL-17A, IL-10 increased most significantly at 12h after CLP, and TNF-α at 24h after CLP. (Fig. 5)

Load of bacteria

CLP induces a mouse model of sepsis, causing mixed infection of multi-bacterial flora in mice. By measuring the number of bacteria in the peripheral blood and spleen of the mice, the bacterial clearance in the mice was evaluated. The results showed that there was no bacterial growth in the peripheral blood and spleen of the mice in the Sham + PBS group at each stage, while the number of colonies in the other groups was different. Number of bacterial colonies in peripheral blood and spleen of mice in CLP + 1µg IL-17A group at 12h, 24h and 7 days after CLP were lower than those in CLP + PBS group (all P < 0.05). However, compared with the mice in the CLP + Anti-IL-17A group, although the peripheral blood colony count was lower, it was only statistically significant at 7 days after CLP (P < 0.05). In addition, the proliferation rate of bacteria in spleen tissue homogenate was significantly faster than that in peripheral blood. (Fig. 6)

Sepsis is a systemic inflammatory response or immune disorder involving the failure of multiple organs in the body[25]. From being first mentioned more than 2,700 years ago to today becoming a disease with high morbidity and mortality worldwide, sepsis has affected more than 30 million people globally each year, and any infection has the potential to progress to sepsis[3].

Interleukin are released during infection and are the most important cytokine in infection. They contain large amounts of proteins secreted by white blood cells and endothelial cells that help promote cellular signaling for immune cell activation, proliferation, death, and/or movement. They are artificially divided into pro-inflammatory interleukin and anti-inflammatory interleukin. Pro-inflammatory interleukins are thought to be responsible for cell activation, tissue damage and necrosis, whereas anti-inflammatory interleukins are designed to inhibit and eventually reverse inflammatory processes[26]. Based on previous findings, the inflammatory response mediated by IL-17A has been established to be essential for host protection and survival against infection[5, 6]. As an important part of the host immune response, it also plays an indispensable role in sepsis, and many studies have shown that increased levels of IL-17A can be found in both laboratory and human sepsis[6, 8]. Nevertheless, the role of IL-17A in sepsis has been disputable. In our experiments, different does of IL-17A were administered exogenously to septic mice, using the analogy at different time points as the baseline, the effects on multi-organ function and prognosis of septic mice were analyzed from multiple perspectives.

In this study, we found that exogenous administration of appropriate does of IL-17A could remarkably improve the 7-day survival rate of septic mice, which may be related to its protective effect on CLP-induced lethal inflammatory response and multiple organ dysfunction. The host's first line of defense against infection are neutrophils, once an infection occurs, neutrophils will quickly migrate to the site of infection to destroy invading pathogens through various mechanisms, such as phagocytosis and oxidation outbreak, neutrophils extracellular trap, and carry out microbial killing[27]. At 12h after CLP, IL-17A concentration in septic mice reached a peak. At this time, we found that the neutrophil count in the peripheral blood of the mice in the CLP + 1µg IL-17A group was apparently higher than that in the CLP + PBS group. This is consistent with the higher concentration of IL-17A at the node in the CLP + PBS group, as IL-17A is able to recruit neutrophils through induction of CXC chemokines (CKs) and antimicrobial proteins at the site of infection, facilitating their migration to the site of infection[28], resulting in decreased neutrophil counts in peripheral blood. Moreover, IL-17A can not only promote the indirect recruitment of neutrophils, but also directly activate the bactericidal action of neutrophils and macrophages[29], through these methods to make the host resistant to the invasion of pathogens.

According to this theory, insufficient IL-17A production is related with elevated host susceptibility to widespread acute, chronic, and recurrent infections, and high concentrations of IL-17A are more conducive to host prognosis, however, this is not always the case. Our experimental results showed that although the IL-17A concentration of mice in CLP + PBS group was higher than that in CLP + 1µg IL-17A group 12h after CLP operation, the function of each organ was poor and the 7-day survival rate was lower. This may be related to the fact that CKs can induce leukocytes to gather at the site of infection and r and also release immune cells from the bone marrow or spleen, once gathered, leukocytes will clear away bacteria and dead cells and generate inflammation, but they will also be activated and then spread inflammation directly throughout the body. Lack of CKs or their receptors results in a quasi-immunosuppressive state and makes the body more susceptible to infection-induced lethality[30–32]. In contrast, we observed higher IL-17A concentrations in mice in the CLP + 1µg IL-17A group than in the CLP + PBS group at 24h and 7 days after CLP, while the neutrophil count was lower. Previous studies have shown that under normal physiological conditions, neutrophils maintain their homeostasis through apoptosis. However, during sepsis, neutrophils undergo a variety of functional changes, including decreased migration, altered antimicrobial activity, delayed apoptosis, resulting in immune dysfunction and persistent inflammation, making changes that contribute to the exacerbation of sepsis and the development of secondary complications[33, 34]. We consider that a certain does of IL-17A may help to improve the abnormal apoptosis of neutrophils. Of course, due to the peculiarities of neutrophil counts in infection, such as a marked increase in neutrophil counts during infection, usually correlates with the overall severity of the infection, but in a relatively severe form of sepsis, neutrophil apoptosis is delayed, which would limit the usefulness of neutrophil counts in certain circumstances[35].Therefore, the diagnostic and prognostic power of neutrophil count alone is poor for sepsis, but multiple studies[36–38] have shown that NLR has been regarded as a reliable biomarker of sepsis, with elevated levels independently associated with poor prognosis in patients with sepsis. This is also consistent with our results. In addition, a meta-analysis on the prognostic value of PLR in sepsis[39] showed that high PLR predicts worse prognosis. These are consistent with our results.

Disruption of inflammatory balance is the most critical feature of sepsis pathogenesis[40]. Macrophages phagocytosis of pathogens and production of a variety of pro-inflammatory cytokines are reported to be the first acute host response to pathogens. Previous studies have shown that the first sign of sepsis is an imbalance of inflammatory cytokines, accompanied by overproduction of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6[41]. The production of pro-inflammatory cytokines can trigger cytokine storms, or can stimulate the innate immune system[42, 43]. Cellular pathways that trigger the immune system leads to the over release of cytokines, chemokines, and other inflammatory mediators such as prostaglandins. Cytokines regulate inflammation by transporting immune cells to the site of infection, thereby preventing infection by controlling local areas. Furthermore, endothelial dysfunction may also be associated with irregular cytokine release, which leads to vasodilation and increased capillary permeability[40]. Studies have shown that inflammatory cytokines such as IL-6, TNF-α and IL-10 may be involved in the pathology of sepsis[40].

Of these, IL-6 can stimulate the protein synthesis and regulate the activation of immune cells in the acute phase of sepsis[44], or it can bind to its homologous receptor, the IL-6 receptor (IL-6R), and subsequently activate the downstream Akt pathway, which is Induces endothelial rupture and exacerbates organ damage during sepsis[45]. And there are studies showing that the severity of sepsis was associated with higher levels of IL-6, with worse outcomes at the highest levels of IL-6[46, 47]. TNF-α is thought to be a master regulator of inflammation, and is released into the circulatory system in the early stages of sepsis, exacerbating systemic inflammatory responses[48]. In addition, TNF-α has been shown to play a major role in mediating sepsis-induced damage to vital organs, such as the lung, because of its role in the escalation of inflammation and activation of necrosis, pyroptosis, and apoptotic cell death processes[48–52]. A meta-analysis of TNF-α[53] also showed that immunotherapy with anti-TNF monoclonal antibodies reduced overall mortality in patients with severe sepsis (pre-shock), while anti-TNF therapy may reduce overall mortality in patients with shock or high IL-6 (> 1000pg/ml), but this trend was not statistically significant. As for IL-10, Jensen et al[54] in 2021 investigated the role of IL-10 production in a CLP model, their study found that mice deficient in IL-10 experienced weight loss, increased mortality, and prolonged disease symptoms after CLP-induced sepsis. However, Oberholzer A et al[55] found that sustained release of IL-10 may lead to sepsis-induced immunosuppression, thereby may increase susceptibility to secondary microbial infections. Moreover, blocking IL-10 has been reported can reverse sepsis-induced immunosuppression and improve the survival rate of sepsis mouse models[56]. In our research, we found that the levels of IL-6, TNF-α and IL-10 were positively correlated in the peripheral blood of septic mice, and exogenous administration of appropriate does of IL-17A could decrease the levels of these inflammatory factors, which keep the levels of various inflammatory factors in a relatively balanced state, has a protective effect on CLP-induced lethal inflammatory response and multiple organ damage in mice, thereby improving the prognosis of septic mice.

As is known to all that elevated levels of ALT and AST are associated with impaired liver function, and increased expressions of Cre and BUN are also considered to be markers of kidney damage. The liver and kidney function indexes of mice after CLP were higher than those in the sham operation group, and reached the peak at 24h after CLP. We found that appropriate levels of IL-17A could reduce these test indexes and then improve the liver and kidney function of septic mice. At the same time, the pathological results can also support this point of view. Compared with the CLP + PBS group, the pathological scores of the mice in the CLP + 1µg IL-17A group were significantly lower, which may be related to the higher bacterial clearance rate. Because we found that the severity of organ damage increased with the number of bacteria. In addition, bacteria in spleen tissue homogenate multiplied much faster than those in peripheral blood.

This study has certain limitations. First, we only measured the levels of inflammatory factors in the peripheral blood of septic mice, and did not detect the levels of inflammatory factors and inflammatory cells in various organs; Secondly, the sample size at 12h and 24h after CLP is relatively small, which will lead to a greater possibility of error in the results; In addition, our experiments explored the effect of exogenous IL-17A on the organ function of mice through organ function indicators and pathological sections, but its specific mechanism still needs to be further explored.

In conclusion, our data suggest that exogenous administration of appropriate dose of IL-17A is beneficial for improving outcomes in septic mice by increasing bacterial clearance and attenuating multiple organ dysfunction in septic mice. Moreover, the effects of various inflammatory factors on the prognosis of septic mice are not simply determined by their levels, and a relatively balanced state is more conducive to the survival of septic mice.

Data availability statement

All data generated or analyzed during the course of this study are included in this article.

Acknowledgments

This research was partially funded by the National Natural Science Foundation of China in 2018 (No.81971873).

Disclosure

The author reports no conflicts of interest in this work.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Haining Meng, Weifeng Xie and Xiangqi Meng. The first draft of the manuscript was written by Yonghui Liang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics statement

The animal study was reviewed and approved by Ethics Committee of Qingdao Municipal Hospital.

Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM et al: The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2016, 315(8):801–810.
Fleischmann C, Scherag A, Adhikari NK, Hartog CS, Tsaganos T, Schlattmann P, Angus DC, Reinhart K, International Forum of Acute Care T: Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations. Am J Respir Crit Care Med 2016, 193(3):259–272.
Rocheteau P, Chatre L, Briand D, Mebarki M, Jouvion G, Bardon J, Crochemore C, Serrani P, Lecci PP, Latil M et al: Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy. Nat Commun 2015, 6:10145.
Rouvier E, Luciani MF, Mattei MG, Denizot F, Golstein P: CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene. J Immunol 1993, 150(12):5445–5456.
Song X, Zhu S, Shi P, Liu Y, Shi Y, Levin SD, Qian Y: IL-17RE is the functional receptor for IL-17C and mediates mucosal immunity to infection with intestinal pathogens. Nat Immunol 2011, 12(12):1151–1158.
Li Y, Wei C, Xu H, Jia J, Wei Z, Guo R, Jia Y, Wu Y, Li Y, Qi X et al: The Immunoregulation of Th17 in Host against Intracellular Bacterial Infection. Mediators Inflamm 2018, 2018:6587296.
Ge Y, Huang M, Yao YM: Biology of Interleukin-17 and Its Pathophysiological Significance in Sepsis. Front Immunol 2020, 11:1558.
Chamoun MN, Blumenthal A, Sullivan MJ, Schembri MA, Ulett GC: Bacterial pathogenesis and interleukin-17: interconnecting mechanisms of immune regulation, host genetics, and microbial virulence that influence severity of infection. Crit Rev Microbiol 2018, 44(4):465–486.
Pool R, Gomez H, Kellum JA: Mechanisms of Organ Dysfunction in Sepsis. Crit Care Clin 2018, 34(1):63–80.
Flierl MA, Rittirsch D, Gao H, Hoesel LM, Nadeau BA, Day DE, Zetoune FS, Sarma JV, Huber-Lang MS, Ferrara JL et al: Adverse functions of IL-17A in experimental sepsis. FASEB J 2008, 22(7):2198–2205.
Freitas A, Alves-Filho JC, Victoni T, Secher T, Lemos HP, Sonego F, Cunha FQ, Ryffel B: IL-17 receptor signaling is required to control polymicrobial sepsis. J Immunol 2009, 182(12):7846–7854.
Accardo Palumbo A, Forte GI, Pileri D, Vaccarino L, Conte F, D'Amelio L, Palmeri M, Triolo A, D'Arpa N, Scola L et al: Analysis of IL-6, IL-10 and IL-17 genetic polymorphisms as risk factors for sepsis development in burned patients. Burns 2012, 38(2):208–213.
Wu HP, Shih CC, Chu CM, Huang CY, Hua CC, Liu YC, Chuang DY: Effect of interleukin-17 on in vitro cytokine production in healthy controls and patients with severe sepsis. J Formos Med Assoc 2015, 114(12):1250–1257.
Paraschos MD, Patrani M, Pistiki A, Katsenos C, Tsaganos T, Netea MG, Giamarellos-Bourboulis EJ, Mandragos K: Defective cytokine production early after multiple traumas: Modulation in severe sepsis. Cytokine 2015, 76(2):222–226.
Ahmed Ali M, Mikhael ES, Abdelkader A, Mansour L, El Essawy R, El Sayed R, Eladawy A, Mukhtar A: Interleukin-17 as a predictor of sepsis in polytrauma patients: a prospective cohort study. Eur J Trauma Emerg Surg 2018, 44(4):621–626.
Han Y, Li X, Gao S, Liu X, Kang L, Li X, Lang Y, Li X, Sun M, Gai Z et al: Interleukin 17 is an important pathogenicity gene in pediatric sepsis. J Cell Biochem 2019, 120(3):3664–3671.
Rendon JL, Choudhry MA: Th17 cells: critical mediators of host responses to burn injury and sepsis. J Leukoc Biol 2012, 92(3):529–538.
Delano MJ, Ward PA: Sepsis-induced immune dysfunction: can immune therapies reduce mortality? J Clin Invest 2016, 126(1):23–31.
Jin L, Batra S, Douda DN, Palaniyar N, Jeyaseelan S: CXCL1 contributes to host defense in polymicrobial sepsis via modulating T cell and neutrophil functions. J Immunol 2014, 193(7):3549–3558.
Ogiku M, Kono H, Hara M, Tsuchiya M, Fujii H: Interleukin-17A plays a pivotal role in polymicrobial sepsis according to studies using IL-17A knockout mice. J Surg Res 2012, 174(1):142–149.
Zhang Y, Zhang YY, Li TT, Wang J, Jiang Y, Zhao Y, Jin XX, Xue GL, Yang Y, Zhang XF et al: Ablation of interleukin-17 alleviated cardiac interstitial fibrosis and improved cardiac function via inhibiting long non-coding RNA-AK081284 in diabetic mice. J Mol Cell Cardiol 2018, 115:64–72.
Drechsler S, Weixelbaumer KM, Weidinger A, Raeven P, Khadem A, Redl H, van Griensven M, Bahrami S, Remick D, Kozlov A et al: Why do they die? Comparison of selected aspects of organ injury and dysfunction in mice surviving and dying in acute abdominal sepsis. Intensive Care Med Exp 2015, 3(1):48.
Harchegani AL, Hemmati AA, Nili-Ahmadabadi A, Darabi B, Shabib S: Cromolyn Sodium Attenuates Paraquat-Induced Lung Injury by Modulation of Proinflammatory Cytokines. Drug Res (Stuttg) 2017, 67(5):283–288.
Li Y, Cao Y, Xiao J, Shang J, Tan Q, Ping F, Huang W, Wu F, Zhang H, Zhang X: Inhibitor of apoptosis-stimulating protein of p53 inhibits ferroptosis and alleviates intestinal ischemia/reperfusion-induced acute lung injury. Cell Death Differ 2020, 27(9):2635–2650.
Huang M, Cai S, Su J: The Pathogenesis of Sepsis and Potential Therapeutic Targets. Int J Mol Sci 2019, 20(21).
Chousterman BG, Swirski FK, Weber GF: Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol 2017, 39(5):517–528.
Witter AR, Okunnu BM, Berg RE: The Essential Role of Neutrophils during Infection with the Intracellular Bacterial Pathogen Listeria monocytogenes. J Immunol 2016, 197(5):1557–1565.
Ishigame H, Kakuta S, Nagai T, Kadoki M, Nambu A, Komiyama Y, Fujikado N, Tanahashi Y, Akitsu A, Kotaki H et al: Differential roles of interleukin-17A and – 17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity 2009, 30(1):108–119.
Lu YJ, Gross J, Bogaert D, Finn A, Bagrade L, Zhang Q, Kolls JK, Srivastava A, Lundgren A, Forte S et al: Interleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog 2008, 4(9):e1000159.
Cai S, Batra S, Lira SA, Kolls JK, Jeyaseelan S: CXCL1 regulates pulmonary host defense to Klebsiella Infection via CXCL2, CXCL5, NF-kappaB, and MAPKs. J Immunol 2010, 185(10):6214–6225.
Lionakis MS, Swamydas M, Fischer BG, Plantinga TS, Johnson MD, Jaeger M, Green NM, Masedunskas A, Weigert R, Mikelis C et al: CX3CR1-dependent renal macrophage survival promotes Candida control and host survival. J Clin Invest 2013, 123(12):5035–5051.
Chousterman BG, Boissonnas A, Poupel L, Baudesson de Chanville C, Adam J, Tabibzadeh N, Licata F, Lukaszewicz AC, Lombes A, Deterre P et al: Ly6Chigh Monocytes Protect against Kidney Damage during Sepsis via a CX3CR1-Dependent Adhesion Mechanism. J Am Soc Nephrol 2016, 27(3):792–803.
Resende CB, Borges I, Goncalves WA, Carneiro R, Rezende BM, Pinho V, Nobre V, Teixeira MM: Neutrophil activity in sepsis: a systematic review. Braz J Med Biol Res 2020, 53(12):e7851.
Gao L, Shi Q, Li H, Guo Q, Yan J, Zhou L: Prognostic value of the combined variability of mean platelet volume and neutrophil percentage for short-term clinical outcomes of sepsis patients. Postgrad Med 2021, 133(6):604–612.
Agnello L, Giglio RV, Bivona G, Scazzone C, Gambino CM, Iacona A, Ciaccio AM, Lo Sasso B, Ciaccio M: The Value of a Complete Blood Count (CBC) for Sepsis Diagnosis and Prognosis. Diagnostics (Basel) 2021, 11(10).
Huang Z, Fu Z, Huang W, Huang K: Prognostic value of neutrophil-to-lymphocyte ratio in sepsis: A meta-analysis. Am J Emerg Med 2020, 38(3):641–647.
Meshaal MS, Nagi A, Eldamaty A, Elnaggar W, Gaber M, Rizk H: Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) as independent predictors of outcome in infective endocarditis (IE). Egypt Heart J 2019, 71(1):13.
Kumarasamy C, Sabarimurugan S, Madurantakam RM, Lakhotiya K, Samiappan S, Baxi S, Nachimuthu R, Gothandam KM, Jayaraj R: Prognostic significance of blood inflammatory biomarkers NLR, PLR, and LMR in cancer-A protocol for systematic review and meta-analysis. Medicine (Baltimore) 2019, 98(24):e14834.
Wang G, Mivefroshan A, Yaghoobpoor S, Khanzadeh S, Siri G, Rahmani F, Aleseidi S: Prognostic Value of Platelet to Lymphocyte Ratio in Sepsis: A Systematic Review and Meta-analysis. Biomed Res Int 2022, 2022:9056363.
Doganyigit Z, Eroglu E, Akyuz E: Inflammatory mediators of cytokines and chemokines in sepsis: From bench to bedside. Hum Exp Toxicol 2022, 41:9603271221078871.
Hawiger J, Veach RA, Zienkiewicz J: New paradigms in sepsis: from prevention to protection of failing microcirculation. J Thromb Haemost 2015, 13(10):1743–1756.
Takeuchi O, Akira S: Pattern recognition receptors and inflammation. Cell 2010, 140(6):805–820.
D'Elia RV, Harrison K, Oyston PC, Lukaszewski RA, Clark GC: Targeting the "cytokine storm" for therapeutic benefit. Clin Vaccine Immunol 2013, 20(3):319–327.
Uciechowski P, Dempke WCM: Interleukin-6: A Masterplayer in the Cytokine Network. Oncology 2020, 98(3):131–137.
Zegeye MM, Lindkvist M, Falker K, Kumawat AK, Paramel G, Grenegard M, Sirsjo A, Ljungberg LU: Activation of the JAK/STAT3 and PI3K/AKT pathways are crucial for IL-6 trans-signaling-mediated pro-inflammatory response in human vascular endothelial cells. Cell Commun Signal 2018, 16(1):55.
Gouel-Cheron A, Allaouchiche B, Guignant C, Davin F, Floccard B, Monneret G, AzuRea G: Early interleukin-6 and slope of monocyte human leukocyte antigen-DR: a powerful association to predict the development of sepsis after major trauma. PLoS One 2012, 7(3):e33095.
Kellum JA, Kong L, Fink MP, Weissfeld LA, Yealy DM, Pinsky MR, Fine J, Krichevsky A, Delude RL, Angus DC et al: Understanding the inflammatory cytokine response in pneumonia and sepsis: results of the Genetic and Inflammatory Markers of Sepsis (GenIMS) Study. Arch Intern Med 2007, 167(15):1655–1663.
Parameswaran N, Patial S: Tumor necrosis factor-alpha signaling in macrophages. Crit Rev Eukaryot Gene Expr 2010, 20(2):87–103.
Sheikh MS, Huang Y: Death receptor activation complexes: it takes two to activate TNF receptor 1. Cell Cycle 2003, 2(6):550–552.
Challa S, Chan FK: Going up in flames: necrotic cell injury and inflammatory diseases. Cell Mol Life Sci 2010, 67(19):3241–3253.
Wang S, El-Deiry WS: TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 2003, 22(53):8628–8633.
Conos SA, Chen KW, De Nardo D, Hara H, Whitehead L, Nunez G, Masters SL, Murphy JM, Schroder K, Vaux DL et al: Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner. Proc Natl Acad Sci U S A 2017, 114(6):E961-E969.
Lv S, Han M, Yi R, Kwon S, Dai C, Wang R: Anti-TNF-alpha therapy for patients with sepsis: a systematic meta-analysis. Int J Clin Pract 2014, 68(4):520–528.
Jensen IJ, McGonagill PW, Butler NS, Harty JT, Griffith TS, Badovinac VP: NK Cell-Derived IL-10 Supports Host Survival during Sepsis. J Immunol 2021, 206(6):1171–1180.
Oberholzer A, Oberholzer C, Moldawer LL: Interleukin-10: a complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug. Crit Care Med 2002, 30(1 Suppl):S58-63.
Hiraki S, Ono S, Tsujimoto H, Kinoshita M, Takahata R, Miyazaki H, Saitoh D, Hase K: Neutralization of interleukin-10 or transforming growth factor-beta decreases the percentages of CD4 + CD25 + Foxp3 + regulatory T cells in septic mice, thereby leading to an improved survival. Surgery 2012, 151(2):313–322.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Exogenous administration of appropriate dose of IL-17A helps to improve multiple organ dysfunction and improve survival in septic mice

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Material And Methods

Animals

Group of mice

Cecal ligation and puncture (CLP) procedure and management

Sample Collection and Treatment

Survival observation experiment

Complete blood count, kidney and liver function index test

Enzyme linked immunosorbent assay (ELISA) detection

Histological Processing

Colony load detection

Statistical analysis

Results

survival of mice

Behavioral performance and abdominal condition of mice

Blood Cell Count

Multiple organ function effects

Changes in the levels of sepsis-associated inflammatory cytokines

Load of bacteria

Discussion

Conclusion

Declarations

Data availability statement

Acknowledgments

Disclosure

Author contributions

Ethics statement

References

Additional Declarations

Status:

Version 1