IL-6 knockout alleviates inammatory response and improve acute lung injury via regulating macrophage polarization in sepsis

Sepsis is a fatal disease with a high rate of morbidity and mortality, during which acute lung injury is the earliest and most serious complication. Macrophage plays a crucial role in the initiation and progress of sepsis. This study meant to explore the effect of IL-6 knockout in CLP induced sepsis. In this study, cecal ligation and puncture (CLP) was performed on wildtype and interleukin 6 (IL-6) knockout C57 mice. General condition and death rate of sepsis mice were observed. organ samples (lungs, livers, kidneys and hearts) and serum were collected for histology observation and inammatory cytokine detection. Lung tissue injury detection were conducted via lung injury score, wet/dry ration and protein concentrations measurement of Bronchoalveolar lavage uid (BALF). In in vivo studies, RAW264.7 macrophages were transfected with IL-6 specic siRNA and treated with LPS. After exposed to IL-6 specic siRNA and LPS, expression of inammatory cytokines interleukin 1 (IL-1), tumor necrosis factor-(TNF-), IL-6 and interleukin 10 (IL-10) were detected by RT-qPCR, concentration of IL-1 and TNF- in culture supernatant were detected by ELISA and M1 and M2 markers were detected by western blot and ow cytometry.


Background
Sepsis, a life-threatening organ dysfunction induced by a dysregulated host response to infection, is a common disease in the Intensive Care Unit (ICU) [1,2]. With a high rate of morbidity and mortality, sepsis features complex pathogenesis and serious condition, which is closely associated with multiple organ dysfunction syndrome (MODS) and even death [3,4]. Among all the complications of sepsis, acute lung injury is the earliest and most severe one for abundant blood ow and vulnerable structure [2,5]. During the early stages of sepsis, excessive in ammatory factors and reactive oxygen species would damage pulmonary capillary endothelium and increase vascular permeability, leading to in ammatory cell in ltration and further damage [3,6]. Despite the progresses in diagnostic approach and supportive treatment, sepsis accounts for nearly 40% death in ICU [7]. In US, sepsis affects more than 1.7 million people every year, of which approximately 270,000 dies [2,8]. Besides, sepsis put great economic burden on both patients and health system. The total medical cost for sepsis is as high as 24 billion U.S. dollars, and the number continues to climb year by year [1,8].
Emerging studies indicated that macrophages play a crucial role in occurrence and development of sepsis by initiating the in ammation cascade reaction [4,6]. Macrophages can be divided into two categories roughly: classically activated macrophages (M1) and alternatively activated macrophages (M2) [9,10]. M1 macrophages are involved in resistance to pathogens by releasing pro-in ammatory cytokines, while M2 polarized macrophages help to repair and remodel tissue, as well as resisting parasites and tumor growth [10,11]. M1 macrophages can recognize invading pathogens signals and induce the expression of wide varieties of in ammatory mediators, including TNF-α, IL-1, IL-6 and reactive oxygen species (ROS) [12,13]. There is no doubt that these mediators are helpful for elimination of pathogen, but excessive in ammatory cytokines and ROS lead to tissue damage [14]. Consequently, inhibition of M1 polarization of macrophages downregulation of in ammatory cytokines secretion is a therapeutic target of sepsis [15].
IL-6 is one of the most important proin ammatory cytokines secreted by M1 macrophages in early stage of cytokine storm [16]. Researches indicated that IL-6 level is closely related to progression and prognosis of sepsis, organ dysfunction and death [17]. Blood IL-6 level in most sepsis patients increased signi cantly at early stage [18,19]. Hence, IL-6 has been regarded as a biomarker in clinic to anticipate and monitor state of sepsis [20]. However, how IL-6 exerts effects on the progression of sepsis remains unclear. So far con icting data on the blockage of IL-6 in animal models of sepsis were reported. In the present study, we explored the role of IL-6 in sepsis by regulating macrophage polarization in vivo and in vitro. These data con rmed that IL-6 knockout can attenuate lung in ammation and promote macrophages toward M2 types. Such ndings may offer a new way on the treatment of sepsis.

Animal model
Cecal ligation and puncture (CLP) was conducted to induce sepsis as previously described [12]. 24 WT C57 mice (12 for CLP group and 12 for sham group) and 12 IL6 knock out mice went through the process of CLP. Brie y, mice were anesthetized with iso urane before operation. The ligated part of the cecum was punctured through with a 21-gauge needle. After repositioning of the bowel, the abdomen was closed layer by layer using a 4.0 surgical suture and metallic clips. Sham animals underwent the same procedure without ligation and puncture of the cecum. Clinical disease score (CDS) ( Table.1) and muridae sepsis score (MSS) ( Table.2) were conducted on CLP induced sepsis mice. 24h after operation, the mice were sacri ced at different time points to obtain serum, lung, liver, heart, and kidney samples for in ammatory cytokines measurement and histological observation.

Histology and tissue processing
Mice were sacri ced at different time points (0, 6, 12, 24 h) after operation, and organ samples (lungs, livers, kidneys and hearts) were collected, xed in 10% buffer formalin for 24 h and embedded in para n using standard procedures. Embedded samples were sectioned and stained with hematoxylin and eosin for morphological analysis.

Lung injury measurement
The entire lungs were collected for lung wet/dry ratio (W/D) measurement in order to assess pulmonary edema after the mice were executed in 0, 6, 12, 24 h. Bronchoalveolar lavage was performed as following steps. The lungs were injected with 0.9% saline (0.5 mL) through the main bronchus and repeated for three times. The BALF was centrifuged for 10 min at 1200 rpm. The supernatants were collected and stored at -80℃. Protein concentration in BALF was determined by a protein detection kit.

Flow cytometry assay
Cells collected from BAL uid were detected by ow cytometry. First, cells were collected from BAL uid by centrifuge at 300g for 10 minutes and then incubated with anti-mouse F4/80, CD206, and CD86 antibody (BD, USA) for 30 minutes at 4℃. Then, cells were analyzed on a ow cytometer (BD, USA) and data were analyzed with FlowJo software.

Western blot analysis
Protein samples from lung tissue were extracted by RIPA lysis buffer, and the concentration of which were detected by BCA kit. The protein samples were separated by electrophoresis in a 10% polyacrylamide gel and transferred onto PVDF membranes. Then the membranes were blocked in 5% non-fat dried milk with Tris-buffered solution containing 0.1% Tween-20 (TBST) at room temperature for 1h and incubated overnight at 4℃ with Arg-1 and iNOS primary antibody (CST, USA). Then, membranes were incubated for 1h with secondary antibody (Boster, China). The results were visualized with ECL on Tanon 5500 Imaging System (Shanghai, China). ImageJ was used to quantify densities of the bands, while GraphPad Prism was used for graphs.

Statistical analysis
The data sets used one-way ANOVA and then compared the mean groups with the student-Newman -Coles multiple comparison test. The proportional-risk model was used for statistical analysis of survival studies. P < 0.05 was considered signi cant.

Results
3.1 IL-6 Knockout (KO) improved the survival rates of the septic mice.
CLP were conducted on 20 WT C57 mice (10 for CLP group and 10 for sham group) and 10 IL-6 knockout mice. CDS was applied for evaluation of septic mice at 24 and 48 h after CLP. In both 24h and 48h following CLP, the CDS was signi cantly higher in septic animals compared to sham group (Fig. 1A), indicating that we successfully constructed the septic model. MSS of IL-6 KO sepsis mice is lower than that of WT group (Fig. 1B). Meanwhile, the survival rates were observed in the consecutive 72h after operation. Results indicated that WT mice in CLP group died more than half (6/10) at 24h after operation. While in IL-6 KO group, the death rate was signi cantly decreased (2/10) compared with CLP groups. And at 48h after operation, 8/10 WT mice in CLP group died, while 4/10 IL-6 KO mice in CLP group died. This result indicated that knockout of IL-6 in CLP mice could signi cantly improve the survival rates (p < 0.05) (Fig. 1C).
3.2 IL-6 Knockout alleviated lung lesion and dysfunction. 24 h after operation, mice in different group were sacri ced for lung injury assessment. Lung tissue were collected for histological detection,while lung injury score, lung tissue wet/dry ratio and protein concentration of BALF detection were conducted for lung injury assessment. As indicated in Fig. 2A, lung tissues in IL-6 +/+ +CLP mice displayed obvious in ammatory in ltration, interstitial edema and alveolar structural damages, while those phenomena were remarkedly improved in the IL-6 −/− +CLP group.
Serum and BALF were collected from mice in different treatment groups for in ammatory cytokine detection by ELISA. As is shown in Fig. 3A, in ammatory cytokines (IL-1β, TNF-α, and IL-6) expression in IL-6 +/+ + CLP mice were signi cantly increased in serum than that of sham group. While in IL-6 −/− mice, concentrations of these in ammatory cytokines were all decreased signi cantly than IL-6 +/+ + CLP group. Consistent with this, same phenomena were observed in the BALF (Fig. 3B). In lung tissues, CD206 and iNOS immuno uorescence staining were performed to observe macrophage in ltration. As is shown in Fig. 3C, number of iNOS + (M1) macrophages in IL-6 +/+ + CLP was much larger than that of sham group. And macrophage M1 polarization of IL-6 KO mice were reduced compared with WT mice. In the meantime, M2 polarization of IL-6 −/− + CLP group was increased than that of IL-6 +/+ + CLP. Hence, aforementioned results suggested that IL-6 knockout distinctly prevented the upregulation of proin ammatory cytokines and promoted anti-in ammatory trait.
In in vivo studies, 1µg/ml LPS and IL-6 speci c siRNA were used to stimulate RAW264.7 macrophages.
In ammatory cytokines IL-1β, IL-6, TNF-α and IL-10 were detected by RT-qPCR. The mRNA levels of these in ammatory cytokines were signi cantly increased by LPS stimulation. To investigate the role of IL-6 in in ammatory response, we constructed IL-6 speci c siRNA. After transfection for 24 h, IL-6 expression was successfully decreased at both the transcriptional and translational levels (Fig. 4A). Hence, we treated siRNA transfected RAW264.7 cells with LPS. ELISA detection revealed the increase of TNF-α, IL-1β concentration in culture supernatant when IL-6 was inhibited (Fig. 4B). RT-qPCR results indicated that LPS induced up-regulation of in ammatory cytokines TNF-α, IL-1β and IL-6 were ameliorated by IL-6 interference (Fig. 4C), while expression of IL-10 was increased. And as showed in Fig. 4D, when IL-6 was silenced by siRNA, iNOS protein expression was overwhelmingly increased, while Arg-1 was decreased.

Discussion
Sepsis is characterized by systemic in ammatory response syndrome which often associated with MODS and even death [21]. Besides, the morbidity and mortality rates of sepsis remained stubbornly high despite the progresses in diagnostic approach and supportive treatment [2,3]. Consequently, it is urgent to discover effective therapeutic targets and intervening measures. Wide arrays of studies con rm that macrophages play an important role in sepsis and other diseases via mediating in ammatory producers [22,23]. Macrophages can be classi ed as 2 phenotypes: classically activated (M1) macrophages, contributing to tissue damage by producing excessive reactive cytotoxic oxidants to destroy pathogens, and alternatively activated(M2) macrophages, suppressing in ammation and promoting repair [9,10]. At the beginning of in ammatory response phase, macrophages are activated by invading pathogens, leading to excessive in ammatory cytokine storm [22,24]. These in ammatory cytokines without doubt are helpful eliminating pathogens, but these undue productions can be pernicious, leading to a "double-edge sword" property that eliminating pathogens at the expense of organ injury [6]. Based on understanding of molecular mechanisms underlying sepsis, novel therapies have been designed to suppress macrophage induced cytokine storm and transform macrophages from M1 to M2 [12]. In in vivo studies, we revealed that interference of IL-6 would alleviate LPS induced in ammatory response. When expression of IL-6 was inhibited, expression of in ammatory cytokines,such as IL-1β and TNF-α were signi cantly decreased, and ratio of M1 macrophage declined while M2 increased. IL-6 plays vital roles in host defense against acute infections via activating acute-phase reactions and immune responses [20]. In sepsis and other infected lesions, IL-6 is produced by monocytes and macrophages when stimulated with bacterial, viral or fungal components such as lipopolysaccharide and peptidoglycan with PAMPs [25]. IL-6, as a soluble mediator, sends out in ammatory signals from localized lesions to the whole body and provides an SOS signal to trigger the host defense against emergent events such as infection and trauma [19]. Concentration of IL-6 in blood as a prognostic factor has been evaluated in several studies. And results showed that IL-6 has a high sensitivity and speci city for the detection of sepsis or early sepsis [4,26]. In this study, we constructed sepsis models via Cecal ligation and puncture (CLP), and observed in ammatory response and tissue damage in lung tissue of WT and IL-6 KO mice. Results indicated that when expression of IL-6 were inhibited, expressions of in ammatory cytokines in CLP induced sepsis mice were decreased and the Muridae Sepsis Score were decreased. 24 h after operation, the damage of tissues, particularly the lung, was signi cantly improved, and the death rate during 48 h after operation was declined in IL-6 KO mice than that of WT. IL-6 is an important in ammatory mediator which plays a crucial role in inducing in ammatory cascade reaction via several signal pathways, including JAK1/STAT3 pathway [27]. When expression of IL-6 was inhibited, CLP induced in ammatory response and organ damage were improved.
Lung tissue is one of the most easily affected organs by sepsis [28]. In sepsis patients, acute lung injury and acute respiratory distress syndrome (ARDS) are the most common and serious complication, the incidence of which is as high as 40% and the mortality is about 40-50% [2,29]. Sepsis-induced ARDS refers to damage of alveolar epithelial cells and capillary endothelial cells caused by excessive in ammatory cytokines and ROS, which leads to pulmonary edema, diffused interstitial and alveolar damage [30]. The occurrence and development of ARDS are closely associated with pulmonary edema caused by endothelial damage and increased vascular permeability [31,32]. Integrality of alveolar barrier is essential to prevent pulmonary edema and promote ARDS relief [33]. Therefore, vascular permeability of alveolar epithelium is an important indicator of occurrence and severity of ARDS. In this study, CLP induced sepsis resulted in pulmonary edema, in ammatory cell in ltration and alveolar damage. But in IL-6 KO sepsis mice, above lung injury manifestation was remarkably improved. And lung injury score and wet/dry weight ratio indicated that lung injury especially vascular permeability was mitigated. And when expression of IL-6 was suppressed, in ltration of in ammatory cell, especially M1 macrophage, was decreased CLP induced sepsis mice. Consequently, aforementioned results indicated that when expression of IL-6 was blocked, the in ammatory response of macrophage in CLP induced sepsis was decreased, tissue damage was improved and survival rate of mice was increased.

Conclusion
In this study, we found that inhibition of IL-6 could mitigate LPS induced in ammatory cytokines storm in macrophages and polarize macrophages to anti-in ammatory M2 phenotype. And in CLP induced sepsis model, knockout of IL-6 could relieve the in ammation cytokines storm in serum and BALF, reduce in ltration of in ammatory macrophage and improve lung tissue damage in lung which increase survival rate of mice. Collectively, we illustrated a novel potentially therapeutic mechanism that inhibition of IL-6 could alleviate the LPS induced in ammatory cytokines and increase ratio of M2 macrophages, hence improve the in ammatory reaction and the lung injury in sepsis.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated during the current study are not publicly available due to limitations of ethical approval involving the patient data and anonymity but are available from the corresponding author on reasonable request.

Competing interests
No potential conflicts of interest were disclosed.  Tables   Table 1  The   heart, and kidney tissues in CLP treated wild type and IL-6 knockout C57 mice. Scale bar: 50 μm. B. Lung injury score of CLP treated wild type and IL-6 knockout C57 mice, **: P ≤ 0.01. C. Lung wet/dry ration of CLP treated wild type and IL-6 knockout C57 mice, **: P ≤ 0.01. D. Protein concentrations of BALF in CLP treated wild type and IL-6 knockout C57 mice 24 h after operation. IL-6 knockout mitigate CLP induced in ammatory cytokine storm. A. ELISA results of IL-1β, TNF-α and IL-6 contents in the mice serum collected from CLP treated wild type and IL-6 knockout C57 mice. **: P ≤ 0.01. C. ELISA results of IL-1β, TNF-α and IL-6 contents in the mice BALF collected from CLP treated wild type and IL-6 knockout C57 mice. **: P ≤ 0.01. iNOS and CD206 expressions in the lung tissues of CLP treated wild type and IL-6 knockout C57 mice detected by immuno uorescence. Blue: DAPI; Green: iNOS; Red: CD206. Scale bar: 50 μm.