Ulinastatin Activated The ERK5/Mer Signaling Pathway To Promote Macrophage Efferocytosis And Ameliorate Lung Inammation

Purpose:Ulinastatin (UTI) is an endogenous protease inhibitor with potent anti-inammatory, antioxidant and organ protective effects. The inhibitor has been reported to ameliorate inammatory lung injury but precise mechanisms remain unclear. Methods: An in vivo model of lung injury has been constructed by intratracheal infusion of lipopolysaccharide (LPS). The number of neutrophils and the phagocytosis of apoptotic neutrophils were observed by Diff- Quick method. Lung injury was observed by HE staining .BALF cells were counted by hemocytometer and concentrations of protein plus inammatory factors were measured with a BCA test kit. During in vitro experiments, RAW264.7 cells were pretreated with UTI (1000 and 5000U/ mL), stained with CellTracker TM Green B0DIPY TM and HL60 cells added with UV-induced apoptosis and PKH26 Red staining. The expression of ERK5\Mer related proteins was detected by western blot and immunouorescence. Results: An in vivo model of lung injury has been constructed by intratracheal infusion of lipopolysaccharide (LPS). UTI treatment enhanced the phagocytotic effect of mouse alveolar macrophages on neutrophils, alleviated lung lesions, decreased the pro-inammatory factor and total protein content of BALF and increased levels of anti-inammatory factors. in vitro experiments (cid:0)UTI enhanced the phagocytosis of apoptotic bodies by RAW264.7 cells in a dose-dependent manner. Increased expression levels of ERK5 and Mer by UTI were shown by Western blotting and immunouorescence. Conclusions: UTI mediated the activation of the ERK5/Mer signaling pathway, enhanced phagocytosis of neutrophils by macrophages and improved lung inammation. The current study indicates potential new clinical approaches for accelerating the recovery from lung inammation.


Introduction
Ulinastatin (UTI) is an endogenous protease inhibitor, present in blood and urine, which inhibits the activity of hydrolases, such as elastase and cathepsin. UTI has the effects of stabilizing lysosomal membranes, inhibiting the excessive release of in ammatory mediators and protecting tissues and organs [1][2][3]. It is widely applied to pancreatitis, sepsis, shock and other diseases [4][5][6][7]. The organ protective effect of UTI can be only partly explained by its protease inhibitory action and its antiin ammatory mechanism requires further elucidation.
Acute lung injury (ALI) is a response to a variety of primary diseases. Its pathogenesis is unclear but acute non-controllable in ammation is a common factor in its occurrence and development [8,9]. When viruses and endotoxins invade lung tissues, macrophages are activated resulting in enhanced phagocytic activity, release of anti-in ammatory factors and a reduced in ammatory response to promote tissue repair [10]. Such a sequence of events highlights the role of macrophages in in ammatory recovery, including the phagocytosis and clearance of apoptotic cells, a process termed efferocytosis [11,12]. Extracellular signal-regulated kinase 5 (ERK5) is a widely expressed member of the MAPK subfamily [13,14]. ERK5 is known to regulate IL-4-induced differentiation of m2-type macrophages and to in uence immune regulation and tissue remodeling [15]. The kinase has anti-in ammatory and immunomodulatory effects which make it a potential therapeutic target for a variety of critical in ammatory diseases [16]. Mer is a macrophage receptor, one of three members of the TAM family (Tyro3, Axl and Mer) [17], that stimulates clearance of apoptotic neutrophils [18]. Mer is known to play a synergistic role with TIM-4 or Axl to enhance efferocytosis and prevent continued necrosis of apoptotic in ammatory cells thereby reducing in ammation and restoring tissue homeostasis [19][20][21].
The current study examines the role of UTI in promoting the resolution of lung in ammation by enhancing cellular burial by macrophages. The effect of UTI on cell burial and the involvement of the ERK5/Mer signaling pathway in in vitro and in vivo has been investigated.  Mouse peritoneal macrophage cell-line, RAW264.7, and human acute myeloid leukemia cell-line, HL60, were purchased from Wuhan Procell Life Science and Technology Co., LTD. RAW264.7 cells were cultured in high glucose DMEM supplemented with 10 % (v/v) FBS and HL60 cells were cultured in RPMI 1640 medium supplemented with 12 % (v/v) FBS, 100 U/ mL penicillin and 100 μg/ mL streptomycin and in humidi ed air with 5 % CO 2 at 37 ℃. UTI was dissolved in PBS and further diluted with cell culture media.

Specimen collection
The left lung was isolated immediately after anesthesia and xed with 4 % paraformaldehyde. The upper lobe of the right lung was isolated, weighed and baked at 60 ℃ for 72 hours. Bronchoalveolar lavage uid (BALF) was collected into 2 ml pre-cooled phosphate buffered saline (PBS) and the total number of cells were determined immediately. BALF was centrifuged and the supernatant was stored at -80 ℃ for determination of total protein and in ammatory factor levels. The cell pellet was immediately resuspended in PBS and numbers of neutrophils were determined by Diff-Quick staining solution.
In ammatory response A wet-dry ratio was calculated for the lung tissue to indicate lung exudation during LPS-induced injury.
The wet weight was determined immediately after collection and tissue dried in an oven for 72 hours before determination of the dry weight. BALF cells were counted by hemocytometer and concentrations of protein plus in ammatory factors were measured with a BCA test kit. The BALF cell pellet was resuspended in PBS and cells were evenly distributed on a glass slide using a cell slicer before estimation of neutrophil number by Diff-Quick dye staining, as previously described [24].

Phagocytosis by alveolar macrophages in vivo
BALF was centrifuged and cell pellets resuspended in PBS with 30 % FBS to a density of 5×10 5 cells/ mL, as described previously [25]. 200 μL cell suspension was added to each sling hole and evenly distributed on the slide with a cytoprep (MOTIC, Xiamen, China). Diff-Quick dye was added, according to the manufacturer's instructions. After staining, 300 macrophages were randomly selected under the microscope and numbers of apoptotic bodies within the macrophages were divided by 300 to obtain the phagocytic rate.
Preparation of apoptotic cells HL60 cells were suspended in 10 ml RPMI1640 medium and seeded into a Petri dish, 9 cm from the ultraclean cell for 30min UV irradiation. After incubation at 37°C for 3 h, apoptotic HL60 cells were stained with PKH26 Red.

Macrophage phagocytosis in vitro
In vitro phagocytosis was assessed as described previously [21]. Brie y, RAW264.7 cells were seeded into 24-well plates at 10 5 cells/ml. After adhesion, cells were pretreated with either 1000 U/mL or 5000U/mL UTI for 3 h[26]. Cells were stained with CellTracker TM Green B0DIPY TM dye to a nal concentration of 5 μM for 30 min. Cells were washed 3 times with PBS and 10 6 cells/well apoptotic HL60 cells were added with incubation in a cell incubator overnight. Wells were washed 3 times with PBS and anti-uorescence quench agent added. Phagocytosis was observed under a uorescence microscope.

Immuno uorescence
The cell experiment was rst divided into 0 U/ mL, 1000 U/ mL and 5000 U/ mL groups. Mouse peritoneal macrophages RAW264.7 cells were seeded into 24 well plates at 10 5 / mL. After cell adherence, the cells were pretreated with ulinastatin at different concentrations for 3 h. ERK5 inhibitor BIX02189 was used to divide the cells into blank control group, BIX02189 group, 5000 U/ mL group and 5000 U/ mL +BIX02189 group. The cells were pretreated with ERK5 inhibitor BIX02189 for 1 h and then 5000 U/mlUTI for 3 h. Cells were washed 3 times with PBS and 4 % paraformaldehyde added to x the cells at 4 ℃ overnight.
After washing 3 times with PBS, primary antibodies raised against Mer (1:100) were added for overnight incubation at 4 ℃. Fluorescently conjugated secondary antibodies (AlexaFluor488) were used for staining and cells were incubated at room temperature in the dark for 1h. After 3 washes with PBS, DAPI was used for nuclear staining, slides observed by uorescence microscope equipped with Nikon DS-U3 imaging system and uorescence intensity quanti ed with Image-J software.

Western blotting
The cells were grouped as before. Total protein was extracted and protein loading buffer added. After boiling and denaturation, SDS-PAGE electrophoresis was carried out, proteins were transferred to a PVDF membrane and incubated with ERK5 primary antibody (1:1000) at 4 ℃ overnight. Goat anti-rabbit uorescent secondary antibody (1:30,000) was added, incubated at room temperature for 1 h and bands developed by uorescence scanner. Image-J software was used to determine the gray value of each band and relative expression levels of ERK5 protein represented by the ratio of the ERK5 gray band to the GAPPH band.
Statistical analysis SPSS 23.0 software was used for statistical analysis. Student's t test was used to compare means ± standard deviations (x±s), for two groups with normal distribution and ANOVA and SNK test to compare multiple groups. A value of p< 0.05 was considered statistically signi cant.

UTI ameliorates LPS-induced pneumonia injury
UTI is known to have an organ protective role [6,7,21]. A pneumonia lung damage model was established by intratracheal infusion of LPS with histopathological damage assessed by H&E staining. Lung wet/dry (W/D) ratios, BALF protein level, neutrophil count, in ammatory factor IL-6, MPO and anti-in ammatory factor TGF-β levels were measured on days 1, 3, 5 and 7 to evaluate the degree of pulmonary edema and secretion of pro-in ammatory and anti-in ammatory factors. Signi cant lung injury resulted from LPS treatment (Fig. 1A), including alveolar septal thickening, pulmonary edema and in ammatory cell in ltration. Markers of lung damage were reduced by UTI treatment (Fig. 1A), and the lung histopathological score was reduced (Fig. 1B). LPS infusion caused a gradual increase in PMN count (Fig. 1C), protein level (Fig. 1D), pulmonary edema (Fig. 1E), MPO level (Fig. 1F), pro-in ammatory factor IL-6 ( Fig. 1G) and anti-in ammatory factor TGF-β (Fig. 1H) in BALF to a peak at day 3. A gradual decrease was then observed. Administration of UTI accelerated the regression of neutrophil in ltration, pulmonary edema and pro-in ammatory factors on days 3, 5 and 7 and promoted secretion of anti-in ammatory factors. These results provide evidence that UTI ameliorates LPS-induced pulmonary in ammatory damage.
The necrotic role of macrophages is essential for the prompt clearance of apoptotic neutrophils and the resolution of in ammation [28,29]. In vitro and in vivo phagocytosis experiments were designed to investigate whether UTI ameliorates lung in ammatory injury through macrophage burial. Phagocytotic activities of alveolar macrophages on neutrophils were enhanced in LPS mice treated with UTI compared with untreated LPS mice and with control mice (Fig. 2B). In vitro experiments showed enhancement of RAW264.7 cell phagocytosis of apoptotic HL60 cells after pretreatment with 1000 U or 5000 U UTI compared with untreated cells (Fig. 2A). Phagocytic stimulation by UTI was dose-dependent. These ndings demonstrate that treatment with UTI enhanced macrophage phagocytosis of apoptotic cells and improved LPS-induced pneumonia injury both in vitro and in vivo.

UTI increased macrophage ERK5/Mer expression in a dose dependent manner
ERK5 is a serine protein kinase with anti-in ammatory and immunomodulatory functions and a potential therapeutic target for many critical in ammatory diseases 16,30 . Mer is a macrophage receptor member of the TAM family (Tyr03, Axl, Mer), expressed by m2-type macrophages. Activated Mer and Axl are known to negatively regulate the in ammatory cascade and mediate the phagocytosis of apoptotic cells 18,31 .
RAW264.7 cells were pretreated with UTI and used to investigate the role of the ERK5/Mer signaling pathway in macrophage burial. ERK5 and Mer expression were shown to increase with increasing UTI concentration by Western blot and immuno uorescence assays ( Fig.3A; C). UTI may act to increase Mer expression in the macrophage cell membrane by activation of ERK5, thus enhancing the intracellular burial effect of macrophages. Use of the ERK5 inhibitor, BIX02189, inhibited Mer expression in the cell membrane ( Fig. 3B; C), indicating an effect of UTI on the ERK5/Mer signaling pathway.

UTI enhanced macrophage burial and improved LPS-induced in ammation by activating the ERK5/Mer signaling pathway
The results described above indicate that UTI increased Mer expression by activating ERK5 protein, thus enhancing the phagocytic function of macrophages. The ERK5 inhibitor, BIX02189, was used for both in vivo and in vitro experiments designed to ascertain levels of pathological lung damage by H&E staining and to evaluate rates of macrophage burial. In vivo results demonstrated more severe lung damage, indicated by higher histopathological scores, in mice treated with LPS+UTI and in which BIX02189 was present than in mice with no ERK5 inhibitor (Fig. 4A). In addition, the presence of the inhibitor also increased phagocytosis of apoptotic neutrophils in BALF by alveolar macrophages (Fig. 4B). Experiments conducted in vitro showed that pretreatment with BIX02189 produced a decreased phagocytic response, in terms of both rate and function, to 5000U UTI (Fig. 4C). In summary, UTI increased the expression of Mer in the macrophage cell membrane by activating ERK5 protein and enhanced cytoburial by macrophages, improving LPS-induced in ammatory damage.

Discussion
Acute lung injury (ALI) often results from a systemic in ammatory response, involving the overexpression of in ammatory factors in lung tissue due to pathogenic invasion. During pathogen clearance, immune cells are over-stimulated to release pro-in ammatory factors which cause uncontrolled in ammation and destruction of lung parenchymal cells, resulting in rapid attenuation of respiratory function [9]. As a component of the innate immune system, macrophages phagocytose and kill pathogenic microorganisms and clear damaged and aging cells [10]. Intracellular burial by lung macrophages ensures lung homeostasis, reducing oxidative damage and enhancing tissue repair [21]. Therefore, a clinical intervention which enhances intracellular burial by macrophages would have great value in promoting recovery from lung injury.
The current study generated a mouse model of lung in ammation by means of dripping LPS into the trachea. There was a consequent increase in the W/D ratio of lung tissue, indicating the presence of pulmonary edema. Clear changes to the structure of the lung tissue could be visualized under the microscope. There was evidence of alveolar wall thickening, interstitial diffuse hyperemia and edema, in ltration by in ammatory cells and increased pathological score. These changes establish the successful generation of the mouse model of lung in ammation.
Early administration of UTI has been shown to dampen the excessive in ammatory response by inhibiting release of neutrophil protease, thus reducing levels of oxygen free radicals and superoxide dismutase activity 31 . Many studies have demonstrated the impact of UTI in inhibiting the in ammatory response and regulating innate immunity. Animal studies have shown increased survival rates of septic mice after UTI treatment [33,34] and, in recent years, UTI has been used extensively in the clinical treatment of sepsis. The current study demonstrates that UTI treatment reduced the lung W/D ratio, BALF neutrophil count, total protein content, IL-6 and MPO levels while increasing the anti-in ammatory TGF-β level in the LPS-induced mouse model of lung in ammation. These results indicate a dampening of the in ammatory response after UTI administration. The degree of lung injury, indicated by histopathological score, was reduced, indicating that UTI reduces the lung tissue injury induced by LPS.
Macrophages identify, process and remove abnormal, aging or infected cells through the process of macrophage burial, thereby reducing the lung's in ammatory response and promoting tissue recovery. Du et al. found that when cytolysis of mouse macrophages was stimulated by iso urane, recovery from pulmonary in ammation was promoted [21]. It is known that chromatin condensation and cytoplasmic blistering in apoptotic cells can be visualized under the light microscope, a phenomenon identi ed with the formation of apoptotic bodies which can then be phagocytosed by macrophages. Apoptotic cells generated during the current study displayed these characteristics. RAW264.7 cells were pretreated with . The possibility remains that UTI may increase Mer expression in the macrophage cell membrane by activating ERK5, thereby enhancing intracellular burial by macrophages. The current study demonstrates increased expression of ERK5 in RAW264.7 cells pretreated with UTI. A subsequent increase in Mer expression was also detected. The ERK5 inhibitor, BIX02189, reduced the expression of these cell membrane proteins with the result that pathological damage to lung tissue increased and phagocytosis was reduced both in vivo and in vitro. These ndings demonstrate that, by activating ERK5, UTI treatment has the effect of increasing the expression of macrophage cell membrane proteins associated with cell burial. Therefore, it is likely that UTI enhances the necrotic effect of macrophages through activation of the ERK5 pathway.

Conclusion
In summary, the current study has shown that Ulinastatin has the effect of increasing expression of Mer protein in the macrophage cell membrane via activation of ERK5. The downstream effect is one of enhanced macrophage efferocytosis so as to promote tissue recovery from LPS-induced lung injury.