Ulinastatin promotes macrophage efferocytosis and ameliorates lung inflammation via the ERK5/Mer signaling pathway

Acute lung injury (ALI) is a pneumonic response characterized by neutrophil infiltration. Macrophage efferocytosis is the process whereby macrophages remove apoptotic cells, and is required for ALI inflammation to subside. The glycoprotein ulinastatin (UTI) has an anti‐inflammatory effect during the acute stages of ALI, but its effect on efferocytosis and the subinflammatory stage of ALI is unclear. Extracellular signal‐regulated kinase 5 (ERK5) is a key protein in efferocytosis, and we thus hypothesized that it may be activated by UTI to regulate efferocytosis and the resolution of pneumonia. To test this hypothesis, here we monitored phagocytosis of macrophages through in vivo and in vitro experiments. Pulmonary edema, neutrophil infiltration, protein exudation, and inflammatory factor regression were observed on days 1, 3, 5, and 7 in vivo. RAW264.7 cells were pretreated with different concentrations of UTI and ERK5 inhibitors, and the expression of tyrosine‐protein kinase Mer (Mer) protein on macrophage membrane was detected. UTI increased the phagocytosis of apoptotic neutrophils by macrophages in vitro and in vivo, and promoted the resolution of pneumonia. The protein expression of ERK5 and Mer increased with UTI concentration, while the expression of Mer was down‐regulated by ERK5 inhibitors. Therefore, our results suggest that UTI enhances efferocytosis and reduces lung inflammation and injury through the ERK5/Mer signaling pathway, which may be one of the targets of UTI in the treatment of lung injury.

Acute lung injury (ALI) is a pneumonic response characterized by neutrophil infiltration. Macrophage efferocytosis is the process whereby macrophages remove apoptotic cells, and is required for ALI inflammation to subside. The glycoprotein ulinastatin (UTI) has an anti-inflammatory effect during the acute stages of ALI, but its effect on efferocytosis and the subinflammatory stage of ALI is unclear. Extracellular signal-regulated kinase 5 (ERK5) is a key protein in efferocytosis, and we thus hypothesized that it may be activated by UTI to regulate efferocytosis and the resolution of pneumonia. To test this hypothesis, here we monitored phagocytosis of macrophages through in vivo and in vitro experiments. Pulmonary edema, neutrophil infiltration, protein exudation, and inflammatory factor regression were observed on days 1, 3, 5, and 7 in vivo. RAW264.7 cells were pretreated with different concentrations of UTI and ERK5 inhibitors, and the expression of tyrosine-protein kinase Mer (Mer) protein on macrophage membrane was detected. UTI increased the phagocytosis of apoptotic neutrophils by macrophages in vitro and in vivo, and promoted the resolution of pneumonia. The protein expression of ERK5 and Mer increased with UTI concentration, while the expression of Mer was down-regulated by ERK5 inhibitors. Therefore, our results suggest that UTI enhances efferocytosis and reduces lung inflammation and injury through the ERK5/Mer signaling pathway, which may be one of the targets of UTI in the treatment of lung injury.
Acute lung injury (ALI) is a critical clinical illness of common occurrence and is a pneumonic reaction characterized by neutrophil infiltration [1]. The pathophysiological process of ALI can be roughly divided into the acute inflammatory stage and the subsiding inflammatory stage. In the subsiding phase of inflammation, macrophages directly engulf and clear apoptotic neutrophils to repair damaged tissues, a process called efferocytosis, which promotes the recovery of lung epithelial and endothelial functions and the reconstruction of lung tissue structure. If apoptotic cells are not cleared in time, many intracellular danger signaling molecules are released by secondary cell necrosis, which further hinders tissue repair. Therefore, promoting the regression of pneumonia will shorten the pathophysiological process of ALI and reduce lung injury [2,3].
Ulinastatin (UTI), a commonly used drug in clinical practice, is an endogenous molecule with actions against infectious pathogens and its pharmacological Abbreviations ALI, acute lung injury; ERK5, extracellular signalregulated kinase 5; Mer, tyrosine-protein kinase Mer; UTI, ulinastatin. effects include inhibiting the excessive release of inflammatory mediators scavenging oxygen-free radicals and alleviating lung injury by means of its antiinflammatory effect [4]. Xu et al. [5] showed that Xubijing combined with UTI was an effective sepsis treatment. Zhu et al. [6] found that UTI reduced doxorubicin-induced myocardial injury in SD rats. However, the effect of UTI on the inflammatory resolution period following ALI remains unclear. In view of the critical role of macrophages, especially efferocytosis, in inflammation regression [7,8], we investigated the effect of UTI on efferocytosis.
ERK5 is a member of the MAPK family [9,10]. It can be activated by a series of stimuli (including proinflammatory stimuli), transmitting extracellular stimulation signals to the intracellular, compartment, and having anti-inflammatory and immunomodulatory functions [11,12]. In addition, it has been reported that activation of ERK5 enhances macrophage efferocytosis, thus inhibiting the occurrence of atherosclerosis [13]. These pieces of evidence suggest that ERK5 is a key factor in the regulation of macrophage function. CD36 and Mer receptor tyrosine kinases on the cell membrane of macrophages recognize apoptotic cells via bridging proteins [14][15][16]. We previously demonstrated that isoflurane promotes the phagocytosis of apoptotic neutrophils by macrophages via AMPKmediated ADAM17/Mer signaling pathway [17]. Therefore, we investigated whether UTI regulates Mer receptors through activation of ERK5 and hence efferocytosis through Mer-receptor tyrosine kinases.
This study aimed to determine the effect of UTI on macrophage efferocytosis in the context of ALI. There was a particular, focus on whether UTI affected efferocytosis through the ERK5/Mer signaling pathway, promotes pneumonia resolution, and alleviated lung inflammation and injury.

Animals and laboratory protocols
Male c57bl/6 mice (8 weeks old and weighing 20 AE 2 g; Nanning, Guangxi, China) were purchased from the Experimental Animal Center of Guangxi Medical University (animal certificate number: SYXK9 (GUI) 2014-0003). All animal surgeries were approved by the Ethics Committee of the Experimental Animal Center of Guangxi Medical University (Nanning, China; Approval No. 202008010). In the experimental design, we achieved the minimum necessary amount by optimizing the experimental scheme. In the process of the experiment, we used a relatively mild injection technique and the appropriate amount of adjuvant to reduce the serious side effects on animals and reduce their pain of the animals. Animals were assigned to one of four groups and treated as follows: LPS: 5 mgÁkg À1 intravenous LPS; LPS + UTI: 5 mgÁkg À1 intravenous LPS immediately followed by 50 000 UÁkg À1 intraperitoneal UTI; Control group; UTI: 50 000 UÁkg À1 intraperitoneal UTI. We used ERK5 inhibitor BIX02189 to re-divide the animal experiments into the control group, BIX02189 group, LPS + UTI group, and LPS + UTI + BIX02189 group. BIX02189 group and LPS + UTI + BIX02189 group were intraperitoneally injected with 10 mgÁkg À1 BIX02189 2 h before modeling. In addition, we used XMD8-92 to divide mice into the CON group, XMD8-92 group, LPS + UTI group, and LPS + UTI + XMD8-92 group. Mice in the XMD8-92 group and LPS + UTI + XMD8-92 group were preinjected with XMD8-92(10 mgÁkg À1 ) once a week in advance [18]. Lung tissue and bronchial alveolar lavage fluid (BALF) were collected on days 1, 3, 5, and 7 after LPS-modeling.

Specimen collection
The left lung was isolated immediately after anesthesia and fixed with 4% paraformaldehyde. The upper lobe of the right lung was isolated, weighed, and baked at 60°C for 72 h. Bronchoalveolar lavage fluid (BALF) was collected into 2 mL precooled phosphate-buffered saline (PBS) and the total number of cells was determined immediately. BALF was centrifuged and the supernatant was stored at À80°C for determination of total protein and inflammatory factor levels. The cell pellet was immediately resuspended in PBS and numbers of neutrophils were determined by Diff-Quick staining solution (Soleibao).

Inflammatory 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 were dried in an oven for 72 h before determination of the dry weight. BALF cells were counted by hemocytometer and concentrations of protein plus inflammatory factors were measured with a BCA test kit (Soleibao). The BALF cell pellet was re-suspended 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.

Phagocytosis by alveolar macrophages in vivo
Bronchial alveolar lavage fluid was centrifuged and cell pellets resuspended in PBS with 30% FBS to a density of 5 9 10 5 cellsÁmL À1 , as described previously. Two hundred microliters cell suspension were added to each sling hole and evenly distributed on the slide with cypres (MOTIC, Xiamen, China). Diff-Quick dye was added, according to the manufacturer's instructions [17]. After staining, 300 macrophages were randomly selected under the microscope, and number 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 ultra-clean cell for 30 min UV irradiation. After incubation at 37°C for 3 h, apoptotic HL60 cells were stained with PKH26 Red (Sigma, SLF-MINI26).

Macrophage phagocytosis in vitro
In vitro phagocytosis was assessed as described previously [17]. Briefly, RAW264.7 cells were seeded into 24-well plates at 10 5 cellsÁmL À1 . After adhesion, cells were pretreated with either 1000 or 5000 UÁmL À1 UTI for 3 h [21]. Cells were stained with CellTracker TM Green B0DIPY TM (Invitrogen, C2102) dye to a final concentration of 5 lM for 30 min. Cells were washed three times with PBS and 10 6 cells per well apoptotic HL60 cells were added with incubation in a cell incubator overnight. Wells were washed three times with PBS and an anti-fluorescence quench agent added. Phagocytosis was observed under a fluorescence microscope (Olympus).

Immunofluorescence
The cell experiment was first divided into 0, 1000, and 5000 UÁmL À1 groups. Mouse peritoneal macrophages RAW264.7 cells were seeded into 24 well plates at 10 5 ÁmL À1 . After cell adherence, the cells were pretreated with ulinastatin at different concentrations for 3 h. ERK5 inhibitor BIX02189 (MedChemExpress, HY-12056) was used to divide the cells into blank control group, BIX02189 group, 5000 UÁmL À1 group, and 5000 UÁmL À1 + BIX02189 group. The cells were pretreated with ERK5 inhibitor BIX02189 (15 lm)for 1 h and then 5000 UÁmL À1 UTI for 3 h. In addition, ERK5 inhibitor XMD8-92 (Selleck, S7525, Shanghai, China) was used to divide the cells into control group, XMD8-92 group, 5000 UÁmL À1 group, and 5000 UÁmL À1 + XMD8-92 group. The cells were pretreated with ERK5 inhibitor XMD8-92 (10 lM) [22] for 3 h and then 5000 UÁmL À1 UTI for 3 h. Cells were washed three times with PBS and 4% paraformaldehyde was added to fix the cells at 4°C overnight. After washing three times with PBS, primary antibodies raised against Mer (1 : 100, Abclonal, A5443) were added for overnight incubation at 4°C. Fluorescently conjugated secondary antibodies (1 : 1000, Alexa Fluor 488; Invitrogen: MA700184A488) were used for staining, and cells were incubated at room temperature in the dark for 1 h. After three washes with PBS, DAPI (Sigma-Aldrich) was used for nuclear staining, slides observed by fluorescence microscope (Olympus) equipped with Nikon DS-U3 imaging system, and fluorescence intensity quantified with IMAGE-J software (NIH, New York, NY, USA).

Western blotting
The cells were grouped as before. Total protein was extracted and protein loading buffer (Soleibao) was added. After boiling and denaturation, SDS/PAGE electrophoresis was carried out, proteins were transferred to a PVDF (ISEQ00010, Merck Millipore, Massachusetts, USA) membrane and incubated with ERK5 primary antibody (1 : 1000, Abcam, ab40809) at 4°C overnight. Goat anti-rabbit fluorescent secondary antibody (1 : 30 000, Ray antibody, RM3003) was added, incubated at room temperature for 1 h, and bands developed by fluorescence scanner. IMAGE-J software (NIH) 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 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. The student's t-test was used to compare means AE standard deviations (x AE 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 significant.

Ulinastatin enhanced the macrophage efferocytosis of RAW264.7 cells and alveolar macrophages on apoptotic neutrophils in mice
The process of macrophage phagocytosis and clearance of apoptotic neutrophils is the key to the resolution of pneumonia [23]. Here, the effect of UTI on macrophage efferocytosis was investigated. A dose-dependent increase in phagocytosis of RAW264.7 cells in the range: 0, 1000, and 5000 UÁmL À1 UTI was observed (Fig. 1A,B). In a mouse model of LPS-induced lung injury, the phagocytosis of alveolar macrophages in alveolar lavage fluid in the LPS + UTI group was higher than that in the LPS and CON groups (Fig. 1C,D). These results suggest that UTI enhanced the cellular burial of macrophages. The phagocytosis of RAW264.7 cells increased in a dose-dependent manner when pretreated with 0, 1000, and 5000 UÁmL À1 UTI.

Ulinastatin upregulated surface expression of Mer receptor tyrosine kinase in macrophages
Mer receptor tyrosine kinases on macrophage cell membranes recognize apoptotic cells by means of bridging proteins, and the resolution of inflammation depends on the apoptosis and clearance of recruited neutrophils [14]. The regulation of Mer expression by UTI was investigated. Compared with the 0 UÁmL À1 group, the surface expression of Mer in 1000 and 5000 UÁmL À1 UTI groups significantly increased (Fig. 3). These results suggest that UTI upregulated Mer expression on the macrophage membrane surface.

Ulinastatin upregulated Mer expression by activating ERK5
ERK5 is a serine protein kinase that is activated by phosphorylation and translocated from the cytoplasm to the nucleus, where it exerts anti-inflammatory and immunomodulatory effects [9]. Therefore, p-ERK5 activity was investigated to determine whether UTI enhanced macrophage efferocytosis by mediating Mer expression on the cell surface through modulation of p-ERK5 activity. Compared with the 0 UÁmL À1 group, the 1000 and 5000 UÁmL À1 UTI groups had significantly increased expression of p-ERK5 (Fig. 4A). The ERK5 inhibitor, BIX02189, inhibited the expression of p-ERK5 (Fig. 4B), and the expression of Mer on the macrophage surface was also decreased (Fig. 4D,E). In addition, the use of another ERK5 inhibitor, XMD8-92, show similar results (Fig. 4C) and decreased Mer expression with p-ERK5 inhibition was consistently shown (Fig. 4F,G). In addition, we also examined the effect of ERK5 on Mer at the RNA level. The experimental results showed that there was no change in Mer RNA levels in groups 0, 1000, and 5000 U (Fig. S1A). Similarly, RNA levels of Mer in each group remained unchanged after treatment with ERK5 inhibitor (Fig. S1B,C). Therefore, we speculated that ERK5 might have no effect on the RNA level of Mer after UTI activation. These results suggest that ulinastatin upregulated Mer expression by activating ERK5.
Ulinastatin enhanced macrophage efferocytosis and promoted pneumonia resolution through the ERK5/Mer pathway To further elucidate the critical role of ERK5 in macrophage efferocytosis, ERK5 inhibitors were used in in vitro and in vivo models. Compared with the 5000 UÁmL À1 UTI group, the phagocytosis of RAW264.7 cells in the 5000 UÁmL À1 + BIX02189 group was significantly reduced (Fig. 5A,F). In the LPS-induced lung injury in vivo mouse model, the phagocytic capacity of alveolar macrophages in the bronchoalveolar lavage fluid of the LPS + UTI + BIX02189 group was reduced relative to that in the LPS + UTI group (Fig. 5B,G), and lung histopathological score was reduced (Fig. 5C,H). In addition, use of another ERK5 inhibitor, XMD8-92, resulted in reductions of the phagocytic ability of alve-  olar macrophages in the LPS + UTI + BIX02189 group (Fig. 5D,I) and lung histopathology scores (Fig. 5E,J). In combination, these data suggest that UTI enhanced macrophage efferocytosis and promoted the resolution of pneumonia through the ERK5/Mer pathway.

Discussion
This study investigated the effect of UTI on macrophage efferocytosis and the role of the ERK5/Mer pathway during deactivation of ALI inflammation in vivo and in vitro. Our study confirmed that UTI significantly enhanced macrophage efferocytosis of apoptotic cells and accelerated the resolution of LPS-induced pneumonia. UTI upregulated Mer expression on the macrophage surface by activating ERK5. Inhibition of ERK5 reduced macrophage phagocytosis, slowed the process of pneumonia resolution, and reduced Mer expression on the macrophage surface.
Our results suggest that UTI enhances macrophage endocytosis through the ERK5/Mer signaling pathway, thereby accelerating the resolution of pneumonia. UTI is a serine protease inhibitor with antiinflammatory and immunomodulatory roles in a variety of inflammatory diseases [24,25]. Ju et al. [26] found that UTI improved lung inflammation and injury in rats by inhibiting neutrophil activation, reducing inflammatory cell infiltration, and downregulating inflammatory cytokines. Similarly, Lu et al. [27] showed that UTI inhibited LPS-induced activation of the TLR4/MyD88/NF-jB signaling pathway, thereby reducing inflammation and preventing lung injury. Moreover, Hang et al. [28] found that UTI exerted lung protection by up-regulating the expression of aquaporin in a porcine acute respiratory distress syndrome two-hit model. In addition, UTI stabilizes the lysosomal membrane, inhibits the production of myocardial inhibitory factors, inhibits the release of lysosomal enzymes, scavenges oxygen-free radicals, inhibits the excessive release of inflammatory mediators, improves human microcirculation and tissue perfusion, and plays a role in the function of tissues and organs. There are all effects, Which contribute to the protection of tissues against damage. These studies suggest that UTI may play an important role in lung injury. During the inflammation regression phase of ALI, the inflammation process depends on the apoptosis and clearance of recruited neutrophils. Regulatory mechanisms of macrophage efferocytosis directly affect the recognition, engulfment, and digestion of apoptotic cells, thereby affecting the progression of pneumonic resolution. Therefore, whether UTI promoted recovery from lung injury by accelerating the resolution of ALI inflammation was investigated during this study. These results provide evidence that UTI accelerates the resolution of pneumonia by enhancing macrophage efferocytosis.
Our previous study confirmed that a signaling pathway involving Mer receptors enhances isoflurane-induced macrophage efferocytosis [17], and this study, found that with the increasing UTI concentration, the expression of Mer receptors on the macrophages surface increased. However, it is not clear how UTI mediates Mer receptor tyrosine kinases to enhance efferocytosis [29]. Luiz et al. [29] found that the MEK5/ERK5 signaling pathway mediates IL-4-induced differentiation of M2 macrophages by regulating c-MYC expression. Giurisato et al. [30] found that extracellular regulation of p21 expression mediated by protein kinase 5 promotes macrophage proliferation and tumor growth and metastasis. These studies suggest that ERK5 may be a key factor in the regulation of macrophage function. Does UTI regulate Mer receptors by activating ERK5 and thus regulate macrophage efferocytosis? This study, found that ERK5 expression increased with increasing UTI concentration, and macrophage phagocytosis of apoptotic cells also increased. In the presense of ERK5 inhibitors, Mer expression decreased, and macrophage phagocytosis was also reduced. Furthermore, in the in vivo model of LPSinduced lung injury, it was also found that alveolar macrophage phagocytic apoptosis phagocytic volume decreased in alveolar lavage fluid. Thus, and the pathological injury of lung tissue was aggravated. These results suggest that UTI enhances macrophage efferocytosis through the ERK5/Mer signaling pathway and promotes the resolution of pneumonia.

Conclusion
In conclusion, UTI enhances macrophage efferocytosis through the ERK5/Mer signaling pathway, promotes the regression of pneumonia, alleviates lung injury, and provides a new target and theoretical basis for further development of more effective drugs to treat lung injury.
KQ: methods; collected materials and resources; SPM: data management, interpreted, and supervised the study; YX: resources department, conducted literature analysis; XKD: supervision, project management, fundraising. All authors read and approved the manuscript and scrutinized all data. The authors declare that all data were generated in-house.

Data availability statement
The data that support the findings of this study are available from the corresponding author (duxu-eke@gxmu.edu.cn) upon reasonable request.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.