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Anti-Semaphorin-7A single chain antibody demonstrates beneficial effects on pulmonary inflammation during acute lung injury

Pulmonary inflammation is a primary characteristic of lung injury initiated by the accession of immune cells into the alveolar space. Neutrophil migration serves an important role in pulmonary inflammation mediated by the migration of neutrophils into hypoxic tissue sites. The elimination of pulmonary inflammation is directly associated with rehabilitation in patients with lung injury. Anti-inflammatory treatment is essential following lung injury and ultimately determines patient outcomes. Semaphorin-7A (SEMA-7A) is a member of the Semaphorin family that influences the migration of neutrophils into hypoxic tissue sites, thus promoting inflammation. However, understanding of the role of SEMA-7A serves during lung injury is limited and the immunological function of SEMA-7A during the migration of neutrophils into acute injury sites remains unknown. The present study investigated SEMA-7A expression and constructed a single chain antibody for SEMA-7A (Anti-SEMA-7A) to study its therapeutic efficacy against pulmonary inflammation in a mouse model of acute injury sites. The data indicated that the expression of SEMA-7A was upregulated due to induction by pro-inflammatory cytokines and demonstrated that Anti-SEMA-7A inhibited SEMA-7A expression in vitro and in vivo. The current study also indicated that the production of pro-inflammatory cytokines induced by SEMA-7A in endothelial and epithelial cells enhanced pulmonary inflammation. Anti-SEMA-7A suppressed the transendothelial migration of neutrophils mediated by SEMA-7A. Anti-SEMA-7A treatment neutralized SEMA-7A expression and reduced signs of pulmonary inflammation, leading to the elimination of pulmonary inflammation in rat with acute lung injury. The current study identified Anti-SEMA-7A as a potential agent to interfere with the inflammatory pathway during acute lung injury, which may be the basis for anti-inflammatory strategies to treat lung injuries in the future.


Introduction
Acute lung injury is caused by a variety of direct and indirect injuries of alveolar epithelial and capillary endothelial cells that lead to diffuse pulmonary interstitial, alveolar edema and acute hypoxic respiratory insufficiency (1,2). Acute lung injury is the most common respiratory disease in clinical settings (3). Acute lung injury can be induced in a number of ways, including via stress, trauma, sepsis, seawater aspiration and burns. It frequently leads to respiration distress syndrome, and may result in the development of life-threatening conditions (4). The most severe form of acute lung injury is respiration distress syndrome, which is a leading cause of morbidity and mortality in critical patients with acute lung injury (5,6). The most commonly used treatments for acute lung injury are major surgery and prolonged mechanical ventilation, in addition to the use of anti-inflammatory treatments when inflammation develops in response to pneumonia (7,8). It has been demonstrated that acute lung injury is associated with refractory lung disease and the mortality rates of patients with advanced infectious lung injuries are high (9).
The mechanism of acute lung injury is well known and research indicates that lung injury is aggravated by the migration of neutrophils from the vascular compartment into the alveolar space (10). Pneumonia often occurs in conjunction with acute lung injury as the process of self-propagating inflammation and develops within the alveolar space (11). Pulmonary inflammation is associated with deterioration during acute lung injury. In addition, previous studies have demonstrated that the severity of associated pulmonary inflammation is associated with the extent of alveolar inflammation that occurs, which is a significant factor influencing the outcome of patients with acute lung injury (10,12). Furthermore, it has been indicated that neutrophil infiltration promotes the development of inflammation in the alveolar space, which is regulated by the chemokine system (13). It has also been suggested that the efficacy of the neuronal guidance protein signaling pathway is due to the control and orchestration of acute inflammation by neutrophil migration (11).
Semaphorin7A (SEMA-7A) is a multifunctional membrane-anchored protein, which serves a role in regulating pulmonary fibrosis. SEMA-7A participates in immune and inflammatory responses during acute lung injury (14). It is also a member of the semaphorin family that induces the expression of cytokines in macrophages and monocytes via a glycophosphatidylinositol linkage and serves a significant role during the effector phase of the inflammatory immune response (15). A previous study indicated that SEMA-7A aggravated pulmonary inflammation by inducing pro-inflammatory cytokine production in epithelial and endothelial cells, thus enhancing the condition of pulmonary inflammation and production of cytokines during lung injury (16). However, understanding of the role SEMA-7A serves during lung injury is limited and not well understood regarding the immunological function of SEMA-7A in inducing the migration of neutrophils into acute injury sites.
The present study investigated SEMA-7A expression and constructed an antibody for SEMA-7A (Anti-SEMA-7A). The therapeutic efficacy of pulmonary inflammation during a mouse model of acute injury sites was also assessed. In addition, the production of pro-inflammatory cytokines induced by SEMA-7A in endothelial and epithelial cells was analyzed in vivo. Data indicated that Anti-SEMA-7A inhibited the transendothelial migration of neutrophils mediated by SEMA-7A inactivation. Furthermore, Anti-SEMA-7A significantly alleviated the degree of pulmonary inflammation, leading to the elimination of pulmonary inflammation in rat with acute lung injury.

Materials and methods
Cell culture and reagents. Human endothelial HMEC-1 cells (Cell Center of Nanyang Medicine College, Nanyang, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Alveolar cells were isolated from experimental rats and cultured in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were maintained at 37˚C, 5% CO 2 and saturated humidity.
Cells treatments. Human endothelial HMEC-1 cells (1x10 5 ) were cultured in six-well plates for 12 h at 37˚C. Cell were then incubated with LPS (5 mg/ml, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or PBS for 4 h at 37˚C.
Enzyme-linked immunosorbent assay (ELISA). SEMA-7A concentration levels were analyzed by an ELISA kit according to the manufacturer's protocol (cat no. SEB448Mu; Wuhan Uscn Business Co., Ltd., Wuhan, China). Cleaved Sema-7A from lysed lung cells membranes from rats was evaluated. The results were recorded at 450 nm using an ELISA microplate reader.
The purified protein was collected and gel filtration chromatography (cat no. 17014901; GE Healthcare Life Sciences) was used to further purify Anti-SEMA-7A, as described previously (21).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was extracted from HMEC-1 and lung cells from experimental animals using an RNA Extract kit (cat no. 339390, Qiagen Sciences, Inc., Gaithersburg, MD, USA) according to the manufacturer's instructions. cDNA was synthesized using a RevertAid™ First Strand cDNA Synthesis kit (MBI Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). qPCR was performed using a 50 µl reaction mixture. All forward and reverse primers (Table I) were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Expression of β-actin was used as an endogenous control (Invitrogen; Thermo Fisher Scientific, Inc.). Interleukin (IL)-1β, IL-17A, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine receptor 2 (CXCR2), macrophage inflammatory protein 2 (MIP-2) and keratinocyte chemoattractant (KC) expression was analyzed with the SYBR-Green Master mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. The reaction conditions were performed as follows: 40 cycles of 95˚C for 10 min and 35 cycles of 95˚C for 20 sec and 56.5˚C for 1 min. Each experiment was repeated three times. Relative mRNA expression changes were calculated using the 2 -ΔΔCq method and the results are expressed as the n-fold change compared with the control (22).
Immunofluorescence. Paraffin-embedded tissue sections (4-µm thick) were fixed with 4% paraformaldehyde on slides for 2 h at 37˚C. The sections were washed with PBS three times and then stained with the appropriate antibody: SEMA-7A Concentration of serum albumin. Venous blood samples were collected from the antecubital vein. Serum was obtained by centrifuging at 8,000 x g for 10 min at 4˚C. Serum albumin levels were measured using the bromocresol green dye-binding method on a Roche Modular DP analyzer (Roche Diagnostics, Basel, Switzerland).
Animal study. A total of 60 Specific pathogen-free male SD rats (eight-week old, weighing 280-320 g) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). All animals were housed in a temperature-controlled facility at 23±1˚C and relative humidity of 50±5% with a 12-h light/dark cycle. The current study was approved by the Ethics Committee of The First Affiliated Hospital, Nanyang Medicine College. All rats were then returned to their cages and given food and water. Animals were anesthetized with sodium pentobarbital (40 mg/kg, Sigma-Aldrich; Merck KGaA) by intravenous injection for further analysis. Following anesthesia, the limbs of rats were affixed to a board and necks were shaved, followed by a 1-cm incision. The trachea was punctured with a 25-Ga needle and LPS (500 µg/kg; Sigma-Aldrich; Merck KGaA) was injected into the trachea. Treatments were initiated on day 3 after LPS injection. Rats were divided into two groups (n=20, each) and received treatment of Anti-SEMA-7A (10 mg/ml; Sigma-Aldrich) or the same volume of PBS. The treatments were performed once a day for a total of 10 days. The therapeutic outcome of acute lung injury was evaluated by inflammatory factors Il-1β, IL-17A and TNF-α according to a previous study (23). In addition, Anti-SEMA-7A (10 mg/ml, Sigma-Aldrich) or PBS  Reverse Forward SEMA-7A, Semaphorin-7A; SEMA-7AR, SEMA-7A target receptor IL, interleukin; TNF-α, tumor necrosis factor-α.
(10 mg/ml, Sigma-Aldrich) were transported to lesions using a hybrid nanometer carrier (liposome), following a previously described protocol (24). Animals were sacrificed on day 15 for further analysis. . Data are presented as the mean ± standard deviation of triplicate experiments. Differences among multiple groups were determined using the Student's t-test or one-way analysis of variance followed by a Tukey HSD test and P<0.05 was considered to indicate a statistically significant difference.

Results
Analysis of Semaphorin-7A expression during acute lung injury. On the basis of a previous study (25) demonstrating that SEMA7A regulates neutrophil trafficking during acute pulmonary inflammation, the present study assessed SEMA-7A expression in rats with lung injury induced by LPS (500 µg/kg) in vivo. Levels of SEMA-7A mRNA and protein expression were significantly higher in the pulmonary tissue of rats with lung injury (P<0.05; Fig. 1A and B). In addition, the results in Fig. 1C indicate an increase in SEMA-7A expression in pulmonary tissue in the LPS group compared with the control. Furthermore, SEMA-7A target receptor expression was assessed in pulmonary tissue in rats with lung injury by RT-qPCR (Fig. 1D). Analysis indicated that the expression of SEMA-7A target receptor (SEMA-7AR) was significantly increased in acute pulmonary inflammation in vivo in the LPS group compared with the control group (P<0.01). These findings suggest that SEMA-7A expression is upregulated during acute pulmonary inflammation.

Construction and characteristics of Anti-SEMA-7A in vitro.
Based on the aforementioned report (26), the present study hypothesized that SEMA-7A may serve as a potential therapeutic target molecular for acute lung injury therapy in LPS-induced acute lung injury rats. Therefore, antibody therapy target for SEMA-7A may provide potential novel target therapy for acute lung injury. In the current study, a recombinant single chain antibody liked with Fc was constructed and presented a high affinity for SEMA-7A. Fig. 2A presents the structure of Anti-SEMA-7A, which contains a cell-penetrating peptide (CCP) and Fc fragment to enhance transmembrane ability and stability. Anti-SEMA-7A was expressed by E. coli and the yield was ~42 mg/l. The molecular weight of Anti-SEMA-7A-CPP was ~38 kDa, as determined by SDS-PAGE and ~48 kDa under constant denatured gel electrophoresis (Fig. 2B). In addition, ELISA and western blot analysis assays indicated that Anti-SEMA-7A specifically bound to SEMA-7A ( Fig. 2C and D). The data indicated that Anti-SEMA-7A was purified successfully and possessed the potential to integrate into the corresponding molecules.

Anti-SEMA-7A suppresses the transmigration of neutrophils in endothelial and epithelial cells.
On the basis of the in vitro efficacy of Anti-SEMA-7A, the present study further assessed the role of Anti-SEMA-7A in the LPS-induced acute lung injury rat model. Rats with acute lung injury received Anti-SEMA-7A treatment a total of 7 times. The concentration level of serum albumin was used to determine the therapeutic effects of Anti-SEMA-7A. The results of the present study demonstrated that the number of neutrophils was markedly reduced, along with the expression of Claudin within the pulmonary tissue of Anti-SEMA-7A-treated rats compared with controls. The results in Fig. 4A indicate that the pathogenic condition of rats with acute lung injury was relieved following treatment with Anti-SEMA-7A. Lung sections were prepared for analysis of SEMA-7A and inflammatory factor expression. It was demonstrated that SEMA-7A expression decreased following Anti-SEMA-7A treatment in lung, which did not occur in the lung tissue of rats treated with PBS (Fig. 4B). Results demonstrated that levels of IL-1B, IL-17A and TNF-a are downregulated following Anti-SEMA-7A treatment, compared with controls (Fig. 4C). Furthermore, expression of the chemokines of CXCR2, MIP-2 and KC decreased within the alveolar space of Anti-SEMA-7A-treated rat (Fig. 4D). These findings suggest that Anti-SEMA-7A exhibits beneficial effects on rats with acute lung injury by neutralizing SEMA-7A expression.
Anti-SEMA-7A suppresses ERF activity for EMT signaling pathway. To investigate the potential mechanism of Anti-SEMA-7A in acute lung injury, the activity of ERF as a Ras/ERK mediator in EMT progression was investigated in alveolar cells. Alveolar epithelial cells were isolated from experimental rat that had undergone treatment with Anti-SEMA-7A or PBS. Levels of ERF, ERK, M1-7 and FSF/FKF expression were analyzed in alveolar cells by immunofluorescence. The results indicate that levels of ERF, ERK, M1-7 and FSF/FKF expression were downregulated following treatment with Anti-SEMA-7A (Fig. 5). These findings indicate that Anti-SEMA-7A obstructed the ERF-induced EMT process through the Ras/ERK pathway in alveolar cells.

Discussion
Acute lung injury is a serious acute respiratory disease that presents as the most common type of acute respiratory failure with high mortality and morbidity in critically ill patients (27). Acute lung injury frequently leads directly or indirectly to alveolar epithelial cell injury and capillary endothelial cell damage, which subsequently results in diffuse pulmonary   interstitial, alveolar edema and acute hypoxic respiratory insufficiency (28). Pathological physiological characteristics of acute lung injury are evident decreases in the vital capacity, lung compliance and ventilation/blood flow ratio (29). The clinical characteristics of acute lung injury are progressive hypoxemia and respiratory distress, diagnosed by imaging to identify homogenous exudative lesions (30). Acute lung injury may develop into acute respiratory distress syndrome at severe stages (oxygenation index <200), which may become a clinical problem for patient treatments (31). In the present study, a rat model of acute lung injury induced by LPS was established by inhalation to investigate the primary mechanism and progression of this disease. Pneumonia is the single most common feature for patients with acute lung injury (32). Inflammatory factors contribute to pneumonia and aggravate the condition of patients with acute lung injury (33). Despite pneumonia being identified as the most common risk factor for patients with acute lung injury, there is limited understanding of the molecular mechanism of inflammatory factors in patients with acute lung injury and pneumonia (34). Therefore, investigating the role of inflammatory factors in the progress of pneumonia within the alveolar space in patients with acute lung injury is essential.
SEMA-7A is part of the Semaphorins family of soluble and membrane-anchor proteins originally considered to be involved in the development of axonal signal factors in the nervous system. Previous studies have indicated that SEMA-7A influences polymorphonuclear leukocyte migration during hypoxia and demonstrated that SEMA-7A regulated pulmonary fibrosis, which participates in immune and inflammatory responses during acute lung injury (14,35). Furthermore, it has been reported that overexpression of SEMA-7A promotes the migration of transendothelial neutrophils into the alveoli, resulting in inflammation and stimulating changes in the morphology of lung cells in patients with acute lung injury (14). The most serious threat for patients with acute lung injury is that pneumonia may lead to lung failure. In addition, a previous study has suggested that SEMA-7A may initiate inflammation during acute lung injury induced by different factors, including hypoxia-inducible factor and β1 integrin (36). The etiology of lung injury is frequently associated with the process of inflammation within the lung, which leads to fatal infections deep in the lungs as a sequela of the primary process (37). SEMA-7A expression is associated with the inflammatory process within the lung, indicating that SEMA-7A may be a potential molecular target (38).
In the current study, a chimeric antibody containing cell-penetrating peptide and Fc target was produced for SEMA-7A and the function of Anti-SEMA-7A in an acute lung injury rat model was investigated. The present study indicated that SEMA-7A expression was dysfunctional in the acute lung injury model in vitro and in vivo, consistent with results of a previous study (39). In addition, the current study indicates that levels of IL-1β, IL-17A and TNF-α expression were upregulated in rats with LPS-induced acute lung injury. By contrast, levels of the chemokines CXCR2, MIP-2 and KC were decreased within the alveolar space of Anti-SEMA-7A-treated rat. These inflammatory factors and chemokines may stimulate neutrophil migration into the alveolar space and Anti-SEMA-7A decreased their levels of expression levels, thus inducing a remission effect in acute lung injury (40).
In addition, the data demonstrated SEMA-7A not only induced inflammation but also promoted the neutrophils migration into the alveolar space in vivo. This finding further explains the interaction between SEMA-7A expression and the upregulation of inflammatory factors, which is in accordance with a previous study (41). Despite SEMA-7A inducing the production of cytokines and chemokines in the injury sites of the lung, SEMA-7A also regulated the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells (42). Therefore, the current study suggests that Anti-SEMA-7A inhibits SEMA-7A expression and led to ERF-induced inhibition of EMT in Ras-dependent epithelial cells in vitro and in vivo (43). The hypothesis of the present study was confirmed by the data and results, as it suggested that SEMA-7A promoted EMT progress, leading to neutrophil migration into the alveolar space in vivo. These results may provide a novel target signaling pathway for inhibiting neutrophil migration in patients with acute lung injury.
In conclusion, the current study aimed to determine the therapeutic effects of Anti-SEMA-7A in acute lung injury rats. To the best of our knowledge, this is the first study to use Anti-SEMA-7A targeting of SEMA-7A to analyze the efficacy for rats in a model of lung injury. The results of the current study emphasise the importance of SEMA-7A for the control of the acute inflammatory response and treatment of acute lung injury. The interference of Anti-SEMA-7A with ERF-induced inhibition of EMT in Ras-dependent pathway in epithelial cells suggests its potential association with acute inflammation in lungs, suggesting that it may be developed for clinical applications in the future.