Recombinant human urinary trypsin inhibitor improves the immunological function of splenic dendritic cells in mice with acute lung injury

Abstract

Aim of the study

Acute lung injury (ALI) is a common critical condition in the emergency department and is associated with a high mortality rate. Recombinant human urinary trypsin inhibitor (rhUTI), a serine protease inhibitor compounded by genetic engineering technology, is expected to replace UTI, which has been reported to protect multiple organs against inflammation- and/or injury-induced dysfunction. The aim of the present study was to investigate the immunomodulatory effects of rhUTI on splenic dendritic cells (DCs) in LPS-induced ALI mice.

Materials and methods

RhUTI was administered to mice, and splenic CD11c + DCs were isolated and assessed using flow cytometry for apoptotic or phenotypic analysis. Protein markers and cytokines were determined by western blotting or enzyme-linked immunosorbent assay.

Results

After treatment with rhUTI, lung injury in LPS-induced ALI mice improved and the survival rate of the mice increased. Treatment with rhUTI could markedly upregulate the levels of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class II molecules (MHC-II) on the surface of splenic DC and decreased the apoptosis rate of splenic DCs in LPS-induced ALI mice. In addition, protein levels of markers of endoplasmic reticulum stress (ERS) and ERS-related apoptotic pathways (including GRP78, XBP-1, PERK, caspase-12, and CHOP) were downregulated in the rhUTI-treated group when compared with the LPS-induced ALI group.

Conclusions

These results suggest that rhUTI protects LPS-induced ALI mice by improving the immune response of splenic DCs and inhibiting excessive ERS-mediated apoptosis.

Introduction

Acute lung injury (ALI) is a life-threatening hypoxic respiratory disease characterized by diffuse pulmonary interstitial and alveolar edema with increased permeability of lung epithelial and capillary endothelial cells [1, 2]. Previous studies have shown that immune disorders and abnormal inflammatory responses play an important role in the development of ALI [3, 4]. This immune disorder is closely related to the involvement of various immune cells in innate and adaptive immunity [5]. Among these, dendritic cells are the most important specialized antigen-presenting cells and play an important role in regulating the immune response network. Therapies for the prevention or treatment of ALI remain unclear. Recently, it has been found that bone marrow mesenchymal cells have shown therapeutic potential in anti-inflammatory and cellular repair, but their functional decline associated with aging and the instability of treatment effects have limited their clinical application in the treatment of sepsis-related lung injury [6]. Beyond other supportive care, there is currently no specific drug administration approved for treatment; therefore, it is critical to explore the molecular mechanisms and develop effective therapeutic agents for the treatment of ALI.

Ulinastatin, also known as urinary trypsin inhibitor (UTI), is a multivalent Kunitz-type serine protease inhibitor that was first identified in and purified from human urine in 1982. Both animal and clinical studies have shown that UTI can protect multiple organs against inflammation- and/or injury-induced dysfunction [7–11]. Furthermore, it has been reported that the early application of UTI can reduce the symptoms of ALI [12]. Although UTI has been widely used clinically in some Asian countries to treat pancreatitis, severe infection, septic shock, acute circulatory failure, and multiple organ failure, its protective mechanism requires further investigation [13–15]. Moreover, since UTI are derived from urine, there is a risk of contamination by pathogenic microorganisms and viruses. Thus, the present study was carried out to confirm the therapeutic efficacy of a highly pure UTI protein, rhUTI, in LPS-induced ALI mice and to investigate the potential effects and underlying mechanisms of rhUTI on immunoregulatory effects [16].

Materials and methods

Animals

Adult male C57BL/6 mice were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). They were 6–8 weeks, weighing 20 ± 2 g. All animals were housed in separate cages under climate-controlled conditions (24 ± 1 °C and 40–80% relative humidity) and left to acclimatize for at least 7 days before being used for experiments. LPS (20 mg; Sigma, St. Louis, MO, USA) in 50 ml of phosphate-buffered saline (PBS) was instilled directly into the tracheas of mice sedated with isoflurane (Baxter, Deerfield, IL, USA) using a modified feeding needle. To ensure an unbiased allocation of experimental animals to different groups, we employed a simple randomization method. This process includes numbering, random number generation, and group assignment. To prevent selection bias, the randomization process was performed by researchers who were not involved in subsequent experimental procedures or outcome evaluation. The allocation list is kept confidential and sealed until the end of the study, ensuring that the investigator in charge of the experiment is not aware of the group assignment. Euthanasia of experimental surviving mice was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals to ensure a humane and painless procedure. Mice were briefly anesthetized with isoflurane to minimize stress and ensure a smooth transition to unconsciousness. The animals were subjected to cervical dislocation to ensure complete and irreversible cessation of life, as recommended by the American Veterinary Medical Association (AVMA) guidelines. According to the experimental requirements, a full-thickness incision was made on the abdominal skin of the mice after euthanasia; the required organs were extracted, and the abdomen was closed by full-thickness sutures after the operation.

Medium and reagents

Lipopolysaccharide (LPS) and Concanavalin A (ConA) were purchased from Sigma-Aldrich (St. Louis, MO., USA). rhUTI was purchased from Wanxing Biological Pharmaceutical Co. Ltd. (Shanghai, China). Ulinastatin for Injection (National Drug Approval NO.H19990134, lot 031211103) was purchased from Techpool Bio-Pharma Co., (Guangdong, China). The mouse visceral tissue lymphocyte separation liquid reagent kit (Clone 1ts1092p) was purchased from the Tianjin Hao Yang Biology manufacturer. Ltd, (Tianjin, China). Fetal calf serum (FCS) was purchased from Gibco Life Technologies (Grand Island, NY, USA). RPMI 1640 and PBS were purchased from Solarbio Biology Inc. (Beijing, China). CD11c (N418) microbeads (mouse, clone 130-052-001), anti-mouse CD80-allophycocyanin (APC) (clone 130-102-584), and CD86-phycoerythrin (PE) (clone 130-102-804) were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). Anti-mouse CD11c PE-cy7 (clone E07516-1635) and anti-mouse major histocompatibility complex class II (MHC-II) (clone E06239-1634) were purchased from eBioscience (San Diego, CA, USA). Annexin V-PE apoptosis detection kit I (lot 559763) C/EBP homologous protein was purchased from BD/PharMingen (San Diego, CA, USA). Anti-mouse (CHOP) (clone 2895P) and caspase-12 rabbit (clone 2202S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin (clone sc-47778)-conjugated goat anti-mouse IgG (H + L) (clone C1308) and horseradish peroxidase-labelled goat anti-rabbit IgG (H + L) (clone C1309) were purchased from Pulilai Co. (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-12 p70 (lot EM006), IL-10 (lot EM005), IL-4 (lot EM003), interferon (IFN)-γ (lot EM007) were purchased from ExCell Biology Inc. (Shanghai, China).

Histological evaluation

To evaluate LPS-induced ALI, the mice were sacrificed under isoflurane 48 h after LPS treatment. The lung wet/dry weight ratio was calculated to evaluate ALI severity. The lung was obtained immediately after dissection, and tissue was weighed on a weighing disk (wet weight), dried in a constant temperature oven at 60 °C, and weighed again (dry weight). The left middle lobe of the right lung was used for histological evaluation and immunohistochemistry, whereas the lower lobe was frozen in liquid nitrogen for further analysis. The lungs were fixed in 4% paraformaldehyde, embedded in paraffin, cut into sections, and stained with hematoxylin and eosin (H&E). Four sections were taken for each tissue sample, and four images were taken for each section. A pathologist blindly scored each lung injury objectively according to the following criteria: alveolar congestion, hemorrhage, neutrophil infiltration into the airspace or vessel wall, and thickness of the alveolar wall/hyaline membrane formation. Each category was graded on a 4-point scale: 0, no injury, 1 = injury up to 25% of the field, 2 = injury up to 50% of the field, 3 = injury up to 75% of the field; and 4, diffuse injury [17, 18]. The lung injury score for each animal was calculated as the mean of four lung sections under a light microscope (200 × magnification; Olympus, Tokyo, Japan). We also used blinding in histological assessments.

Purification and isolation of splenic CD11c + DCs and CD4 + T cells

Mice were sacrificed, and spleens were harvested. Mononuclear cells were obtained by passing spleens through a 30 μm stainless steel mesh twice and then subjected to Ficoll–Paque density gradient centrifugation. Splenic CD11c + DCs and CD4 + T cells were isolated from mononuclear cells by positive selection using MicroBeads and a MiniMACS™ Separator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with a positive selection MS column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The selected cells were pelleted by centrifugation (300 × g, 10 min), the supernatant was discarded, and the cell pellet was resuspended in the desired volume of RPMI 1640 medium containing 10% FCS and then cultured at 37 °C in 5% CO2 in humidified air overnight for recovery. The purity of isolated CD11c + DC and CD4 + T cells was verified by flow cytometric analysis (BD Bioscience, Mountain View, CA, USA). First, stain the cells with fluorescent antibodies specific to CD11c and CD4. Then run the stained cells on a flow cytometer and analyze the data. Set appropriate gates based on scatter plots to identify target cell populations. Calculate the percentage of CD11c + cells and CD4 + cells within the gated populations. High percentages indicate high purity of the isolated cells.

Cell culture and stimulation

The cells were subsequently cultured in RPMI 1640 medium containing 10% FCS at 37 °C in 5% CO₂ in humidified air. DCs were stimulated with LPS or rhUTI (LPS, 1 μg/mL; rhUTI: 2700 U/mL) for 24 h [16]. CD4 + T cells were incubated with the T-cell mitogen Con A (5 μg/mL) for 18 h before mixing with DCs at a ratio of 150:1, and co-cultured for 72 h. After culture and stimulation as indicated earlier, cells were collected for phenotypic analysis with flow cytometry, assessment of cytokines in cell culture supernatants by ELISA, and measurement of proliferation by the Cell Counting Kit-8 assay (CCK-8 is a colorimetric method for detecting cell viability, which uses the water-soluble tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)−3-(4-nitrophenyl)−5-(2,4-disulfophenyl)−2H-tetrazolium monosodium salt) to be reduced to an orange formazan dye by the action of dehydrogenase in the cells. The amount of formazan dye generated is proportional to the number of viable cells, and cell viability can be quantitatively assessed by measuring the absorbance at 450 nm).To observe the expression of phenotypic markers, DCs were reacted for 15 min at 4 °C in 100 μL of PBS. After washing cells with 1 × permeabilization buffer twice, the cells were stained with the anti-mouse/rat CD80, CD86, and MHC-II antibodies for 30 min at 4 °C in the dark. In all experiments, isotype controls were included using an appropriate PE- or fluorescein isothiocyanate (FITC)-conjugated irrelevant monoclonal antibody of the same Ig class. After washing twice, the cells were analyzed by flow cytometry using a fluorescence-activated cell sorter (FACS) (BD Biosciences, Mountain View, CA, USA) [19].

Apoptosis determination

The percentage of apoptotic cells was determined using simultaneous annexin V-PE and 7-AAD staining. The assay was performed according to the manufacturer’s instructions. Briefly, DCs (1 × 10⁵) were collected, washed twice with cold PBS, and resuspended in 100 μL of binding buffer, to which 5 μL annexin V-PE and 5 μL 7-AAD were added. After incubation for 15 min in the dark at room temperature, the cells were diluted with 400 μL of binding buffer and analyzed by flow cytometry within 1 h using a FACScan (BD Biosciences, Mountain View, CA, USA) [20].

Western blotting

Cells were washed with ice-cold PBS, suspended in lysis buffer containing 1 mM sodium orthovanadate (A phosphatase inhibitor, aiming to maintain the phosphorylation state of the protein) supplemented with a protease inhibitor mixture (Promega, Madison, WI, USA), and then centrifuged at 12,000 × g for 15 min at 4 ℃. The protein levels were quantified using a bicinchoninic acid protein assay kit (Pulilai Co., Beijing, China). Forty micrograms of protein per lane in loading buffer were separated by 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and electro-transferred onto nitrocellulose membranes. After blocking with 10% skim milk overnight at 4 ℃ and incubating for 6 h at room temperature with anti-glucose-regulated protein (GRP)78, X-box binding protein 1 (XBP-1), pancreatic endoplasmic reticulum kinase (PERK), C/EBP homologous protein (CHOP), caspase-12 antibodies (1:500), or control anti-actin antibodies, the membrane was washed three times with 1 × TBST, followed by incubation with a secondary antibody for 1 h at room temperature. The membrane was washed three times, as described above, and proteins were detected using an ECL system (Amersham Biosciences, Uppsala, Sweden). Protein bands were quantified by densitometry using ImageJ software (National Institutes of Health) [21].

Cytokine levels measured by ELISA

The levels of IL-12, TNF-α, IL-4, IL-1β, IL-10, IFN-γ, MPO, and MDA were measured using ELISA according to the manufacturer’s instructions. The color reaction was terminated by adding 100 μL of orthophosphoric acid. The plates were read using a microplate reader (Spectra MR; Dynex, Chantilly, VA, USA). The concentrations of cytokines in the samples were calculated based on the standard curves generated using purified recombinant cytokines. All samples were serially diluted twofold in duplicate for analysis [22].

Statistical analysis

Data were analyzed using the SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). The primary endpoint of each experiment was the survival rate. Data are expressed as mean ± standard deviation (SD). Differences in survival rates were analyzed using Kaplan–Meier survival plots and log-rank tests. One-way ANOVA and unpaired Student’s t test were used to determine significant differences between groups. Statistical significance was set at P ≤ 0.05. The Chi-square test was used for survival studies.

Results

Treatment with an optimal dose of rhUTI enhanced the survival rate of mice after LPS-induced ALI

In the survival experiment, the number of mice in each group was 36.To investigate whether rhUTI might have positive effects on the survival rate of mice with ALI, C57BL6 mice were treated with rhUTI (250,000 U/kg, dilute with normal saline, intramuscular injection) 1 h before and 12 h, 24 h after stimulation with 5 mg/kg of LPS and the survival rate was carefully monitored for 7 days [16]. As expected, the results (Fig. 1) showed that the LPS + rhUTI group was more resistant to LPS-induced mortality. In the LPS group, approximately 78% of the mice died within 7d. In contrast, rhUTI administration significantly increased the 7 d survival rate compared with that in the LPS group (44.4% vs. 22.2%) (P < 0.05). We assessed the levels of inflammatory cytokines, including IL-12 and TNF-α, in the serum of the mice. As shown in Fig. 1B, C, treatment with rhUTI resulted in a significant lowering in IL-12, TNF-α levels (Fig. 1B, C), (all P < 0.05). As shown in Fig. 1D, E, treatment with rhUTI resulted in a significant increase in IL-1β, IL-10 levels, (P < 0.05).

Fig. 1.

Protective effect of recombinant human urinary trypsin inhibitor (rhUTI) in mice with acute lung injury (ALI). A Mice with ALI were treated with rhUTI (250,000 U/kg) 1 h before and 12 h, 24 h after stimulation with lipopolysaccharide (LPS; 5 mg/kg). Survival was monitored every day for 7 days after LPS treatment (n = 36/group). Survival was analyzed by log-rank (Mantel–Cox) test. B–E Inflammatory mediators in the serum of LPS-induced ALI mice were quantified using enzyme-linked immunosorbent assay (ELISA). * Represents statistical significance between the test and sham groups at the same time point (P < 0.05)

Effect of rhUTI on LPS-induced lung histological changes

We next examined the effect of rhUTI on LPS-induced ALI by assessing the histological changes in lung tissues 48 h after LPS administration. As shown in Fig. 2, alveoli, bronchus and tube in the lung tissue of the sham group were clear, and no obvious abnormalities are found. The above indicated the lung tissues of the sham group exhibited normal structures, with no histopathological changes. However, the lung tissues of the LPS group exhibited extensive pathological changes, the vesicle cells were swollen and detached, and a large number of inflammatory cells could be seen with severe alveolar hemorrhage, which manifested as inflammatory cell infiltration and severe pulmonary edema. In contrast, LPS-induced histopathological lung damage was effectively alleviated by rhUTI. The specific performance of rhUTI treatment group is as follows: a small number of inflammatory cells were seen infiltrating the tissue. Occasionally, individual alveolar parietal cells are swollen, and the tissue is mildly bruised. No other significant abnormalities.

Fig. 2.

Effect of rhUTI on the histopathology of the lung tissues of LPS-induced ALI mice. C57BL/6 mice were treated with rhUTI (250,000 U/kg) or saline 1 h before and 12 h, 24 h after stimulation with 5 mg/kg of LPS. Mice were euthanized with ether and the lungs from each experimental group were examined for histopathology. Representative images for the histological changes in the hematoxylin and eosin stained lung sections (magnification 200 ×)

Effect of rhUTI on lung wet/dry weight ratio and ALI score in mice

To evaluate the extent of lung injury, the lung injury score, which was semiquantitatively assessed by a blinded pathologist, further revealed the protective effects of rhUTI on LPS-induced ALI (Fig. 3A). The wet/dry weight ratios of the lungs were measured to evaluate the effects of rhUTI on LPS-induced lung edema. As shown in Fig. 3B, compared to the sham group, the lung wet/dry weight ratio was significantly increased in the LPS group. However, this increase was attenuated in the LPS + rhUTI group by treatment with rhUTI.

Fig. 3.

RhUTI ameliorated LPS-induced ALI in mice. A Effect of rhUTI on lung-injury score. B Effect of rhUTI on the wet/dry weight ratio of lungs in LPS-induced ALI mice. Data have been represented as mean ± SD (n = 15) from three independent experiments. * Represents statistical significance between experiment and sham groups at the same time point (P < 0.05)

Effects of rhUTI on LPS-induced MPO activity and MDA content in the lung

It is well-known that oxidative stress in lung tissues often occurs in ALI and plays a critical role in the pathogenesis of LPS-induced ALI. To investigate the antioxidative effects of rhUTI, we tested the effects of rhUTI on MPO activity and MDA content. In the LPS group, LPS treatment clearly led to an increase in MPO activity and MDA content compared to those in the sham group. However, these LPS-induced increases were attenuated by rhUTI treatment following LPS stimulation (Fig. 4A, B).

Fig. 4.

RhUTI attenuated myeloperoxidase activity. A, B Malonaldehyde content in the lung tissues of LPS-induced ALI mice. Data have been represented as mean ± SD (n = 15) from three independent experiments. * represents statistical significance in the data between the experimental and sham groups at the same time point (P < 0.05)

Phenotypic and functional changes in DCs of mice after LPS-induced ALI, with or without rhUTI treatment.

DCs, which are the most important potent antigen-presenting cells, induce and coordinate host immune responses. We evaluated the function of DCs in ALI mice, with and without rhUTI treatment. The expression of costimulatory molecules on the surface of DCs, including CD80, CD86, and MHC-II, was analyzed. As shown in Fig. 5, splenic DCs in the LPS + rhUTI group expressed higher levels of CD86, CD80, and MHC-II than those in the LPS group, suggesting that rhUTI treatment may contribute to DC activation and maturation after ALI. We also found that rhUTI treatment relieved endoplasmic reticulum stress (ERS) response in splenic DCs in LPS-induced ALI mice. The expressions of GRP-78, XBP-1, and PERK in splenic DCs were detected by western blot analysis. Representative images of the same membranes are shown in Fig. 6.

Fig. 5.

RhUTI promoted the maturation of splenic dendritic cells (DCs) in LPS-induced ALI mice. Splenic DCs were isolated from the mice using CD11c + MicroBeads. The expression of co-stimulatory molecules, including CD80 and CD86, and MHC-II on the surface of DCs was determined using flow cytometry and analyzed by calculating the ratio of mature: normal cells. rhUTI (250,000 U/kg) was administered 1 h before and 12 h,24 h after stimulation with 5 mg/kg of LPS. Data have been represented as mean ± SD (n = 15). * represents statistical significance between data from the experimental and sham groups at the same time point (P < 0.05)

Fig. 6.

RhUTI alleviated the response of splenic DCs to endoplasmic reticulum stress in LPS-induced ALI mice. Splenic DCs were isolated from mice after 48 h of LPS stimulation. We used western blotting to detect the expression of GRP78, XBP-1, and PERK in splenic DCs (A–D). RhUTI (250,000 U/kg) was administered 1 h before and 12 h,24 h after LPS (5 mg/kg) stimulation. Data have been represented as mean ± SD (n = 4). * represents statistical significance between the data from the experimental and Sham groups (P < 0.05)

Effect of rhUTI on the DCs apoptosis

We also assessed the DC functionality based on the percentage of apoptotic cells. The percentage of apoptotic cells was determined by annexin V-PE and 7-ADD staining. As shown in Fig. 7A, B, treatment with 250,000 U/kg rhUTI [16] significantly reduced DC apoptosis (P < 0.01). As shown in Fig. 7C–E, stimulation with 250,000 U/kg rhUTI decreased the levels of C/EBP homologous protein (CHOP), Caspase-12 (all P < 0.05).

Fig. 7.

RhUTI decreased apoptosis of splenic DCs in LPS-induced ALI mice. Splenic DCs were isolated from mice using CD11c + MicroBeads. Apoptosis in splenic DCs was determined using flow cytometry. rhUTI (250,000 U/kg) was administered 1 h before and 12 h,24 h after LPS (5 mg/kg) stimulation. A, B Rate of apoptosis was determined using the sum of area UR (Annexin-V +/7-AAD + indicates late apoptotic cells) and area LR (Annexin-V +/7-AAD- indicates early apoptotic cells). Data have been represented as mean ± SD (n = 6). * represents statistical significance in the data between the experimental and sham groups at the same time point (P < 0.05). C–E Western blotting for the expression of CHOP and caspase-12 in splenic DCs. Representative images from the same blot are shown. Data have been represented as mean ± SD (n = 6). * represents statistical significance in the data between the experimental and Sham groups (P < 0.05)

RhUTI-treated DCs promoted T-cell proliferation and polarization

We further investigated the proliferation and polarization of T cells in response to DCs after treatment with rhUTI. DCs treated with rhUTI were co-cultured with CD4 + T cells isolated from normal mice at a DC:T ratio of 1:150. T-cell proliferation was assessed using a CCK-8 kit. It is well-known that Th1 cells produce IFN-γ, whereas Th2 cells produce IL-4. Subsequently, the culture supernatants were analyzed for cytokine changes. As shown in Fig. 8A, the proliferation rate of T cells was higher when they were co-cultured with DCs from LPS + rhUTI-treated cells than when they were co-cultured with DCs from LPS-treated cells (P < 0.05). After rhUTI treatment, the ability of T cells to secrete IFN-γ increased, and the level of IL-4 was significantly reduced (all P < 0.05) (Fig. 8B, C).

Fig. 8.

RhUTI treatment ameliorated the splenic DC-mediated regulation of T-cell polarization and proliferation. T cells were isolated from normal mice and stimulated with concanavalin A (ConA; 5 μg/mL) for 18 h. DCs were stimulated with LPS or rhUTI (LPS: 1 μg/mL; rhUTI: 2700 U/mL) for 24 h. Splenic DCs were isolated from mice using CD11c + MicroBeads and co-cultured with T cells at a DC:T ratio of 1:150 (T-cell density: 2 × 105 cells/well) and cultured for 72 h. A T-cell proliferation was detected using a cell counting kit-8. B, C ELISA was used to determine IFN-γ and IL-4 levels and Th1/Th2 polarization. Results have been shown as mean ± SD (n = 6). * represents statistical significance in the data between the experimental and LPS-induced ALI mice (P < 0.05)

Discussion

ALI is a life-threatening syndrome characterized by excessive uncontrolled inflammation and apoptosis and is a major cause of morbidity, mortality, and healthcare burden in critically ill patients. Previous studies have shown that following initial cytokine-mediated hyperinflammation, immunosuppression can lead to nosocomial infections and increased mortality, with immune cell dysfunction through the concomitant occurrence of pro- and anti-inflammatory mechanisms [23, 24]. Immunomodulatory molecules that enhance immunity against infections can be considered potential drug candidates.

UTI are used clinically in Asian countries to treat inflammatory diseases by suppressing the generation of superoxide anion radicals, inhibiting the release of inflammatory mediators, activating neutrophils, protecting cells from apoptosis, and improving circulation and coagulation [25–27]. Several studies have indicated that UTI ameliorate excessive inflammation by modulating the quantity and function of regulatory T cells (Tregs) and other immune cells (such as neutrophils, monocytes, and lymphocytes) [28–30]. In other studies, UTI effectively protects the adhesion junction and helps ameliorate the perfusion of kidney capillaries during sepsis by the inhibition of autophagy and the expression of inflammatory factors [31]. Besides, UTI also can improve energy metabolism and ameliorate injury to the gastrointestinal mucosa in the early stage of septic shock [32]. Natural UTI is derived from urine and carry a risk of contamination by pathogenic microorganisms and viruses. High purification of rhUTI is required for its extensive clinical application worldwide. Therefore, the present study focused on the effects of rhUTI on the development of ALI, and the results indicated that rhUTI treatment significantly increased the 7-day survival rate of ALI mice. In addition, rhUTI-treated mice showed significantly improved pulmonary tissue edema, alleviated lung injury, and decreased levels of oxidative stress in vivo. Previous model studies on lung injury had also found that UTI can alleviate pulmonary edema by reducing lung permeability and stimulating alveolar fluid clearance in a rat model of acute lung injury, which is consistent with our experimental conclusions using rhUTI [33]. Meta-analyses of randomized controlled studies in humans have also suggested that UTI improves all-cause mortality and other related outcomes in patients with sepsis or septic shock [34]. Therefore, we believe that rhUTI is also suitable for future clinical applications.

Based on this result, we attempted to determine whether the survival benefit of rhUTI in ALI mice is related to an improvement in the host immunosuppressive state. DCs act as a bridge between the innate and adaptive immune systems and play a key role in the pathogenesis of inflammatory lung diseases, including ALI [35, 36]. Therefore, we investigated the underlying mechanism by which rhUTI regulates DCs function. The results of the present study demonstrated that treatment with rhUTI markedly enhanced the expression of co-stimulatory molecules, including CD80, CD86, and MHC-II, on the surface of mouse splenic DCs. Our findings indicated that stimulation with rhUTI elicited a significant immunostimulatory response in DCs, suggesting that rhUTI may serve as a potential signal for inducing DC maturation. Simultaneously, IL-12 and TNF-α secretion decreased in the serum of rhUTI-treated ALI mice, indicating that rhUTI has an anti-inflammatory effect in ALI mice. In addition, decreased apoptosis of DCs was observed in the rhUTI-treated group. Administration of rhUTI protects DCs from LPS-induced ALI in mice. To further verify the influence of rhUTI on DCs and their capacity to modulate T-cell immunity, we determined the T-cell proliferation and polarization induced by rhUTI-DCs. The results demonstrated that co-culture with DCs pretreated with rhUTI led to a significantly enhanced T-cell proliferative response to the T-cell mitogen (Con A) and reinforced the polarization of T cells toward Th1 cells. This was evidenced by the elevated IFN-γ secretion and decreased IL-4 levels. These data indicate that, induced by rhUTI treatment, DCs acquired a mature phenotype and were capable of antigen presentation and T-cell stimulation. Moreover, the polarization of CD4 + T cells toward Th1 cells creates a favorable environment for the induction of cellular and humoral immunity [37].

This study demonstrated that rhUTI plays a distinct role in the maturation and immune function of DCs. However, the molecular mechanism underlying the immune regulation of DCs by rhUTI and the theoretical basis for its therapeutic benefits remain poorly understood. ERS is involved in the development of many diseases, and the accumulation of misfolded or unfolded proteins in the endoplasmic reticulum can cause apoptosis or cell death [38]. A previous study documented that excessive ERS initiated apoptosis and dysfunction of splenic DCs and was responsible for immunosuppression during severe thermal injury [39]. In the present study, we observed that treatment with rhUTI downregulated GRP78, XBP-1 and PERK (a marker of ERS that acts as a major ER chaperone with anti-apoptotic properties and controls the activation of transmembrane ERS sensors [40]) in splenic DCs. This indicates a notable attenuation of the ERS response after rhUTI stimulation. To determine how rhUTI protects cells from ERS, we used a chemical biology approach to study the ERS-induced apoptosis in rhUTI-treated DCs. The expression of caspase-12 and CHOP, recognized as two key and specific markers of ERS,—induces apoptosis [41]. In the present study, these two markers were inhibited after rhUTI treatment. This indicated that the protective effect of rhUTI was mediated by suppressing CHOP and caspase-12, reducing DCs apoptosis, improving DCs’ maturity, strengthening their functions, mediating T-cell activation and Th1 differentiation, and playing a key role in immune adjustment to further improve the survival rate of ALI mice. These results suggest that rhUTI plays a protective role in DCs via the ERS response pathway, thereby countering ERS-related apoptosis in ALI mice.

However, this study has some limitations. First, we observed that rhUTI treatment reduced the apoptosis of DCs induced by a severe ERS response in LPS-induced ALI mice. Further investigations into how rhUTI modulates ERS signaling pathways for adaptation and the identification of key checkpoints for immunotherapy should be pursued. Second, it may be more informative to investigate dendritic cells in the lung rather than in the spleen, even though the spleen is an accessible source of DCs. In future studies, we will explore the local nature of ALI by extracting dendritic cells from lung tissue in mice to make up for these limitations. Further analysis could provide more direct insights into local immune responses. Finally, there are many studies on UTI but few on rhUTI so far, and our future development direction may focus on various mechanisms of rhUTI in the treatment of sepsis and other diseases, such as rhUTI regulating the immune disorder of dendritic cells in acute lung injury.

Conclusion

In this study, the beneficial effects of rhUTI in ameliorating LPS-induced ALI were explored for the first time and elaborated at multiple levels. First, rhUTI significantly ameliorated lung injury, reduced the level of oxidative stress in the lung tissues, and greatly improved the survival rate of ALI mice. Second, rhUTI improved the immune function of splenic DCs by promoting their maturation of splenic DCs, mediating T-cell proliferation and polarization to Th1, reducing apoptosis of splenic DCs, and possibly providing protection to DCs by inhibiting ERS-mediated apoptosis-related mechanisms. Compared with UTI, rhUTI has a higher purity and lower risk of contamination. In summary, rhUTI is expected to be a more promising and clinically valuable drug for the treatment of ALI, and its potential molecular mechanisms and large-scale clinical trials have more room for exploration, which can provide new research directions and priorities for the treatment of ALI.

Abbreviations

ALI

Acute lung injury

BMSC

Bone marrow mesenchymal stem cell

CHOP

C/EBP homologous protein

DC

Dendritic cell

ERS

Endoplasmic reticulum stress

GRP78

Glucose-regulated protein 78

LPS

Lipopolysaccharide

MHC-II

Major histocompatibility complex class II

PERK

Pancreatic endoplasmic reticulum kinase

UTI

Urinary trypsin inhibitor

XBP-1

X-box binding protein 1

Author contributions

Q conceived and designed the experiments. Li, Wan and S performed experiments. Wang contributed to writing the manuscript. Liu critically revised the manuscript. All authors contributed to manuscript drafting or editing and approved the submitted draft.

Funding

The Science&Technology Development Fund of Tianjin Education Commission for Higher Education (2020KJ191). The Science&Technology Development Fund of Tianjin Education Commission for Higher Education (2020KJ192).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All experimental manipulations were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals with the approval of the Laboratory Animal Welfare Ethics Committee of Tianjin Medical University General Hospital. (Ethical approval number: IRB2021-DWFL-133).

Competing interests

The authors declare no competing interests.