HIP/PAP protects against bleomycin‐induced lung injury and inflammation and subsequent fibrosis in mice

Abstract Hepatocarcinoma‐intestine‐pancreas/pancreatitis‐associated protein (HIP/PAP), a C‐type lectin, exerts anti‐oxidative, anti‐inflammatory, bactericidal, anti‐apoptotic, and mitogenic functions in several cell types and tissues. In this study, we explored the role of HIP/PAP in pulmonary fibrosis (PF). Expression of HIP/PAP and its murine counterpart, Reg3B, was markedly increased in fibrotic human and mouse lung tissues. Adenovirus‐mediated HIP/PAP expression markedly alleviated bleomycin (BLM)‐induced lung injury, inflammation, and fibrosis in mice. Adenovirus‐mediated HIP/PAP expression alleviated oxidative injury and lessened the decrease in pulmonary superoxide dismutase (SOD) activity in BLM‐treated mice, increased pulmonary SOD expression in normal mice, and HIP/PAP upregulated SOD expression in cultured human alveolar epithelial cells (A549) and human lung fibroblasts (HLF‐1). Moreover, in vitro experiments showed that HIP/PAP suppressed the growth of HLF‐1 and ameliorated the H2O2‐induced apoptosis of human alveolar epithelial cells (A549 and HPAEpiC) and human pulmonary microvascular endothelial cells (HPMVEC). In HLF‐1, A549, HPAEpiC, and HPMVEC cells, HIP/PAP did not affect the basal levels, but alleviated the TGF‐β1‐induced down‐regulation of the epithelial/endothelial markers E‐cadherin and vE‐cadherin and the over‐expression of mesenchymal markers, such as α‐SMA and vimentin. In conclusion, HIP/PAP was found to serve as a potent protective factor in lung injury, inflammation, and fibrosis by attenuating oxidative injury, promoting the regeneration of alveolar epithelial cells, and antagonizing the pro‐fibrotic actions of the TGF‐β1/Smad signaling pathway.


| INTRODUC TI ON
Pulmonary fibrosis (PF) is a progressive pathological process characterized by epithelial damage, aberrant proliferation of mesenchymal cells, and the formation of fibrotic foci and is a common outcome of various pulmonary diseases. Numerous factors are involved in its development, including dust, smoking, drugs, infection, auto-immunity, and radiation. A population of patients with connective tissue diseases was characterized by PF. 1 Oxidative/antioxidative imbalance [2][3][4][5][6] and the excessive production of pro-inflammatory and pro-fibrotic cytokines 2,3,7-11 are critically involved in the pathogenesis of PF. Accumulation of myofibroblasts (MFBs) is well recognized as the pivotal event in the PF pathogenesis. 1 To date, few therapeutic measures have been developed to resolve established PF or hinder PF progression to respiratory failure. 12 Thus, efforts are required to uncover the mechanisms underlying PF and further explore novel therapeutic targets for this disease.
In this study, we first measured the expression of HIP/PAP and its mouse counterpart, Reg3B, in human and mouse fibrotic lung tissues. Then, we investigated the effects of recombinant adenovirus-mediated HIP/PAP expression on bleomycin (BLM)-induced lung injury, inflammation, and fibrosis in mice and explored the possible underlying mechanisms.

| Ethics
This study was conducted in accordance with the ethical guidelines of the Helsinki Declaration for experiments involving humans and was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China). All animal experiment protocols were approved by the Institutional Animal Ethics Committee of Xi'an Jiaotong University.

| Human specimens
Surgically resected, paraffin-embedded human fibrotic lung tissue specimens (10 cases) and pathologically normal para-tumor lung tissue specimens (10 cases) were obtained from the Department of Pathology, the First Affiliated Hospital of Xi'an Jiaotong University, with the approval of the Institutional Review Board. The clinical information of the human specimens is listed in Supplementary Table S1.

| Animals
Specific pathogen-free, 6-week-old male ICR mice, weighing 25-30 g were provided by the Experimental Animal Center, School of Medicine, Xi'an Jiaotong University. The mice were housed under pathogen-free conditions under a 12 hours light/dark cycle at constant temperature (22 ± 2°C) and humidity, with free access to water and standard laboratory chow. All mice were acclimatized to the abovementioned conditions for one week before initiating experiments. All efforts were undertaken to minimize the suffering of the mice.

| Verification of adenovirus transduction
Replication-incompetent adenoviruses carrying HIP/PAP (AdHIP/ PAP) and green fluorescence protein (AdGFP) were prepared as described previously 18 using the AdEasy adenovirus vector system (Stratagene, La Jolla, CA, USA). Plaque assays using HEK293 cells were performed to determine the titers of transduced adenoviruses, expressed as plaque-forming units per milliliter (pfu/mL).
To verify the transduction efficiency of repeated intraperitoneal (i.p.) adenovirus injection, twelve mice were equally assigned to the AdGFP and AdHIP/PAP groups. Mice of each group were given an i.p. injection of AdGFP (5 × 10 9 pfu) or AdHIP/PAP (5 × 10 9 pfu) on day 0. Two mice from each group were randomly euthanized on day 7 and day 14, respectively. The remaining mice were injected again with AdGFP (5 × 10 9 pfu) or AdHIP/PAP (5 × 10 9 pfu) on day 14 and were sacrificed on day 21. Another two mice were set aside as controls administered an i.p. injection of normal saline (NS) and were euthanized on day 0. The lungs of euthanized mice were harvested for further examination.
Genomic DNA (gDNA) was extracted from lung tissues using a DNeasy Kit (Qiagen, Valencia, CA, USA). One microgram (μg) of the extracted gDNA was used to determine the abundance of the viral vector CMV promoter in lung tissues by PCR amplification.

| Adenovirus-mediated HIP/PAP expression upon bleomycin (BLM)-induced lung injury and fibrosis
To observe the expression of Reg3B in mouse lung after BLM treatment, thirty mice were equally divided into normal saline (NS, n = 5) and bleomycin (BLM, n = 25) groups. At day 0, the mice were intratracheally (i.t.) instilled with BLM (50 μL, 2.4 mg/mL) or an equal volume of NS as described previously. 23 Five mice from the BLM group were euthanized on days 3, 7, 14, 21, and 28, while all the mice in the NS group were euthanized on day 3. Mouse lungs were collected for haemotoxylin and eosin (H&E) and picrosirius red staining, western blotting, and other experiments.
To investigate the effects of HIP/PAP on acute lung injury and fibrosis, eighty mice were equally assigned into four groups (n = 20): NS, NS + BLM, AdGFP + BLM, and AdHIP/PAP + BLM.
Mice in the two Ad groups were i.p. administered the corresponding recombinant adenovirus (AdGFP or AdHIP/PAP, 5 × 10 9 pfu in F I G U R E 1 HIP/PAP (Reg3B) expression is elevated in human and mouse fibrotic lung tissues. Immunohistochemistry showed that type II alveolar epithelial cells were positively stained with anti-HIP/PAP antibody in normal human lung tissues. HIP/PAP expression was drastically elevated in fibrotic human lung tissues, with the majority of the HIP/PAP-positive signals found in seemingly hyperplastic alveolar epithelial cells (A). Expression of Reg3B in mouse lung tissues was upregulated at both the mRNA (B) and protein (C) levels from day 3 post-BLM installation and increased steadily until day 28. Immunohistochemical staining showed that the expression patterns of Reg3B in normal and fibrotic mouse lung tissues are similar to those in the normal and fibrotic human lung tissues (D). Error bars indicate SD. Scale bars = 100 μm 0.25 mL) for the first time, while mice in the other two groups were dosed with an equal volume of NS. Two days later (Day 0), the mice were i.t. instilled with NS or BLM. Ten mice in each group were sacrificed on day 3. The remaining mice received the second i.p.
administration of adenoviruses or NS on day 12 (two weeks after the first adenovirus administration) and were sacrificed on day 28, when the lungs and serum were harvested for subsequent experiments. The mice were weighed during BLM modeling, and their lung coefficient was calculated (lung coefficient = lung wet weight/ body weight × 100).

| HIP/PAP on SOD expression in mouse lungs
To test the effect of adenovirus-mediated HIP/PAP expression on SOD expression in mouse lungs, fifteen mice were equally allocated into normal saline (NS), AdGFP, and AdHIP/PAP groups and treated as described above. Seven days later, all mice were euthanized and their lungs were collected. See Section 2.5 for a description of the measurement of SOD expression.

| Bronchoalveolar lavage (BAL)
BAL was carried out on day 3 following BLM administration.
Immediately after the mice were sacrificed, their lungs and trachea were extracted en bloc, and a 20G intravenous catheter was inserted into their trachea. One milliliter of PBS was instilled into the lungs and withdrawn three times via the catheter. More than 85% of the fluid was recovered as bronchoalveolar lavage fluid (BALF), which was then centrifuged at 1000 rpm for 10 minutes at 4°C. The supernatants were collected and stored at −80°C for protein quantification and ELISA. The precipitate was washed with red blood cell lysis buffer (eBioscience, San Diego, CA, USA) and resuspended in 500 µL of PBS for counting total white blood cells.

| Measurement of hydroxyproline content
Pulmonary hydroxyproline content was determined using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's protocols and expressed as milligrams per gram tissue (mg/gram).

| Enzyme-linked immunosorbent assay (ELISA)
BALF HMGB1, TNF-α, IL-1β, and IL-6 levels were determined by ELISA using commercially available kits (HMGB1 ELISA kit,  at 37°C and 5% CO 2 . Three biological replicates and three technical replicates were set for each experiment.

| Cell proliferation assay
Cells were inoculated into 96-well plates at 500 cells per well and allowed to adhere for 24 hours. Then, the complete medium was replaced with DMEM or F-12K containing 2% FBS and various concentrations of rHIP/PAP (final concentrations of 0, 125, and 250 ng/mL), and cells were cultured for 72 hours. Cell viability was assessed using the cell counting kit-8 (CCK-8, Dojindo, Kyushu, Japan) assay at 24, 48, and 72 hours, in accordance with the manufacturer's instructions.

F I G U R E 2
AdHIP/PAP alleviates BLM-induced lung injury and inflammation in mice. Mice in the AdHIP/PAP + BLM group lost less body weight than those in the other two BLM groups. * P < 0.05 vs the NS + BLM or AdGFP + BLM groups and P < 0.01, vs the NS group (A). Similarly, AdHIP/PAP significantly alleviated the increase in the mouse lung coefficient on both day 3 and day 28 after BLM installation (B). Adenovirus-mediated HIP/PAP expression obviously mitigated lung injury and inflammation, as indicated by H&E staining of mouse lungs (C), BALF protein concentration determination (D), and cell counting (E) on day 3 after BLM instillation consistently showed that the anti-inflammatory action of HIP/PAP is substantiated by the decreased expression of CD45 in mouse lung, as indicated by qRT-PCR and immunohistochemistry results (F, G). Error bars indicate SD. Scale bars = 100 μm

| SOD expression
A549 and HLF-1 cells were seeded into 6-well plates at a density of 5 × 10 4 cells per well and allowed to adhere for 24 hours; then, the medium was replaced with serum-free medium supplemented with rHIP/PAP (125 ng/mL). Seventy-two hours later, the cells were harvested, lysed with normal saline for assessing SOD activity or with TRIzol for assessing RNA extraction for qRT-PCR. The culture medium was also used for the determination of SOD activity.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cultured cells and mouse lung tissues using the TRIzol reagent (Thermo, Life Technologies,

| Western blotting
Total protein was extracted from lung tissues or collected cells, frac-

| Histological staining, immunohistochemistry, and immunofluorescence
Formalin-fixed, paraffin-embedded mouse lung tissues were subjected to H&E and picro-sirius red staining to assess pulmonary architectural alterations and collagen deposition. Lung fibrosis was graded in a blinded fashion using previously established criteria. 24 Immunohistochemistry was carried out using the streptavidin/

| Statistical analysis
Statistical analyses were performed using GraphPad Prism software version 6 (GraphPad Software Inc, La Jolla, CA, USA). The data are presented as mean ± standard deviation (SD). Differences among groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test. P < 0.05 was considered statistically significant.

| HIP/PAP (Reg3B) expression was elevated in human and mouse fibrotic lung tissues
Immunohistochemical staining showed that in normal human lung tissues, only a few type II alveolar epithelial cells were positively stained with the anti-HIP/PAP antibody. Contrarily, in fibrotic human lung tissues, a marked increase in HIP/PAP-positive cell numbers, the majority of which were seemingly hyperplastic alveolar epithelial cells, led to a significant increase in the average IOD compared with that in normal tissues ( Figure 1A). In BLM-treated mice, qRT-PCR and western blotting consistently demonstrated that the lung expression of Reg3B was markedly elevated starting from day 7 after BLM treatment and increased thereafter ( Figure 1B,C). The increased Reg3B expression in fibrotic mouse lung tissue was also confirmed by immunohistochemistry, which showed that type II alveolar epithelial cells were weakly or moderately immunostained with the anti-HIP/PAP antibody in normal mouse lung tissues, while numerous hyperplastic epithelial cells were strongly immunostained with the anti-HIP/PAP antibody in fibrotic mouse lung tissues ( Figure 1D). In addition, we tested the effect of TGF-β1, the pivotal  Figure S1).

| Intraperitoneal (i.p.) administration of adenovirus efficiently transduced mouse lung tissue
To verify the transduction of recombinant adenoviruses in mouse lungs, the CMV-IE sequence was detected using PCR. The CMV-IE sequence was found to be amplified in the lung tissues of AdGFP and AdHIP/PAP mice ( Figure S2A). Moreover, western blotting showed that expression levels of HIP/PAP and GFP following the second injection of the recombinant adenoviruses were comparable to those observed following the first injection ( Figure S2B). These results clearly indicated that realizing prolonged ectopic expression by repeated i.p. injection of recombinant adenoviruses was feasible. In addition, histological examination, BALF protein concentration determination and cell counting, and lung tissue MPO activity measurement did not reveal any obvious lung injury after either the first or the second administration of the recombinant adenoviruses (data not shown).

| HIP/PAP protected mice from BLM-induced lung injury and inflammation
The body weights of mice continuously decreased and their lung coefficient was markedly increased after BLM treatment; AdHIP/ PAP significantly attenuated these changes (Figure 2A,B  AdHIP/PAP markedly alleviated these changes in the mouse lung ( Figure 2C). Accordingly, BLM-induced increases in BALF protein content ( Figure 2D) and total cell number ( Figure 2E) were significantly attenuated by AdHIP/PAP.
While tissue MPO activity, an indicator of oxidative injury as well as neutrophil infiltration, was elevated by BLM treatment, this increase was also ameliorated by HIP/PAP (Table 1) (Table 2).
Lieu et al proposed that HIP/PAP might exhibit superoxide dismutase-like and glutathione reductase-like activities. 17 In our study, however, HIP/PAP did not exhibit any in vitro SOD-like activity in a nitro-blue tetrazolium (NBT) assay (data not shown). We found that the total SOD activity in mouse lung tissue was markedly decreased by BLM treatment and that this decrease was significantly alleviated by AdHIP/PAP administration ( Figure 3A). In addition, both SOD

| HIP/PAP attenuated BLM-induced lung fibrosis in mice
Pulmonary fibrogenesis started as early as three days after BLM instillation, as indicated by the markedly increased pulmonary expression of Col1A2 and Col3A1 ( Figure 4A,B). Twenty-eight days after BLM treatment, the pulmonary hydroxyproline content was markedly increased ( Figure 4C). H&E and picro-sirius red staining followed by histological examination showed that normal lung architecture disappeared, lots of spindle-shaped fibrotic cells clumped together, and bulky collagen fibers accumulated ( Figure 4D), leading to a significantly increased fibrosis score ( Figure 4E). Similar to the expression of Col1A2 and Col3A1, pulmonary TGF-β1, plateletderive growth factor (PDGF)-A, -B, -C, connective growth factor (CTGF) and plasminogen activator inhibitor (PAI)-1 levels were markedly increased starting from day 3 ( Figure 4F) following BLM treatment and were sustained until day 28 ( Figure 4G). All the above fibrotic changes were significantly alleviated by AdHIP/PAP, while AdGFP showed no significant effect ( Figure 4A-G).
The expression level of α-SMA, a marker for MFBs, in the AdHIP/ PAP + BLM group was significantly lower than that in the other two BLM groups on both day 3 and day 28 ( Figure 5A). The expression profile of α-SMA on day 28 was also verified by western blotting ( Figure 5B) and immunohistochemistry ( Figure 5C).

| HIP/PAP suppressed the proliferation and attenuated the TGF-β1-induced activation of HLF-1 cells
The CCK-8 assay showed that rHIP/PAP significantly inhibited the growth of HLF-1 cells ( Figure 6A). HLF-1 cells presented distinct resistance to H 2 O 2 injury. The viability of HLF-1 cells was not significantly decreased until the H 2 O 2 concentration was raised to 800 μmol/L ( Figure 6B). qRT-PCR revealed that rHIP/PAP did not significantly affect basal expression of α-SMA, vimentin, and E-cadherin, but markedly attenuated the rhTGF-β1-induced elevation of α-SMA and vimentin and decreased expression of E-cadherin in HLF-1 cells ( Figure 6C). The regulatory effect of rHIP/PAP on α-SMA expression in HLF-1 cells was further confirmed by immunofluorescence ( Figure 6D). Similar results were also observed for Col1A2, Col3A1, CTGF, PDGF-B, and TGF-β1 expression in HLF-1 cells ( Figure 6C, Table 3). These results indicated that HIP/PAP exerted its functions, at least partly, by antagonizing TGF-β1. Western blotting showed that rHIP/PAP remarkably mitigated rhTGF-β1-induced phosphorylation of Smad 2/3 but did not affect the basal levels of pSmad2/3 in HLF-1 cells, confirming the antagonizing effect of HIP/PAP on TGF-β1 ( Figure 6E).

| HIP/PAP promoted the proliferation of alveolar epithelial cells, protected them from H 2 O 2induced apoptosis, and suppressed their TGF-β1induced epithelial-mesenchymal transition (EMT)
The effects of HIP/PAP on proliferation, H 2 O 2 -induced apoptosis, and TGF-β1-induced EMT were tested in A549 and HPAEpiC. The Col1A2, and Col3A1, as well as the rhTGF-β1-induced downregulation of E-cadherin expression in A549 cells ( Figure 7D). The opposing effect of rHIP/PAP on rhTGF-β1-induced EMT was further verified by immunofluorescence ( Figure 7E). In addition, rHIP/PAP mitigated the rhTGF-β1-induced, but not the basal, expression of TGF-β1, CTGF, PDGF-B and -C, and PAI-1 at the mRNA level (Table 3).  (Table 3). These results clearly indicated that HIP/PAP confers resistance to oxidative injury and TGF-β1induced EndoMT in HPMVEC.

| D ISCUSS I ON
The present study demonstrated that HIP/PAP alleviated BLM- or BLM-induced PF in mice. 32 An interesting finding in our present study was that lung fibroblasts exhibited much stronger tolerance F I G U R E 8 HIP/PAP accelerates proliferation, alleviates H 2 O 2 -induced apoptosis, and inhibits TGF-β1-induced EndoMT in HPMVEC. rHIP/PAP at 125 and 250 ng/mL promoted HPMVEC growth to similar extents. * P < 0.05 vs the blank control (A  40 and HMGB1, 33,41 which also exert fibrogenic effects. Similarly, our present study demonstrated that HIP/PAP alleviated BLM-induced lung inflammation as well as the production of pro-fibrotic cytokines, suggesting that its anti-inflammatory potency contributed to its anti-fibrotic effect.
Besides protecting lungs from oxidative injury, our study demonstrated that HIP/PAP promotes the proliferation of alveolar epithelial cells and pulmonary microvascular endothelial cells. The mitogenic effect of HIP/PAP has been well identified in several tissues and cells. 17,18 The proliferation-promoting effect of HIP/ PAP might facilitate the repair of damaged alveoles and the pul- In summary, we found here, that the increased pulmonary production of HIP/PAP presents an adaptive/compensatory reaction for lung fibrosis. Pulmonary overexpression of HIP/PAP mediated by recombinant adenovirus efficiently ameliorated BLM-induced murine lung injury and fibrosis via multiple activities, suggesting a protective role of HIP/PAP in PF.

ACK N OWLED G EM ENT
We thank Honghong Li, Department of Pathology, the First Affiliated Hospital of Xi'an Jiaotong University, for his kind assistance in the preparation of tissue sections.

CO N FLI C T O F I NTE R E S T
The authors confirm that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.