High-mobility group box1 peptide ameliorates bronchopulmonary dysplasia via suppressing inflammation and fibrosis in a mouse model

doi:10.21203/rs.3.rs-892162/v1

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High-mobility group box1 peptide ameliorates bronchopulmonary dysplasia via suppressing inflammation and fibrosis in a mouse model

https://doi.org/10.21203/rs.3.rs-892162/v1

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Bronchopulmonary dysplasia (BPD) is a chronic lung disorder that affect approximately 40% of preterm infants, with no established curative therapy. The administration of mesenchymal stem cells (MSCs) to BPD patients has shown promising outcomes. Previously, we demonstrated that a synthesized peptide originating from high mobility group box-1 protein (HMGB1) induces a regenerative cascade through activating endogenous MSCs. Here, we tested whether the HMGB1 peptide can ameliorate BPD-related lung injury. In a mouse BPD model established via hyperoxia exposure, three shots of HMGB1 peptide significantly improved survival and suppressed inflammation and fibrosis in the lung. Single-cell RNA-sequencing of the lung further showed that the peptide significantly suppressed a hyperoxia-induced inflammatory signature in macrophages and fibrotic signature in fibroblasts. These changes in the transcriptome were also confirmed at the protein level. Taken together, our data show that treatment with the HMGB1 peptide suppressed inflammation and fibrosis, thus preventing BPD progression. This study serves as a foundation for the development of new effective therapies for BPD.

Molecular Biology

Biological Chemistry

Stem Cell & Developmental Cell Biology

peptide

lung

hmgb1

suppressed

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that typically affects infants delivered at a gestational age of < 30 weeks with a birth weight < 1500 g ^[1], a group that covers approximately 40% of preterm infants ^[2]. The disease is associated with significant mortality in the neonatal intensive care unit ^{[3, 4]}. Additionally, survivors continue to show decreased lung capacity and are at increased risk of respiratory illnesses (e.g., asthma) and neurodevelopmental disorders ^[5–7]. The etiology of BPD is multifactorial, but a major factor is supplemental oxygen and invasive ventilation, common treatments for preterm infants; these treatments tend to induce inflammation and trauma that eventually cause interstitial fibrosis in the lung ^[8]. In addition to fibrosis, impaired alveolarization is another important histopathological characteristic of BPD ^[9].

Although surfactant replacement therapies and ventilation are common strategies for managing BPD-related lung injury, they have limited efficacy ^[10]. Relatively new approaches using stem cells have shown promise as potential curative therapies ^[11], particularly, treatments using mesenchymal stem cells (MSCs) ^[12–15]. In hyperoxygen-exposed rodent models of BPD, MSC administration improved alveolarization and ameliorated pulmonary hypertension, lung inflammation, fibrosis, angiogenesis and apoptosis ^[12]. However, the clinical application of MSC therapy is hampered by difficulties in preparing a large number of MSCs with consistent quality ^[16].

Previously, we showed that high mobility group box 1 (HMGB1), released from injured tissue, induced regeneration through activating platelet-derived growth factor receptor alpha-positive bone-marrow MSCs (PDGFRα + BM-MSCs) ^{[17, 18]}. HMGB1-induced circulating PDGFRα + BM-MSCs exhibited tissue-regenerative activity for mesenchymal and epithelial tissues in the mouse skin graft model of recessive dystrophic epidermolysis bullosa (RDEB), a genetic blistering skin disease with devastating epithelial injury ^[17]. Our previous work also identified and synthesized a critical domain in HMGB1 that induces regeneration. Through increasing PDGFRα + BM-MSCs accumulation in cardiac tissue, the synthesized peptide has therapeutic potential in dilated cardiomyopathy and myocardial infarction ^{[19, 20]}. Currently, we are evaluating the efficacy of the HMGB1 peptide in a phase II clinical trial for RDEB patients (UMIN ID: UMIN000026645). Our work suggests that the HMGB1 peptide activates endogenous MSCs to induce tissue regeneration without requiring large-scale MSC production and transplantation. For clinical use, the cost- and time-saving benefits are considerable.

In this study, we examined whether systemic administration of HMGB1 peptide improves alveolarization and ameliorates lung fibrosis in a mouse model of BPD. We also performed single-cell RNA sequencing (scRNA-seq) to investigate molecular-level changes in response to the treatment.

HMGB1 peptide treatment ameliorated BPD

We used a well-characterized mouse BPD model created with a hyperoxic chamber ^[¹²^,^21,22^] (Figure 1a). Treatment of hyperoxic mice with HMGB1 peptide (O₂+ HMGB1 group) improved survival rates to 89.3%, whereas treatment with normal saline (O₂+ NS group) resulted in a reduced survival rate of 75.0% (Figure 1b). However, these survival rates did not differ significantly between the O₂+ NS and O₂+ HMGB1 groups.

On postnatal day 14, O₂+ HMGB1 group had a significantly higher mean body weight than the O₂+NS group (P < 0.05), indicating that HMGB1 peptide treatment ameliorated BPD-induced weight loss (Figure 1c).

Alveolar structure is impaired in BPD, with hyperoxia hampering proper oxygen exchange ^[^21,22^]. Hematoxylin and eosin staining revealed irregular alveolar structures in the hyperoxia group (Figure 1d). We also found that HMGB1 peptide treatment significantly shortened MLI compared with control treatment, suggesting that HMGB1 peptide treatment improved alveolar structure (Figure 1e).

Single cell RNA-seq on lungs treated with HMGB1 peptide

We used scRNA-seq to characterize how HMGB1 peptide affected lung integrity. The UMAP plot represents transcriptome patterns of all detected cells (Figure 2a). After conventional quality control and filtering, we recovered 6104, 4314, and 7584 cells from the RA + NS, O₂ + NS, and O₂ + HMGB1 groups, respectively. We identified 15 cell clusters with marker gene analysis and cell type information from previous publications (Figure 2b) ^[²³^]. The detected cell types were in agreement with existing lung studies. Cell proportion analysis indicated an increase in neutrophils and macrophages among hyperoxia groups, suggesting that scRNA-seq captured BPD-related inflammatory events (Figure 2c). However, cell proportion analyses did not provide mechanistic insight into how the HMGB1 peptide ameliorated BPD symptoms.

HMGB1 peptide caused differential gene expression and suppressed BPD-induced inflammation in macrophage fraction

Our differential expression analysis revealed that HMGB1 peptide significantly altered gene expression, compared with control treatment (Figure 3a). Gene Ontology analysis indicated that these differentially expressed genes enriched the IL-1 signaling pathway, suggesting that HMGB1 peptide modified the inflammatory status of macrophages (Figure 3b).

We then re-clustered macrophages to further visualize how their inflammatory status changed (Figure 3c), specifically examining IL-1β and IL-6, with well-characterized involvement in lung inflammation (Figure 3d,e) ^[^24,25^]. IL-1β was most prominently expressed in sub-cluster 1 of the macrophage fraction, and the proportion of this sub-cluster decreased upon HMGB1 peptide treatment. We observed the same suppression pattern in IL-6.

Protein quantification revealed that hyperoxia elevated both IL-1β and IL6, but HMGB1 peptide treatment significantly decreased both inflammatory cytokines, supporting our RNA data. These findings indicate that HMGB1 peptide reduced inflammatory signatures in the macrophage fraction at both the RNA and protein levels.

HMGB1 peptide treatment inhibited lung fibrosis

To investigate how HMGB1 peptide affected fibrosis, we isolated and re-clustered lung fibroblasts (Figure 4a). The cell proportion analysis did not indicate any significant changes upon treatment (Figure 4b). We then focused on sub-clusters 1 and 3, representing the myofibroblast fraction expressing Acta2 (Figure 4c). Analyses of differentially expressed genes and Gene Ontology terms clarified that HMGB1 peptide altered the expression pattern of fibrosis-related genes (Figure 4d).

We then specifically assessed Ccn2, a gene known to promote lung fibrosis (Figure 4e). As expected, Ccn2 was upregulated following hyperoxia exposure. Interestingly, HMGB1 peptide suppressed Ccn2 expression, implying that the treatment can also inhibit fibrosis. To validate these RNA-seq results, we measured the amount of soluble collagen to evaluate fibrosis degree. Interestingly, HMGB1 peptide treatment significantly suppressed lung soluble collagen, which had increased under hyperoxia. These results clearly indicate that HMGB1 peptide ameliorated BPD-related fibrosis in the lungs.

In this study, we showed that systemic administration of HMGB1 ameliorated BPD-related lung injury in a mouse model. HMGB1 peptide rescued BPD-related decreases in body weight and survival. The treatment also restored alveolar structure. The likely mechanisms were significant inhibition of inflammation in the macrophage fraction and of fibrosis in the myofibroblast fraction.

Mesenchymal stem cells secrete IL-1 receptor antagonist (IL-1Ra), an IL-1 family glycoprotein that binds to and inhibits both IL-1β and IL-1R, leading to anti-inflammatory and antifibrotic effects during lung injury ^{[26, 27]}. Several studies have demonstrated that IL-1 is strongly associated with BPD pathophysiology. For instance, IL-1β expression is elevated in hyperoxia-induced lung injury ^[28]. Furthermore, perinatal pulmonary expression of this cytokine disrupts alveolar septation, causing abnormalities in alpha-smooth muscle actin, as well as elastin deposition in the septa of distal airspaces ^[29]. Experiments with a rodent model of ventilator-induced lung injury demonstrated that IL-1Ra treatment reduces protein permeability during pulmonary edema and neutrophils in bronchoalveolar lavage fluid ^[30]. In some cases, however, cell therapy with MSCs is more effective in reducing inflammation. Although they do not generate IL-1Ra, MSCs produce multiple anti-inflammatory proteins, including tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) and prostaglandin E2 (PGE2). TSG-6 inhibits the transendothelial migration of neutrophils to exert a protective effect ^[31], whereas PGE2 inhibits the transition of fibroblasts to myofibroblasts and upregulates anti-inflammatory cytokine IL-10 ^[32].

A member of the CCN family, Ccn2 is a downstream mediator of TGF-β and regulates multiple fibroblast behaviors that are responsible for fibrosis development, including fibroblast adhesion, migration, proliferation, differentiation, and matrix production ^[33].

Ccn2-/- mice have hypoplastic lungs with reduced cell proliferation and increased apoptosis, indicating that Ccn2 is required for normal lung development ^[34]. However, patients with BPD exhibited dramatically increased Ccn2 expression in the thickened alveolar septa of their lungs ^[35]. Furthermore, results from a double-transgenic mouse model with doxycycline-inducible Ccn2 overexpression demonstrated that excess Ccn2 disrupts alveologenesis and capillary formation while inducing fibrosis ^[36]. Therefore, this protein is heavily involved in BPD-induced lung fibrosis, suggesting that the condition could be prevented through inhibiting Ccn2 expression with systemic administration of HMGB1 peptide.

Our study has several limitations. First, we were unable to identify the exact mechanism of HMGB1’s protection against lung damage. Further, although we confirmed anti-inflammatory and anti-fibrotic effects in the lung, we could not demonstrate the accumulation of MSCs in the injured lung. Second, we did not evaluate lung function, even though HMGB1 peptide treatment is expected have a positive effect, given the results of previous cell therapies with MSCs ^{[37, 38]}.

In conclusion, systemic administration of HMGB1 peptide exerted anti-inflammatory effects on macrophages and anti-fibrotic effects on fibroblasts in a mouse model of BPD. Because there is currently no cure for BPD, our results are extremely important for developing novel therapies. We expect that the HMGB1 peptide will be applied clinically in the future.

Animal model.

All procedure involving animals followed guidelines of and were approved by the Animal Committee of the Osaka University Graduate School of Medicine. All authors complied with the ARRIVE guidelines.

Time-dated pregnant C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan) at E14–E15 days of gestation. Animals were housed in individual cages with 12 h light-dark cycles. A standard rodent diet and water were provided ad libitum. Within 12 h of birth, newborn pups were pooled and randomly allocated to three experimental groups: room air treated with normal saline as vehicle (normoxic condition, RA + NS group) (n = 14), hyperoxia exposure treated with normal saline as vehicle (O₂+ NS group) (n = 14), and hyperoxia exposure treated with HMGB1 peptide (O₂+ HMGB1 group) (n = 14). Pups in the two hyperoxic conditions were housed in chambers of 90% oxygen from birth to postnatal day 14 with a brief interruption for animal care (< 10 min/day). Oxygen levels were monitored using a ProOx P110 monitor (Bio-Spherix, Redfield, NY, USA). Each nursing dam was assigned seven pups to standardize nutritional status. Dams were rotated between hyperoxia and room-air litters every 24 h to avoid oxygen toxicity. Pups received treatment with HMGB1 peptide or normal saline (control) respectively on postnatal days 4, 8, and 12. Body weight was measured on postnatal days 4, 8, 12 and 14. Mouse pups were sacrificed on postnatal day 14 for detailed evaluation.

HMGB1 peptide.

As previously reported ^[^19,20,39^], StemRIM (Osaka, Japan) employed solid-state synthesis to produce the HMGB1 peptide from the MSC mobilization domain of human HMGB1. The synthetic HMGB1 peptide was dissolved in distilled water to a concentration of 1 mg/mL prior to systemic administration.

Systemic administration of HMGB1 peptide or normal saline

Glass needles (diameter: 130 µm) for systemic administration were created using a puller and grinder. HMGB1 peptide or normal saline (each 5 mL/kg/day) was administered on postnatal days 4, 8, and 12 under isoflurane anesthesia, following published procedures ^[^40,41^]. For postnatal day 4, injections occurred via the temporal vein. On postnatal days 8 and 12, a skin incision was made from the anterior to the external auditory meatus and the posterior to the lateral canthus, exposing the facial vein for injection. Neonatal injections were performed under a microscope to visualize vessels. After injection, pups were warmed and returned to their cages.

Tissue preparation

Pups were euthanized on postnatal day 14 using isoflurane. The chest and abdominal were then opened to cut the abdominal aorta and vena cava. Lung tissue was harvested for morphometric and biochemical analyses. For morphometric analysis, the left lung was fixed using intratracheal instillation of 10% buffered formalin and kept in the solution for 24 h at room temperature before being embedded in paraffin. The right lung was weighed and stored in a deep freezer for biochemical analysis.

Lung morphometric analysis

Lung morphometric analysis was performed as described previously ^[⁴²^,⁴³^]. Briefly, sections (5 μm thick) sliced from paraffin blocks were stained with hematoxylin and eosin. Degree of alveolarization was estimated using the mean linear intercept (MLI), an indicator of mean alveolar diameter. The value is calculated as total length of lines drawn across the lung section divided by number of intercepts encountered. A minimum of two sections per sample and six non-overlapping microscopic fields (20× magnification) per section were evaluated.

ELISA

Frozen right lung samples were homogenized in lysis buffer with protease inhibitor. Cellular debris were removed from lysates with centrifugation at 10,000 × g and 4°C for 10 min. Supernatant protein content was quantitated using the bicinchoninic acid (BCA) method and bovine serum albumin as the standard. Inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels were measured in the lung using the R&D Mouse Quantikine ELISA kit following manufacturer protocol (R&D Systems, Minneapolis, MN, USA).

Collagen measurements　

Collagen measurements were taken from frozen right lung samples using a soluble collagen assay kit (QuickZyme Bioscience, Leiden, NLD), following manufacturer protocol. Lungs were homogenized in 1 mL of a 0.5 M acetic acid solution containing 1 mg pepsin (Nacalai Tesque, Kyoto, Japan) per 10 mg tissue. Each sample was incubated with constant shaking for 24 h at 4°C. Lysates were purified using centrifugation at 3,000 × g and 4°C for 10 min. The BCA method was again used to quantify supernatant protein content, with bovine serum albumin as the standard.

Lung isolation and tissue dissociation for scRNA-seq

Six lungs (n = 2/group) were harvested on postnatal day 14 and rinsed with Dulbecco’s PBS (Nacalai, kyoto, Japan). Lungs were rapidly dissected in RPMI-1640 medium (Nacalai, Kyoto, Japan) with 10% fetal bovine serum and finely minced in a Petri dish on ice. Tissue pieces were placed in 5 mL of enzyme mixture and digested with a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). The enzyme mixture contained the following: 50 µL of dispase (5000 U/mL) (Corning, NY), 100 uL of collagenase (100 mg/mL) (Wako, Osaka, Japan), 192 µL of elastase (26 mg/mL) (Worthington Biochemical Corporation, NJ), 10 µL of DNase I (5 U/uL) (Takara, Shiga, Japan), and 4648 µL of RPMI-1640 medium with 2% fetal bovine serum. Dissociated cells were passed through a 70 μm cell strainer and centrifuged at 300 ×g for 5 min. The supernatant was discarded and the pellet suspended in red blood cell lysis buffer (BioLegend, San Diego, CA). Cells were counted from the suspension.

Library preparation and sequencing of scRNA

Live cells were sorted and collected using a BD FACSAria III instrument (Becton Dickinson, NJ, USA). Cells were washed twice with PBS containing 0.1% bovine serum albumin and suspended at 1 × 10⁶cells/mL. Single cell suspensions were prepared using Chromium Next GEM Single Cell 3 GEM, Library & Gel Bead Kit v3.1 (10× Genomics, California, USA). Gene expression libraries were prepared following manufacturer protocol. A target of 6000 cells was used to generate the libraries. Libraries were sequenced on the NextSeq 2000 platform using NextSeq 2000 P3 Reagents (100 cycles) (Illumina, CA). The read length was set to 28 (read 1) + 8 (i7) + 0 (i5) + 91 (read 2) bases.

Bioinformatics

Processing of raw sequencing reads

Raw sequencing reads from the libraries were processed using 10× Genomics Cell Ranger v5.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) aligning reads to the mm10 build of the mouse genome. Default parameters were used for all samples. Raw sequencing reads were demultiplexed and mapped using Cell Ranger. Doublets were detected and removed using the Python package scrublet ^[⁴⁴^].

Data quality control, integration, and clustering

All processing steps were performed in Monocle3 v0.2.3.0 (R package) (https://cole-trapnell-lab.github.io/monocle3/docs/starting/) ^[⁴⁵^]for every sample. The threshold for minimum read count per cell was 500 reads. Thresholds for minimum gene number per cell were 39 (RA group) and 56 (O₂+ NS group and O₂+ HMGB1 group). All mitochondrial and sex chromosomal genes were removed from the analysis. Samples were assembled into a single monocle-cds object. Principal component analysis (PCA) and UMAP were used to display genetic distance and relatedness between cell populations. The preprocess_cds() function was used to normalize gene expression and calculate principal components (PC). The top 15 PCs were used for UMAP embedding. Cell types were annotated with R package SingleR v1.4.0. Default settings were used for parameters except for number of dimensions = 15 in preprocess_cds() and resolution = 10^-5 in cluster_cells().

Differential expression analysis and functional analysis

The R package edgeR v3.34.0 with a standard workflow was used to identify differentially expressed genes under hyperoxia or BPD treatment ^[⁴⁶^]. The inclusion criterion was an expression rate > 10% of cells in the cluster. A false discovery rate (FDR) < 0.05 was considered significant.

Gene set enrichment analysis was performed in enrichR v2.1 (R package)^[⁴⁷^]. (https://maayanlab.cloud/Enrichr/). Differentially expressed genes (FDR < 0.05 and |log₁₀FC| > 1.5) were assessed for their enrichment of pathways in the WikiPathway_2019_Mouse reference.

Statistical analysis

Data are expressed as means ± SEM. Survival curves were compared using Kaplan-Meier analysis followed by a log-rank test. Between-group differences were compared using the Steel-Dwass test. All data were analyzed in JMP Pro 15 (SAS Institute, Cary, NC, USA). Significance was set at P < 0.05.

Data Availability

All sequencing data used in this study were deposited in GEO and are available under the accession number GSE181497.

Acknowledgements

We thank E.T. and Li. YT. for their technical assistance.

Author contributions

T.H. performed experiments; collected, analyzed, and interpreted data; and wrote the manuscript. T.S. designed the study; analyzed and interpreted data; and wrote the manuscript. T.M. and M.F. analyzed and interpreted data. T.K., M.N. M.H., and K.Y. performed experiments and collected data. M.E. and K.T. conceived and designed the study; interpreted data; and helped write the manuscript. T.T. and T.K. interpreted data and helped write the manuscript. T.K. and K.T. approved the final draft. All authors reviewed the manuscript.

Additional information

Competing interests

K.T. is a scientific founder and stockholder of StemRIM and received research funding from StemRIM. T.S. is a stockholder of StemRIM. T.K., M.N. M.H. and K.Y. are employees of StemRIM. This study was funded by StemRIM and supported by Japan Agency for Medical Research and Development under Grant Number JP19lm0203018, and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan under Grant Number T18K092280, T21K077960, and JP19H03682. The funding agencies had no role in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.

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Competing interest reported. K.T. is a scientific founder and stockholder of StemRIM and received research funding from StemRIM. T.S. is a stockholder of StemRIM. T.K., M.N. M.H. and K.Y. are employees of StemRIM. This study was funded by StemRIM and supported by AMED under Grant Number JP19lm0203018, and JSPS KAKENHI Grant Number T18K092280, T21K077960, and JP19H03682. The funding agencies had no role in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.

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High-mobility group box1 peptide ameliorates bronchopulmonary dysplasia via suppressing inflammation and fibrosis in a mouse model

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