
==== Front
Stem Cell Res Ther
Stem Cell Res Ther
Stem Cell Research & Therapy
1757-6512
BioMed Central London

39278922
3933
10.1186/s13287-024-03933-8
Research
Sex-related differences in efficacy of bone marrow-derived high aldehyde dehydrogenase activity cells against pulmonary fibrosis
Inada Shugo 1
http://orcid.org/0000-0002-0035-674X
Nakashima Taku tnaka@hiroshima-u.ac.jp

1
Masuda Takeshi 1
Shimoji Kiyofumi 1
Sakamoto Shinjiro 1
Yamaguchi Kakuhiro 1
Horimasu Yasushi 1
Iwamoto Hiroshi 1
Fujitaka Kazunori 1
Hamada Hironobu 2
Hattori Noboru 1
1 https://ror.org/03t78wx29 grid.257022.0 0000 0000 8711 3200 Department of Molecular and Internal Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, 734-8551 Japan
2 https://ror.org/03t78wx29 grid.257022.0 0000 0000 8711 3200 Department of Physical Analysis and Therapeutic Sciences, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551 Japan
15 9 2024
15 9 2024
2024
15 3045 3 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Although bone marrow-derived cells with high aldehyde dehydrogenase activity (ALDHbr) have shown therapeutic potential against various diseases in animal studies, clinical trials have failed to show concurrent findings. We aimed to clarify the optimal conditions for the efficacy of ALDHbr cells by using a murine bleomycin-induced pulmonary fibrosis model.

Methods

We intravenously transferred male or female donor C57BL/6 mice-derived ALDHbr cells into recipient C57BL/6 mice under various conditions, and used mCherry-expressing mice as a donor to trace the transferred ALDHbr cells.

Results

Pulmonary fibrosis improved significantly when (1) female-derived, not male-derived, and (2) lineage (Lin)-negative, not lineage-positive, ALDHbr cells were transferred during the (3) fibrotic, not inflammatory, phase. Consistent with the RNA-sequencing results, female-derived Lin−/ALDHbr cells were more resistant to oxidative stress than male-derived cells in vitro, and transferred female-derived Lin−/ALDHbr cells were more viable than male-derived cells in the fibrotic lung. The mechanism underlying the antifibrotic effects of Lin−/ALDHbr cells was strongly associated with reduction of oxidative stress.

Conclusions

Our results indicated that Lin−/ALDHbr cell therapy could ameliorate pulmonary fibrosis by reducing oxidative stress and suggested that their efficacy was mediated by sex-related differences. Thus, sex-awareness strategies may be important for clinical application of bone marrow ALDHbr cells as a therapeutic tool.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-024-03933-8.

http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science 20K08519 Nakashima Taku Ryokufukaiissue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

Idiopathic pulmonary fibrosis is an interstitial lung disease with a poor prognosis despite the use of therapeutic agents [1]. Men show higher mortality rates for pulmonary fibrosis than women [2]; however the reasons underlying these sex-related differences remain unknown [3]. Although antifibrotic agents are available to treat idiopathic pulmonary fibrosis, these agents cannot completely stop disease progression [4, 5]. Therefore, new therapeutic approaches are being explored.

Reactive oxygen species (ROS) are highly reactive oxygen metabolites such as hydrogen peroxide (H2O2). Exogenous oxidizing agents such as cigarette smoke, toxins, hyperoxia, asbestos fibres, drugs and radiation also induce the production of ROS [6]. Oxidative stress induces lung fibrosis through deposition of collagen, upregulation of profibrotic mediators such as transforming growth factor (TGF)-β1, and differentiation of fibroblasts into myofibroblasts [6]. Therefore, antioxidants such as N-acetyl-l-cysteine (NAC) may serve as therapeutic agents for pulmonary fibrosis and are being investigated in various clinical trials [7]. Cell therapy for pulmonary fibrosis represents another line of investigation [8]. In particular, bone marrow (BM)-derived mesenchymal stromal cells have been reported to be effective for pulmonary fibrosis in pilot studies [9, 10].

Aldehyde dehydrogenase (ALDH) is an enzyme that protects the body from oxidative stress by oxidizing aldehyde to carboxylic acids [11, 12]. ALDH is known to be highly expressed in undifferentiated cell populations, namely stem and progenitor cells of various cell lineages, including hematopoietic and mesenchymal cells [11, 13, 14]. Viable cells of these cell populations with high aldehyde dehydrogenase activity (ALDH-bright [ALDHbr] cells) can be collected by using specific reagents and flow cytometry techniques [15]. BM-derived ALDHbr cells have been enriched for hematopoietic stem cells [13] and ALDHbr cell therapy has been reported to ameliorate various diseases in mouse models. For example, human lineage-negative BM ALDHbr (Lin−/ALDHbr) cells have been shown to augment the recovery of perfusion and increased blood vessel density in murine ischemic limbs [14]. In addition, Lin−/ALDHbr cells have been shown to improve systemic hyperglycemia and augment insulin secretion by increasing islet size and vascularization in streptozotocin-treated mice [16]. However, autologous Lin−/ALDHbr cell therapy failed to show effectiveness in humans [17, 18]. Although the reasons underlying this lack of efficacy are unknown, we hypothesized that one possible reason may be the high proportions of male donors, who constituted 73% [17] and 69% [18] of the study populations in these clinical trials.

We had previously suggested that lung-resident/non-hematopoietic (CD45−) ALDHbr cells could be useful for treating pulmonary fibrosis, since intravenous transfer of lung-derived CD45−/ALDHbr cells successfully suppressed murine bleomycin-induced pulmonary fibrosis [19]. However, for practical application to human pulmonary fibrosis, lung-resident ALDHbr cells may not be the best cell source, since large amounts of donor lung tissue are required to collect sufficient CD45−/ALDHbr cells. On the other hand, if BM ALDHbr cells are proven to be effective against pulmonary fibrosis, they can be a more suitable cell source since BM cells are easier to collect than lung-resident cells. Therefore, this study aimed to determine whether BM ALDHbr cells suppress bleomycin-induced pulmonary fibrosis, and further reveal the mechanisms and optimal conditions for ALDHbr cells to exert their efficacy.

Methods

Animals

This study was performed in accordance with the experimental procedures approved by the Animal Ethics Committee of Hiroshima University (A21-93 and 2021-59) and was in line with the ARRIVE guidelines 2.0. C57BL/6J mice (8–10-week-old young mice and 52-week-old aged mice) were purchased from Charles River Laboratories, Japan (Yokohama, Japan). In experiments to compare the number of transferred viable cells, we also used C57BL/6 Gt (ROSA)26Sor < tm1.1 (H2B-mcherry) Osb > heterozygotic and homozygotic mice (mCherry mouse, BRC No. RBRC06036, RIKEN, Tokyo, Japan) as a donor population [20]. The mice were housed in pathogen-free rooms with a controlled environment under a 12-h light–dark cycle and maintained on laboratory chow with free access to food and water. The mice were euthanized by inhalation of isoflurane (Fujifilm Wako Junyaku, Osaka, Japan) until one minute after breathing stops.

Bleomycin‑induced pulmonary fibrosis

We used bleomycin models recommended for the preclinical assessment of potential therapies for pulmonary fibrosis [21]. Mice were anesthetized with a combination of medetomidine hydrochloride (0.3 mg/kg body weight, Kyoritsu Seiyaku, Tokyo, Japan), midazolam (4 mg/kg body weight, Sandoz K.K., Tokyo, Japan), and butorphanol tartrate (5 mg/kg body weight, Meiji Seika Pharma, Tokyo, Japan). Then, the mice were administered bleomycin (Nippon Kayaku, Tokyo, Japan) through oropharyngeal aspiration (OA), as described previously [22]. Since female mice were more resistant to bleomycin-induced pulmonary fibrosis than male mice [23], the dose of bleomycin was adjusted to 2.0 mg/kg in saline for female mice and 1.5 mg/kg for male mice. The mice were randomly assigned to bleomycin or control groups using sequential allocation method. For the biochemical analysis of lung fibrosis, the hydroxyproline content in murine left lungs was evaluated as described previously [24].

Histological analysis

Right murine lungs were inflation-fixed with a 2% formalin solution. After embedding in paraffin wax, the tissues were stained with Masson’s trichrome stain. Immunohistochemical staining for mCherry was performed with anti-mCherry rabbit polyclonal antibody (dilution factor 1:400; ab167453, Abcam, Cambridge, UK) as the primary antibody, and peroxidase-conjugated anti-rabbit goat IgG polyclonal antibody (ready to use; #424144, Nichirei, Tokyo, Japan) as the secondary antibody. Positive and negative controls were the same as reported previously [19].

Cell isolation

BM cells were isolated from murine femur, tibia, and pelvis [25]. To isolate lung cells, the lung tissues were resected and minced, and incubated in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 1.0 mg/mL collagenase A (Roche Diagnostics, Basel, Switzerland) at 37 °C for 30 min. For flow cytometry, red blood cells were lysed using ACK lysis buffer (Thermo Fisher Scientific), and the cell suspensions were resuspended in 2 mL of phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 2 mM ethylenediaminetetraacetic acid (Sigma-Aldrich).

Antibodies and ALDH staining

All antibodies were purchased from BioLegend, San Diego, CA, USA. Cell suspensions were blocked with anti-CD16/32 antibody (FcγR, clone 93). ALDH activity was assessed using the ALDEFLUOR™ Kit (STEMCELL Technologies Inc., Vancouver, Canada) in accordance with the manufacturer’s protocol. ALDEFLUOR reagent and diethylaminobenzaldehyde (DEAB, provided in the ALDEFLUOR™ Kit), a specific inhibitor of ALDH, allow the discrimination of cells with low and high ALDH activity (ALDHdim and ALDHbr) using flow cytometry [26]. We also evaluated ALDHbr cells for the expression of (1) CD45, a hematopoietic cell marker; (2) Lin, an antibody cocktail against mature hematopoietic cell markers; and (3) cKit/CD117 and Sca-1, hematopoietic stem and progenitor cell markers. 4′,6-diamidino-2-phenylindole (DAPI) was used to detect viable cells. For morphological examination, ALDHbr and ALDHdim cells were transferred on slides by using a cytospin centrifuge and subsequently stained using Diff-quik (Sysmex, Hyogo, Japan).

Flow cytometric analysis and cell sorting

Flow cytometric analysis of BM and lung cells was performed using the FACS Aria II system (BD Biosciences, San Jose, CA, USA) and LSRFortessa X-20 (BD Biosciences) in accordance with a previously described method [26, 27]. To collect Lin−/ALDHbr cells more efficiently, mature hematopoietic cell populations (Lin+) were preliminarily depleted using magnetic-activated cell sorting (MACS) cell separation with Anti-Biotin MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s protocol. The collected data were analyzed using FACSDiva software (BD Biosciences) and FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Cell transfer to recipient mice

Recipient C57BL/6 mice belonging to the bleomycin group received sorted 1.0 × 105 Lin−/ALDHbr cells derived from another healthy donor mouse intravenously via the tail vein 3 days (inflammatory phase) or 10 days (fibrotic phase) after treatment with bleomycin (ALDHbr group). Mice in the control group were injected with PBS (PBS group) or Lin−/ALDHdim (ALDHdim group) at the same dose through the tail vein. The number of transferred cells was determined on the basis of previous studies [14, 16]. The sample size was mainly decided by the amount of collected cells, since the therapeutic effect of BM Lin−/ALDHbr cell therapy was unknown and we could not predetermine the cohort size. The recipient mice were euthanized on day 21 after bleomycin administration, and lung and bronchoalveolar lavage fluid (BALF) were collected for mRNA and protein expression analyses, flow cytometry, and histological evaluation. To confirm the viable number of the transferred cells, mCherry+ Lin−/ALDHbr cells were transferred intravenously into recipient mice and the right lung were analyzed by flow cytometry. In assessments to confirm that the transferred cells were viable and hematopoietic, DAPI−/CD45+/mCherry+ cells were considered to be the donor-derived cells. Cell sorting from mCherry-expressing donor mice and analysis of transferred donor mCherry+ cells was performed using the SORP Aria (BD Biosciences) and LSRFortessa X-20 (BD Biosciences) systems, respectively. Immunohistochemical staining of the lungs was also performed to confirm localization pathologically.

RNA-sequencing analysis

Mice that received Lin−/ALDHdim cells or Lin−/ALDHbr cells on day 10 after bleomycin administration were euthanized on day 14, and their right upper lungs were homogenized with 1 mL of TRIzol reagent (Life Technologies, Grand Island, NY, USA). Sorted female and male Lin−/ALDHbr cells were also suspended in 500 µL of TRIzol reagent. Total RNA from the lung and BM cells was extracted using the RNeasy Mini Kit (QIAGEN, Venlo, Netherlands) and underwent transcriptome analysis with 3′ untranslated region RNA-sequencing at the Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan [28]. Differentially expressed genes were plotted into a Hallmark gene set using GSEA Desktop (ver. 4.2.3; https://www.gsea-msigdb.org/gsea/index.jsp). To explore biological functions, Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed [29]. These transcriptome data were analyzed with integrated Differential Expression and Pathway (iDEP) analysis [30].

Cytotoxicity assay

Sorted 5.0 × 103 Lin−/ALDHbr cells were suspended in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin streptomycin stock solution (Thermo Fisher Scientific), seeded onto 96 well-culture plates, and incubated with hydrogen H2O2 (Sigma-Aldrich) for 1 h at 37 °C in a 5% CO2 incubator. Then, the number of viable cells was examined by trypan blue exclusion assay (Sigma-Aldrich).

ELISA

BALF samples were centrifuged at 1500 rpm for 5 min, and the supernatant was stored at − 80 °C. Right lung samples were homogenized in 1.0 mL of PBS and centrifuged at 8000 × g for 10 min, and the supernatant was stored at − 80 °C. Total glutathione (GSH) in the lung or BALF was measured using a Total Glutathione Quantification Kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) in accordance with the manufacturer’s instructions. TGF-β1 levels in the BALF were measured using Mouse TGF-beta 1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA).

Polymerase chain reaction and agarose gel electrophoresis

RNA from the sorted cells and lung tissue was extracted and reverse-transcribed into complementary DNA using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (PCR) was performed using the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems) and the TaqMan Gene Expression Assays (Applied Biosystems), as described previously [31]. The expression of Actb (β-actin, Mm02619580_g1; Applied Biosystems) was used as an internal control. The primers (Applied Biosystems) used for quantitative real-time PCR were as follows: ALDH1a1, ALDH1a2, ALDH1a3, ALDH1a7, ALDH1b1, ALDH1l1, ALDH2, ALDH3a1, ALDH4a1, ALDH7a1, ALDH8a1, ALDH18a1, Nrf-2 (nuclear factor-erythroid 2-related factor 2), STRA6 (stimulated by retinoic acid 6), CRABP1 (cellular retinoic acid-binding protein 1), CRABP2, Rara (intranuclear retinoic acid receptor A), Rarb, Rarg. To distinguish the mCherry-heterozygotic and homozygotic mice from wild-type mice, DNA from mouse tail was extracted using the DNeasy Blood and Tissue Kit (QIAGEN). The extracted DNA was amplified by PCR and bands were confirmed using electrophoresis, as described previously [19].

Statistical analysis

We conducted experiments to test our hypothesis at least three times and representative results are presented. All statistical analyses were performed by several authors who completed the 'Utilization of Data Literacy in Medicine' program in Medical Science at Hiroshima University using JMP Pro 17 software (SAS Institute Inc., Cary, NC, USA). Data were expressed as the median with interquartile range. Mann–Whitney U and Kruskal–Wallis tests were used for comparisons between groups. Correlation coefficients for parameters were calculated using Spearman’s rank correlation coefficient analysis. Statistical significance was set at p < 0.05.

Results

Characteristics of BM ALDHbr cells

BM ALDHbr cells were identified by comparing the results obtained with ALDEFLUOR reagent plus DEAB to those obtained with ALDEFLUOR reagent alone (Fig. 1A), as described previously [19]. To characterize BM ALDHbr cells, we divided the BM cell population into CD45+ and CD45− cells (Fig. 1B), or Lin+ and Lin− cells (Fig. 1C). In each fraction, the percentage of ALDHbr cells was determined. Unlike in the lungs [19], no ALDHbr cells were observed in the CD45− fraction (Fig. 1D), and all ALDHbr cells in the BM were CD45+ (Fig. 1E). In contrast, both Lin+/ALDHbr (Fig. 1F) and Lin−/ALDHbr (Fig. 1G) cells were present in the BM. BM Lin+/ALDHbr cells were also CD11b+, indicating the presence of monocytic or granulocytic cell populations (Additional file 1A). BM Lin−/ALDHbr cells were partially cKit-positive but Sca1-negative, indicating the presence of a myeloid cell population containing myeloid progenitor cells (Additional file 1B). In fact, both Lin−/cKit+/Sca1+ hematopoietic stem cells (also known as KSL) and Lin−/cKit+/Sca1− myeloid progenitor cells, but not Lin−/cKit−/Sca1+ lymphoid progenitor cells, in the BM contained certain percentages of ALDHbr cells (Additional file 1C). BM Lin−/ALDHbr cells were DAPI-negative and confirmed to be viable cells (Fig. 1H), and were slightly bigger than BM Lin− cells with low aldehyde dehydrogenase activity (Lin−/ALDHdim, Additional file 1D). Moreover, old mice had lower proportions of Lin−/ALDHbr cells in the BM cell population than young mice (Additional file 1E).Fig. 1 Antifibrotic effects and characteristics of BM ALDHbr in bleomycin-induced pulmonary fibrosis. A–H Detection of the cell population with high aldehyde dehydrogenase activity (ALDHbr) in murine bone marrow (BM) cells using flow cytometry. A BM ALDHbr cells defined in the whole BM cells by comparing samples stained with ALDEFLUOR reagent (ALDH) plus diethylaminobenzaldehyde (DEAB) versus those stained with ALDEFLUOR reagent alone. (B and C) Whole BM cells divided into B hematopoietic (CD45+) and nonhematopoietic (CD45−) fractions or C mature lineage (Lin)-positive hematopoietic (Lin+) and Lin− fractions. D–G Proportion of ALDHbr cells in the D CD45−, E CD45+, F Lin+, and G Lin− fractions. H Viability of BM Lin−/ALDHbr cells assessed using 4′,6-diamidino-2-phenylindole (DAPI). I Experimental scheme of comparison of treatments for bleomycin-induced pulmonary fibrosis. O.A., oropharyngeal administration. BLM, bleomycin. ALDHdim, Lin− BM cells with low aldehyde dehydrogenase activity. J, K Comparison of J hydroxyproline levels and K Masson’s trichrome staining results in lung tissue sections among the without-BLM, PBS, ALDHdim, and ALDHbr groups on day 21 (n = 6–14/group). Data represent mean ± SEM. Scale bar: 400 μm. Kruskal–Wallis tests were used for comparisons between groups

Antifibrotic effects of female Lin−/ALDHbr cells on bleomycin-induced pulmonary fibrosis

To clarify the antifibrotic effects of Lin−/ALDHbr cells on pulmonary fibrosis, we first collected BM Lin−/ALDHbr cells and Lin−/ALDHdim cells from healthy mice using fluorescence-activated cell sorting (FACS). Pre-depletion of differentiated cells using MACS yielded enriched Lin−/ALDHbr cells (Additional file 2A) and reduced the time required for FACS to approximately one-sixth (Additional file 2B). We compared the antifibrotic effects of PBS, female BM Lin−/ALDHdim cell, and female Lin−/ALDHbr cell injections via the tail vein in female mice 10 days after bleomycin administration (Fig. 1I). Murine body weight did not differ significantly among the bleomycin administrated groups (Additional file 3A). The levels of hydroxyproline, the recommended indicator of lung collagen content, were significantly lower in the ALDHbr group than in the PBS and ALDHdim groups (Fig. 1J). Furthermore, consistent with the hydroxyproline levels, the ALDHbr group showed a clear reduction in histological lung structural damage and fibrosis (Fig. 1K). Ashcroft scores were also significantly lower in the ALDHbr group than in the PBS and ALDHdim groups (Additional file 3B). In addition, TGF-β1 levels in the BALF tended to be lower in the ALDHbr group than in the PBS group (Additional file 3C). These results indicated that female Lin−/ALDHbr cells had antifibrotic effects on bleomycin-induced pulmonary fibrosis.

Conditions required for the antifibrotic effects of BM ALDHbr cells

Since BM ALDHbr cells contain hematopoietic stem and progenitor cells [13], and female hematopoietic stem cells show superior proliferative potential than male cells [32], we further sought to identify the ideal conditions, including sex-related differences, for the antifibrotic effects of ALDHbr cells. When male-derived, instead of female-derived, Lin−/ALDHbr cells were intravenously transferred into male mice 10 days after bleomycin administration, the hydroxyproline levels in the ALDHbr group showed no difference in comparison with those in the PBS and ALDHdim groups (Fig. 2A). To confirm whether the antifibrotic effect depends on the recipient sex, male-derived Lin−/ALDHbr cells were transferred to female mice, which also resulted in no reduction in hydroxyproline levels (Fig. 2B). Moreover, when female-derived Lin+/ALDHbr cells, instead of Lin−/ALDHbr cells, were transferred into female mice 10 days after bleomycin administration, no reduction in hydroxyproline levels was observed in comparison with the PBS group (Fig. 2C). In addition, no reduction in hydroxyproline levels was observed when female-derived Lin−/ALDHbr cells were transferred into female mice 3, instead of 10, days after bleomycin administration (Fig. 2D). Furthermore, no reduction in hydroxyproline levels was observed when female-derived Lin−/ALDHbr cells were intratracheally, instead of intravenously, transferred into female mice 10 days after bleomycin administration (Fig. 2E). These results confirmed that the antifibrotic effects were observed when female-derived Lin−/ALDHbr cells were transferred intravenously in the fibrotic phase (Fig. 2F).Fig. 2 Conditions required for the antifibrotic effects of BM ALDHbr cells. A–E Comparison of hydroxyproline levels in lung tissue sections between each group on day 21 in various conditions (n = 6–15/group). Mann–Whitney U and Kruskal–Wallis tests were used for comparisons between groups. A Transfer of BM Lin−/ALDHdim and Lin−/ALDHbr cells from male donors to male mice. B Transfer of BM Lin−/ALDHbr cells from male donors to female mice. C Transfer of BM Lin+/ALDHbr cells from female donors to female mice. D Transfer of BM Lin−/ALDHbr cells from female donors to female mice on day 3. E Transfer of BM Lin−/ALDHbr cells from female donors to female mice via OA. F Summary of ideal conditions for the antifibrotic effects of BM ALDHbr cells

Engraftment of the transferred BM ALDHbr cells in the lung

We next examined the engraftment of the transferred donor BM Lin−/ALDHbr cells in the recipient lungs. To distinguish the transferred donor BM Lin−/ALDHbr cells from the recipients’ lung-resident cells, mCherry knock-in mice were used as donors. After confirming that virtually all donor BM cells obtained from the mCherry knock-in mice were mCherry-positive (Fig. 3A), we sorted the BM mCherry+/Lin−/ALDHbr cells for transfer. To determine whether the sex of the recipient affected the engraftment rate of the transferred cells, we first intravenously transferred mCherry+/Lin−/ALDHbr cells from a female donor into female and male mice without bleomycin administration. The engraftment of mCherry+ donor cells in both female and male BM and lungs was assessed using flow cytometric analysis (Fig. 3B, C). The engraftment rate was not affected by the sex of the recipient (Fig. 3D). Subsequently, to compare the role of the donor’s sex on the engraftment rate, BM mCherry+/Lin−/ALDHbr cells from female and male donors were transferred into female mice 10 days after bleomycin administration. As shown in Fig. 3E, F, the engraftment rate tended to increase when female-derived mCherry+/Lin−/ALDHbr cells were used (Fig. 3F). In addition, the engraftment rate in female mice administered bleomycin was significantly greater than that in mice that did not receive bleomycin (Fig. 3G).Fig. 3 Engraftment of transferred BM ALDHbr in the lung. A Representative flow cytometric images of whole BM cells in donor mCherry-expressing mice. B Representative flow cytometric images of mCherry+ in DAPI− (live)/CD45+ cells in bone marrows after transfer of mCherry+ /Lin−/ALDHbr cells. C Representative flow cytometric images of mCherry+ in DAPI−/CD45+ cells in healthy male and female lungs after transfer of mCherry+ /Lin−/ALDHbr cells from female donors. D The engraftment rate of mCherry+ in DAPI−/CD45+ cells in the lungs of male and female mice after transfer of female-derived mCherry+ /Lin−/ALDHbr cells (n = 3–4/group). E Representative flow cytometric images of mCherry+ in DAPI−/CD45+ cells in the lungs of female mice after administration of bleomycin and transfer of PBS or male- or female-derived mCherry+ /Lin−/ALDHbr cells. F The engraftment rate of mCherry+ in DAPI−/CD45+ cells in female lungs after transfer of male- or female-derived mCherry+ /Lin−/ALDHbr cells (n = 6–7/group). G Comparison of DAPI−/CD45+/mCherry+ cell numbers in right female lungs after transfer of female mCherry+ /Lin−/ALDHbr cells to mice administered BLM and those not treated with BLM (n = 4–7/group)

Sex differences in BM ALDHbr cells

To determine why female-derived, but not male-derived, BM Lin−/ALDHbr cells ameliorated pulmonary fibrosis, we first measured the mRNA expression levels of the ALDH subtype in lungs obtained from mice without (Additional file 4A) or with bleomycin administration (Additional file 4B) and sorted Lin−/ALDHbr cells (Additional file 4C). No sex-related differences were observed in the mRNA expression levels of the ALDH subtype between these groups. Next, we performed RNA-sequencing analysis using the RNA extracted from BM Lin−/ALDHbr cells derived from female and male donors. t-distributed stochastic neighbor embedding (t-SNE) showed a clear difference between the BM Lin−/ALDHbr cells derived from female and male donors (Fig. 4A). Analysis of the differentially expressed genes confirmed that expression of the ROS pathway was upregulated in the BM Lin−/ALDHbr cells derived from female donors (Fig. 4B). No significant differences in estrogen receptor gene expression were confirmed (Fig. 4C). On the basis of these results, we performed Kyoto Encyclopedia of Genes and Genomes pathway analysis of glutathione metabolism and found that the BM Lin−/ALDHbr cells from female donors upregulated glutathione-increasing signals and downregulated glutathione-degrading signals in comparison with those derived from male donors (Fig. 4D). These results suggested that under ROS stimulation, BM Lin−/ALDHbr cells derived from female donors showed more antioxidant activity than those derived from male donors. To investigate the resistance of BM Lin−/ALDHbr cells against ROS, cell viabilities were measured after addition of H2O2 to the cell culture medium. We observed that the BM Lin−/ALDHbr cell viabilities decreased in an H2O2 concentration-dependent manner (Fig. 4E). When 10 µM of H2O2 was added to the medium for culturing BM Lin−/ALDHbr cells, the Lin−/ALDHbr cells derived from female donors maintained significantly higher cell viabilities than those derived from male donors (Fig. 4F). These findings suggest that BM Lin−/ALDHbr cells derived from female donors were more resistant to ROS than those derived from male donors and survived longer in an oxidative stress environment.Fig. 4 Sex differences in BM ALDHbr. A–C RNA-sequencing analysis using extracted RNA from female- and male-derived BM Lin−/ALDHbr cells (n = 3/group). A t-distributed stochastic neighbor embedding (t-SNE) for comparison between female- and male-derived BM Lin−/ALDHbr cells. B Differentially expressed gene analysis of female- and male-derived BM Lin−/ALDHbr cells. padj, adjusted p value. C Estrogen receptor genes in female- and male-derived BM Lin−/ALDHbr cells. Esr, estrogen receptor. D Kyoto Encyclopedia of Genes and Genomes pathway analysis of glutathione metabolism in female- and male-derived BM Lin−/ALDHbr cells. GSH, Glutathione; GSSG, Glutathione disulfide; L-Cys-Gly, l-Cysteinyl-glycine; R-S-G, R-S-Glutathione; L-Cys-Gly-S-conjugate, l-Cysteinyl-glycine-S-conjugate; ggt, Gamma-glutamyltransferase; Chac, Glutathione-specific gamma-glutamylcyclotransferase; Gsr, Glutathione disulfide reductase; gpx, Glutathione peroxidase; PRDX, Peroxiredoxin; GST, Glutathione transferase; HPGDS, Prostaglandin-D synthase; LANCL1, LanC-like Glutathione S-Transferase 1. E Viabilities of female BM Lin−/ALDHbr cells exposed to different concentrations of hydrogen peroxide (H2O2) in vitro (n = 3–5/group). F Comparison of cell viabilities between male and female BM Lin−/ALDHbr cells exposed to 10 μM of H2O2 (n = 7/group)

Antifibrotic mechanisms of BM Lin−/ALDHbr cells

To examine how the BM Lin−/ALDHbr cells derived from female donors ameliorated pulmonary fibrosis, we first investigated the expression of retinol-metabolizing pathway-related genes, which were upregulated in lungs transferred with lung CD45−/ALDHbr cells [19]. No differences in the mRNA expression of these genes were observed in lungs treated with BM Lin−/ALDHbr cells than in those treated with Lin−/ALDHdim cells (Additional file 5). We next compared the findings for fibrotic lungs treated with BM Lin−/ALDHbr cells and those treated with Lin−/ALDHdim cells using RNA-sequencing analysis. In the t-SNE, lungs treated with Lin−/ALDHbr and Lin−/ALDHdim cells showed differences with partial overlap (Fig. 5A). When focusing on the antioxidant pathway, the lungs treated with Lin−/ALDHbr cells showed enhanced mRNA signals for transformation of GSH to glutathione disulfide (GSSG) and NAC (Fig. 5B), which reduced the ROS stimulation induced by bleomycin [33, 34]. On the basis of these results, we performed enzyme-linked immunosorbent assay (ELISA) to further measure GSH concentrations in the BALF and lung tissue obtained from mice treated with Lin−/ALDHbr cells. The GSH concentrations in both BALF and lung tissue significantly increased after bleomycin administration but decreased after treatment with BM Lin−/ALDHbr cells (Fig. 5C, D). We observed a positive correlation between GSH and hydroxyproline concentrations in lung tissue (Fig. 5E). Moreover, Nrf-2, a central regulator for oxidation reaction [33], mRNA expression in lung tissue tended to be upregulated in the ALDHbr group when compared to that in the PBS group (Fig. 5F).Fig. 5 Mechanisms underlying the antifibrotic effects of BM ALDHbr. A, B RNA-sequencing analysis using female fibrotic lungs after transfer of female-derived BM Lin−/ALDHdim and Lin−/ALDHbr (n = 5/group). A t-SNE comparing lung tissues after transfer of BM Lin−/ALDHdim and Lin−/ALDHbr cells. B Kyoto Encyclopedia of Genes and Genomes pathway analysis of glutathione metabolism in lungs after transfer of BM Lin−/ALDHdim and Lin−/ALDHbr cells. NAC, N-acetyl-l-cysteine; G6PD, Glucose-6-phosphate dehydrogenase; NAT8, Cysteine-S-conjugate N-acetyltransferase; gclc, Glutamate-cysteine ligase. C, D Comparison of glutathione concentrations in C lung tissue and D bronchoalveolar lavage fluid (BALF) among the without-BLM, PBS, and ALDHbr groups on day 21 (n = 4–5/group). E Correlations between the glutathione concentration and hydroxyproline level in the lung tissue of mice administered bleomycin. Correlation coefficients for parameters were calculated using Spearman’s rank correlation coefficient analysis. F Comparison of nuclear factor-erythroid 2-related factor 2 (Nrf-2) mRNA expression in lung tissue in the PBS and ALDHbr groups (n = 5/group)

Discussion

The findings of this study indicated that treatment with BM Lin−/ALDHbr cells from female, but not male, donors ameliorated murine bleomycin-induced pulmonary fibrosis. BM Lin−/ALDHbr cells from female donors were more tolerant to oxidative stress and showed longer survival than those from male donors, resulting in an enhanced antifibrotic effect. The GSH levels in lung tissues were correlated with the levels of hydroxyproline, the recommended indicator of pulmonary fibrosis, and were significantly decreased by treatment with BM Lin−/ALDHbr cells derived from female donors. Thus, the improvement in pulmonary fibrosis after treatment with BM Lin−/ALDHbr cells derived from female donors may be attributable to a reduction in oxidative stress.

In the present study, BM Lin−/ALDHbr cell therapy showed antioxidant effects against pulmonary fibrosis. The potential underlying mechanisms of ALDHbr cell is inhibition of ROS production and detoxification of reactive aldehydes and drugs [11]. ROS promote the apoptosis of airway epithelial cells, resulting in the production of molecules related to myofibroblast differentiation and collagen deposition against oxidative stress [6]. GSH, which is increased in the lung during bleomycin-induced pulmonary fibrosis [35], exhibits antioxidant effects by conversion into GSSG via enzymatic reactions using enzymes such as glutathione peroxidase (gpx) [35]. Administration of the antifibrotic drug pirfenidone has been shown to increase the expression of Nrf-2 and gpx in lung fibroblasts and decrease ROS [33]. In addition, administration of glutaredoxin has been shown to decrease pulmonary GSH and hydroxyproline levels in murine bleomycin-induced pulmonary fibrosis [35]. Consistent with these findings, BM Lin−/ALDHbr cell therapy was shown to decrease GSH levels in the lungs and BALF, resulting in reduced hydroxyproline levels. Furthermore, ALDHbr tumor stem cells have been shown to be resistant to chemotherapy by decreasing ROS [36], and cardiovascular oxidative stress has been reported to be enhanced in ALDH2-knockout mice [37]. Taken together, these findings imply that the amelioration of pulmonary fibrosis with Lin−/ALDHbr cell therapy was most likely mediated by ROS reduction.

To our knowledge, our study is the first to reveal that BM Lin−/ALDHbr cells derived from female, but not male, donors exert antifibrotic effects. Female sex has been associated with a better prognosis than male sex in coronavirus disease 2019 (COVID-19) [38] and among ICU patients [39] complicated with respiratory disorders. Moreover, male sex is one of the risk factors for the onset, progression, and death of pulmonary fibrosis [40]. In a previous report using human donor-derived ALDHbr cell therapy for murine ischemic disease, both female and male recipient mice showed therapeutic effects [41]. Thus, the sex of the donor, not the recipient, is thought to influence therapeutic effect of ALDHbr cells. Notably, hematopoietic stem cells from female donors have shown superior proliferative potential [32], and female-derived cells have been reported to show more antioxidant activity than male-derived cells [42]. In the current study, the antioxidant glutathione pathway was revealed to be upregulated in female-derived BM Lin−/ALDHbr cells, which proved to be more resistant against oxidative stress. The therapeutic effects in previous clinical trials using BM ALDHbr cells for various diseases were not as significant as those observed in the corresponding animal studies. Since these clinical trials predominantly included male patients who received their own ALDHbr cells [17, 18], our results imply that the male predominance of the study populations may have been one of the reasons for the failure of these clinical trials.

The present study has potential limitations. A major limitation is that all results were obtained from animal experiments. More research is still needed to develop successful strategies focused on cell-based therapies in lung fibrosis [8]. Since allogeneic stem cell transplant requires immunosuppression and has a high mortality rate yet [43], we estimate that it is more appropriate to transplant BM Lin−/ALDHbr cells through autologous transplantation rather than allogeneic transplantation as with previous clinical trials [17, 18]. Although BM is an easier organ to approach than lung, the human ALDHbr cell population is approximately 1% of the total BM cell population [14], and the number of BM ALDHbr cells transferred in this study was equivalent to about 3 × 108 cells in a human adult. If BM ALDHbr cells can be grown in culture while maintaining their ALDH activity and therapeutic efficacy, the application of BM ALDHbr cells for clinical use will be more feasible. Second, we did not transfer BM Lin−/ALDHbr cells into old mice. Indeed, idiopathic pulmonary fibrosis often occurs primarily in older adults [1]. However, there is no evidence that the pathways leading to fibrosis are different in young mice when compared to old mice and no currently compelling reason to recommend either standard or prioritized use of old mice for pharmacological testing [21]. Moreover, no experiments were conducted to modify the dosage or frequency of cell administration. Further studies are needed to validate these results.

In conclusion, this study showed that BM Lin−/ALDHbr cells could be used as a novel and useful therapeutic tool for pulmonary fibrosis. The therapeutic effects of BM Lin−/ALDHbr cells are thought to show sex-related differences, indicating the need to account for these differences in treatment. These results may explain why ALDHbr cell therapy failed in clinical trials, and might be related to the reasons underlying the less frequent exacerbations and better prognosis in women with pulmonary fibrosis than in men.

Conclusions

This study showed that BM Lin−/ALDHbr cells could be used as a novel and useful therapeutic tool for pulmonary fibrosis. The therapeutic effects of BM Lin−/ALDHbr cells are thought to show sex-related differences, indicating the need to account for these differences in treatment. These results may explain why ALDHbr cell therapy failed in clinical trials, and might be related to the reasons underlying the less frequent exacerbations and better prognosis in women with pulmonary fibrosis than in men.

Supplementary Information

Additional file 1. Characteristics of bone marrow cells with high aldehyde dehydrogenase activity (BM ALDHbr). (A) Determination of the proportion of CD11b+ cells among mature hematopoietic (Lin+) ALDHbr cells using flow cytometry. (B) Determination of the proportion of cKit+ cells among BM Lin−/ALDHbr cells using flow cytometry. (C) Proportion of ALDHbr cells among the hematopoietic myeloid progenitor cell (Lin−/cKit+/Sca1−), lymphoid progenitor cell (Lin−/cKit−/Sca1+), and stem cell populations (Lin−/cKit+/Sca1+). (D) Morphology of Lin− BM cells with low and high aldehyde dehydrogenase activity (Lin−/ALDHdim and Lin−/ALDHbr). (E) Comparison of the Lin−/ALDHbr rate in whole BM cells obtained from young and old mice.

Additional file 2. Effect of magnetic-activated cell sorting (MACS) on the enrichment of BM Lin−/ALDHbr. (A) Representative image of BM Lin−/ALDHbr cells before and after MACS. (B) Population of BM Lin−/ALDHbr cells among the analyzed cells before and after MACS.

Additional file 3. Antifibrotic effect of female-derived BM Lin−/ALDHbr in bleomycin-induced pulmonary fibrosis. (A) Comparison of murine weight among the without-BLM, PBS, Lin−/ALDHdim, and ALDHbr groups on day 0, 7, 14 and 21 (n = 6–15/group). BLM, bleomycin. (B) Comparison of fibrosis determined using the Ashcroft score in lung tissue sections among the PBS, Lin−/ALDHdim, and ALDHbr groups on day 21 (n = 6–9/group). I.V., injection via tail vein. (C) Comparison of transforming growth factor β1 (TGF-β1) levels in the bronchoalveolar lavage fluid (BALF) between PBS and ALDHbr groups on day 21 (n = 5/group).

Additional file 4. mRNA expression levels of ALDH subtypes. Real-time quantitative PCR analysis of the mRNA expression levels of ALDH subtypes in (A) lungs obtained from mice without bleomycin administration or (B) lungs with bleomycin administration, and (C) sorted BM Lin−/ALDHbr.

Additional file 5. Retinol-metabolizing pathway-related genes in fibrotic lungs treated with BM Lin−/ALDHdim and Lin−/ALDHbr.

Abbreviations

ALDH Aldehyde dehydrogenase

ALDHbr Cell populations with high ALDH activity

ALDHdim Cell populations with low ALDH activity

BALF Bronchoalveolar lavage fluid

BLM Bleomycin

BM Bone marrow

Chac Glutathione-specific gamma-glutamylcyclotransferase

COVID-19 Coronavirus disease 2019

CRABP Cellular retinoic acid-binding protein

DAPI 4′,6-Diamidino-2-phenylindole

DEAB Diethylaminobenzaldehyde

Esr Estrogen receptor

FACS Fluorescence activated cell sorting

G6PD Glucose-6-phosphate dehydrogenase

gclc Glutamate-cysteine ligase

ggt Gamma-glutamyltransferase

gpx Glutathione peroxidase

GSH Glutathione

Gsr Glutathione disulfide reductase

GSSG Glutathione disulfide

GST Glutathione transferase

H2O2 Hydrogen peroxide

HPGDS Prostaglandin-D synthase

LANCL1 LanC-like glutathione S-transferase 1

L-Cys-Gly L-cysteinyl-glycine

L-Cys-Gly-S-conjugate L-cysteinyl-glycine-S-conjugate

Lin Lineage

MACS Magnetic-activated cell sorting

NAC N-Acetyl-l-cysteine

NAT8 Cysteine-S-conjugate N-acetyltransferase

Nrf Nuclear factor-erythroid 2-related factor

OA Oropharyngeal aspiration

PCR Polymerase chain reaction

PRDX Peroxiredoxin

RALDH Retinal dehydrogenase/retinaldehyde dehydrogenase

RAR Intranuclear retinoic acid receptor

ROS Reactive oxygen species

R-S-G R-S-glutathione

STRA Stimulated by retinoic acid

TGF Transforming growth factor

t-SNE T-distributed stochastic neighbor embedding

Acknowledgements

We would like to thank Ms. Yukari Iyanaga and Mr. Yu Matsumoto (Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University) for assisting with the experiments and Ms. Yoko Hayashi (Natural Science Center for Basic Research and Development, Hiroshima University) for her technical assistance with the flow cytometry analysis. We would also like to thank Editage (www.editage.com) for English language editing. This work was supported in part by the Natural Science Center for Basic Research and Development (NBARD-00093), and the Program of the Network-type Joint Usage/Research Center for Radiation Disaster Medical Science.

Author contributions

SI: collection of data, data analysis and interpretation, manuscript writing. TN: conception and design, collection of data, financial support, data analysis and interpretation, manuscript writing. TM, KS, SS: data analysis and interpretation, reviewing the manuscript for important intellectual content and approving for submission. KY, YH, HI, KF, HH: reviewing the manuscript for important intellectual content and approving for submission. NH: conception and design, financial support, reviewing the manuscript for important intellectual content and approving for submission.

Funding

This study was partially supported by JSPS KAKENHI Grant Numbers 20K08519, and a grant from Ryokufukai, Hiroshima University.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request. Raw RNA sequencing data were deposited in the NCBI for the Biotechnology Information Gene Expression Omnibus database (GEO GSE276258 and GSE276259).

Declarations

Ethics approval and consent to participate

(1) Title of the approved project: Roles of lung and bone marrow ALDH bright stem cells in bleomycin-induced pulmonary fibrosis. (2) Name of the institutional approval committee or unit: Committee on Animal Research at Hiroshima University. (3) Approval number: A21-93 and 2021-59. (4) Date of approval: August 5, 2021.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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