Immunosuppressive effect of mesenchymal stem cells on lung and gut CD8+ T cells in lipopolysaccharide‐induced acute lung injury in mice

Abstract Objectives Acute lung injury (ALI) not only affects pulmonary function but also leads to intestinal dysfunction, which in turn contributes to ALI. Mesenchymal stem cell (MSC) transplantation can be a potential strategy in the treatment of ALI. However, the mechanisms of synergistic regulatory effects by MSCs on the lung and intestine in ALI need more in‐depth study. Materials and methods We evaluated the therapeutic effects of MSCs on the murine model of lipopolysaccharide (LPS)‐induced ALI through survival rate, histopathology and bronchoalveolar lavage fluid. Metagenomic sequencing was performed to assess the gut microbiota. The levels of pulmonary and intestinal inflammation and immune response were assessed by analysing cytokine expression and flow cytometry. Results Mesenchymal stem cells significantly improved the survival rate of mice with ALI, alleviated histopathological lung damage, improved intestinal barrier integrity, and reduced the levels of inflammatory cytokines in the lung and gut. Furthermore, MSCs inhibited the inflammatory response by decreasing the infiltration of CD8+ T cells in both small‐intestinal lymphocytes and Peyer's patches. The gut bacterial community diversity was significantly altered by MSC transplantation. Furthermore, depletion of intestinal bacterial communities with antibiotics resulted in more severe lung and gut damages and mortality, while MSCs significantly alleviated lung injury due to their immunosuppressive effect. Conclusions The present research indicates that MSCs attenuate lung and gut injury partly via regulation of the immune response in the lungs and intestines and gut microbiota, providing new insights into the mechanisms underlying the therapeutic effects of MSC treatment for LPS‐induced ALI.


| INTRODUC TI ON
Sepsis-related acute lung injury (ALI), the early pathophysiological change in acute respiratory distress syndrome (ARDS), has a high mortality rate due to lack of effective therapies. 1,2 ALI/ARDS can progress to multiple organ failure, which is characterized by increased lung permeability, pulmonary oedema and infiltration of inflammatory cells. 3,4 Despite much research, the mortality rate is 36-44%, 5 and there is no effective therapeutic strategy. 6 A connection between the lungs and gut has been demonstrated in human and animal studies. [7][8][9][10] A disturbance in the intestinal bacterial communities will disrupt the integrity of the intestinal barrier, thereby increasing the risk of bacterial translocation, activating systemic immune system and aggravating immune damage to the lung. 11 For instance, stimulation of mouse lungs with lipopolysaccharide (LPS) causes acute changes in the structure and function of intestinal microflora, resulting in bacterial and endotoxin translocation and eventually lung injury, which is an important factor that initiates ARDS. [12][13][14] Under physiological conditions, the intestinal mucosal barrier, gut microbiota and their metabolites together form the intestinal microecology, which maintains in a dynamic balance. 15 However, the mechanism of how intestinal microflora changes affect lung injury during ARDS remains unclear. In addition, pneumonia induces intestinal injury and decreases the proliferation of gut mucosal epithelial cells. 16 The mechanisms are likely related to the local inflammatory factor microenvironment and the immune response. 17 Mesenchymal stem cells (MSCs), which are multipotent and immunoregulatory, have therapeutic potential in lung diseases. 18 They can repair LPS-induced ALI, pneumonia, inflammatory bowel disease (IBD) and systemic sepsis in animal models. 7,19 They protect against ALI by reducing inflammatory cytokine secretion, enhancing macrophage phagocytosis and regulating T-cell differentiation. 20,21 Importantly, systemic administration of MSCs affects the gut epithelium and significantly ameliorates intestinal mucosal inflammation. [22][23][24] The gut epithelium functions as a protective barrier against LPS under normal conditions. Small-intestinal intraepithelial lymphocytes (IELs) are the first line of defence and are activated by inflammatory signals to eliminate infected epithelial cells. 25 The CD8 + T cells infiltrating IELs produce interferonγ (IFNγ), which is correlated with the severity of intestinal damage. 26 Moreover, the activation state of CD4 + T cells reflects the intestinal immune response. 27 However, the mechanisms of MSC and IEL recruitment are unclear.
Although MSCs have an immunomodulatory effect in ALI, 28,29 we propose that they not only have an immunomodulatory effect but also alter the intestinal microbiome in a manner that promotes barrier integrity.
In this study, we investigated the efficacy of MSCs in a mouse

| Animals
Male wild-type 6-to 8-week-old C57 BL/6J mice were purchased from Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China. The same specific pathogen-free room was used to house all mice. All animal experimental procedures were conducted according to a protocol approved by the Ethics Committee of The First Affiliated Hospital of Zhejiang University (No. 2015-130).

| Animal model of LPS-induced acute lung injury
Lipopolysaccharide (derived from Escherichia coli 0111: B4, Sigma-Aldrich, Poole, United Kingdom) was given intratracheally as a model of direct lung injury. The ALI model was induced by 20 mg/ kg LPS as described previously, 30   neomycin sulphate 1 g/L, Sangon Biotech; metronidazole 1 g/L, Sangon Biotech and vancomycin 0.5 g/L, Sangon Biotech) in drinking water for 3 weeks as described. 31 Two days after cessation of the antibiotics, the mice were treated as described above.

| Mouse tissue collection and processing
Mice were sacrificed for removal of the lungs, small-intestinal segments and cecum and colon contents. Portions of the lung and intestinal tissues were fixed in 10% neutral buffered formalin to prepare paraffin-embedded sections, and sliced into 5μm-thick sections.
Another set of mice was sacrificed. The lungs were washed three times with 800 µl ice-cold PBS through a tracheal cannula as described previously, 32 and the bronchoalveolar lavage fluid (BALF) was collected and centrifuged at 800 g for 10 min at 4°C. The supernatant was stored at −80°C for subsequent assay of cytokines and protein concentration, and the cell pellet was resuspended in 200 μl for cell counting and cell smear generation. The total protein content of BALF was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Inc).

| Cytokine analyses
BALF and small-intestinal tissue homogenate supernatants were harvested and analysed for their contents of tumour necrosis factorα (TNFα), interferonγ (IFNγ), interleukin-6 (IL-6), IL-1α and IL-1β by bead-based multiplex LEGENDplex™ assay (Multi-Analyte BALF total cell count peaked in the LPS/PBS group at day 1 and decreased at day 7 and was lower in the LPS/MSC group. The BALF protein concentration peaked at day 3 and decreased at day 7 in the LPS/PBS group and was markedly lower in the LPS/MSC group (mean ± SEM, n = 5 or 6). **P <.01, ***P <.001 by Student's t-test Flow Assay Kit, BioLegend, Koblenz, Germany) according to the manufacturer's instructions.

| Lung and intestinal histology
For histopathologic analyses, formalin-fixed lung and ileum samples were paraffin-embedded, sectioned (5 μm thickness) and stained with haematoxylin and eosin (H&E). The sections were evaluated by an experienced pathologist who was blinded to the group assignment.

| Isolation of pulmonary immune cells, IELs and PPs
Lung tissues were harvested, cut into 1 cm pieces, transferred to a gentleMACS C tube (Miltenyi Biotec, Bergisch Gladbach, Germany) containing an enzyme mixture (Mouse Lung Dissociation Kit, Miltenyi Biotec), homogenized and digested at 37°C for 30 minute. Pulmonary immune cells were obtained after density gradient centrifugation.

| Mass cytometry antibody staining
Mass cytometry was used to evaluate the CD8 + T-cell populations in mouse lung. Pulmonary immune cell suspensions were washed once in 1 ml staining buffer (PBS with 0.5% BSA and 0.02% NaN 3 ) and incubated with 0.25 μM cisplatin (Fluidigm, San Francisco, CA) on ice for 5 minute to label dead cells. Next, the cells were labelled with a mixture of metal isotope-conjugated antibodies against cell surface markers for 30 minute on ice. Then, 0.03 μM Ir nucleic-acid intercalator (Fluidigm) in Fix and Perm Buffer was added, and the cell suspensions were placed at 4°C overnight. The cells were washed twice and incubated with a metal isotope-conjugated intracellular antibody cocktail for 30 minute on ice. The cells were resuspended in distilled water containing 20% EQ. 4 beads (Fluidigm) and acquired on a CyTOF instrument with a Helios system (Fluidigm) at 500 events per second.

| Flow cytometry
The single-cell suspensions of IELs and PPs were stained using standard procedures for cell surface markers. The following mono-  No sequences contained ambiguous bases. Details of the sequencing output, such as raw reads, clean reads and average read lengths, are summarized in supplemental Table S1. Operational taxonomic unit (OTU) clustering was performed using a 99% similarity cut-off to represent one species per OTU. After sequencing data were rarefied, the alpha and beta diversities were calculated, and differences in relative abundance were compared using Statistical Analyses of Metagenomic Profiles (STAMP) software.

F I G U R E 3
Therapeutic effects of MSCs on cytokines in the lung and gut in LPS-induced acute lung injury. The IFNγ, TNFα, IL-1α, IL-1β and IL-6 concentrations were determined at days 1, 3 and 7 in the PBS, LPS/PBS, LPS/MSC groups by bead-based multiplex LEGENDplex™ assay. (A) Inflammatory cytokine concentrations in BALF were significantly increased in the LPS/PBS group, but were lower in the LPS/ MSC group. MSCs decreased the cytokine concentrations in BALF (mean ± SEM, n = 6). (B) Cytokine levels in the small intestine increased, particularly at days 1 and 3, in the LPS/PBS group compared with the PBS group. Administration of MSCs reduced the LPS-induced increased cytokine concentrations in intestinal tissue (mean ± SEM, n = 4). *P <.05, **P <.01, ***P <.001 by Student's t-test

| Statistical analyses
We used the Kaplan-Meier log-rank test to compare survival between groups (GraphPad Prism 8). Two-tailed, unpaired Student's t-tests were performed for comparing two groups. Quantification of immunohistochemical assays was performed using National Institutes of Health image analysis software ImageJ bundled with 64-bit Java 1.8.0_172.
The alpha diversity, including the indexes of Shannon, Chao1 and observed species, was calculated using QIIME (Version 1.9.1).
Unweighted uniFrac principal coordinates analysis and weighted uni- was used to evaluate differences in overall microbiota composition between the groups using weighted and unweighted UniFrac distance matrices. Linear discriminant analysis (LDA) effect size was utilized to evaluate differentially abundant bacterial taxa and predicted function between the animal groups. Data are presented as means ± standard error of the mean (SEM). We set three levels of statistical significance (*P <.05; **P <.01; ***P <.001).

| MSCs improve survival and attenuate lung injury in LPS-induced ALI mice
We constructed a stable ALI mouse model via intratracheal injection of 20 mg/kg LPS. Mouse MSCs were characterized as shown in Figure S1. The ALI mice were administered LPS; after 4 hours, MSCs or PBS was given orotracheally ( Figure 1A). To determine how MSCs ameliorate LPS-induced ALI, mice were divided into three groups: PBS, LPS/PBS and LPS/MSC groups. Twelve days after LPS exposure, the survival rate in the LPS/MSC group was significantly greater than that in the LPS/PBS group (75% vs. 40%; P <.05) ( Figure 1B). The degree of lung injury was assessed based on the lung histology ( Figure 1C), BALF total cell count ( Figure 1D) and BALF protein concentration ( Figure 1E). Diffuse alveolar and interstitial in- concentration in BALF were significantly reduced, by MSCs, confirming that these cells attenuate lung injury in LPS-induced ALI mice ( Figure 1D and 1E).

| MSCs reduce intestinal mucosal injury and improve intestinal barrier integrity
Stimulation of mouse lungs with LPS has been shown to alter microbial diversity and abundance, which plays a vital role in preventing the invasion of pathogenic bacteria and maintaining the immune homeostasis of the gut mucosa as a microbial barrier, 16,36,37

| MSCs attenuate lung and intestinal inflammation caused by LPS
We assayed the levels of IFNγ, TNFα, IL-1α, IL-1β and IL-6 in the lung and small intestine. LPS induced lung inflammation, as evidenced by increased IFNγ, TNFα, IL-1α, IL-1β and IL-6 levels in BALF at days 1 and 3 compared with the PBS group ( Figure 3A).

| Effects of MSC administration on lung and gut immunoregulation
To determine the effects of MSCs on immune responses in the lung and small intestine in ALI, we analysed CD45 + (lymphocyte common antigen) cells in LPS-treated mice. CD8 + T cells in the lungs were analysed by mass cytometry using the reasonable gating strategy.
The populations of CD3 + T cells and Ly6C + CD8 + T cells in lung immune compartments were significantly increased in the LPS/PBS group compared with the PBS group ( Figure 4A). In mice treated with MSCs, the populations of CD3 + T cells and Ly6C + CD8 + T cells were significantly reduced compared with LPS-treated mice at day 7, and F4/80 + CD11b + macrophages were recruited to the injured lung in the LPS/MSC group at day 3 ( Figure 4B). Therefore, MSCs regulate systemic immunity by modulating the CD8 + T-cell response and macrophage recruitment to the injured lung.
Next, we evaluated the numbers and phenotypes of CD45 + cells in small-intestinal IELs and PPs ( Figure 5A). Compared to the PBS group, the numbers of IELs and PPs were decreased in the LPS/PBS group and increased in the LPS/MSC group at day 1 ( Figure 5B). In addition, the population of CD8 + T cells in IELs was drastically increased, but that of CD4 + T and F4/80 + CD11b + macrophages was

| Gut bacterial community composition is altered by MSCs
Lipopolysaccharide-induced lung injury has been shown to alter gut bacterial communities. 12 Thus, we analysed the effects of MSCs To characterize the global differences in microbial communities between groups, principal coordinate analysis (PCoA) was performed on unweighted UniFrac distances and weighted UniFrac distances.
The PCoA plots showed significant separation at days 1 and 3, but not at day 7, between the LPS/PBS and LPS/MSC groups ( Figure 6A), based on weighted UniFrac distances. Furthermore, ANOSIM revealed a significant difference in beta diversity among the groups ( Figure S4A). At day 1, the number of species was decreased in the LPS/PBS compared with the PBS group ( Figure S4B). In addition, the Shannon and Chao1 indices suggested a significantly decreased diversity in the LPS/PBS group ( Figure 6B). In addition, the MSCtreated groups shared more OTUs with the control group than the LPS/PBS group at day 1 ( Figure S4C).
The dominant phyla in all mice were Bacteroidetes, Deferribacteres and Firmicutes (~88-97% combined total relative abundance). Other  Figure S4D. Together, the findings indicate that MSCs restored a healthy intestinal bacterial community composition.

| The impact of gut microbiota depletion on the efficacy of MSCs in ALI mice
The gut bacterial community composition is associated with peripheral immunity homeostasis. 38,39 Therefore, bacterial community dysbiosis may modulate the therapeutic effects of MCSs. We treated C57/BL mice with four broad-spectrum antibiotics (ampicillin, neomycin, metronidazole and vancomycin) (ABX) in drinking water for 3 weeks before LPS injection to deplete the gut microbiota ( Figure 7A). 22%, P <.05) ( Figure 7B), indicating that lung injury was severe during ALI in antibiotic-treated mice and was significantly alleviated after administration of MSCs. Moreover, the survival curve also showed that, compared to the LPS/PBS group, the A-LPS/PBS group had a higher mortality rate (78% vs. 60%, P <.05). Antibiotic administration altered the gut mechanical barrier in mice, which resulted in gut villus separation and gut mucosal damage ( Figure 7C). These changes were evident in the A-LPS/PBS group compared with the LPS/PBS group.
The histopathological changes also showed the same trend in the lung; that is, compared to the LPS/PBS group, the lung of the A-LPS/ PBS group exhibited a more severely distorted structure ( Figure 7C).
A large number of inflammatory cell infiltrations were observed in the pulmonary interstitium, and many red blood cells were observed in the lung vasculature, while leakage occurred in the bronchial lumen at day 3 and day 7 in the A-LPS/PBS group.
The number of IELs decreased significantly at day 1 in the A-LPS/ PBS group and was similar to those in the other groups ( Figure 7D).
The CD3 + T-cell population, in particular CD8 + T, CD8ααTCRγδ + T cells, was dramatically altered ( Figure 7E). In addition, CD8 + T-cell populations among IELs were significantly increased, and those of F4/80 + CD11b + macrophages were reduced, in the A-LPS/PBS group. The percentages of CD8 + T cells and CD8ααTCRγδ + T cells were lower in the A-LPS/MSC group, similar to mice that were not treated with antibiotics ( Figure 7F).
Next, we analysed IELs from the LPS/MSC and A-LPS/MSC groups at three time points. The effect of MSCs on T-cell populations and macrophages was more apparent in antibiotic-treated mice compared with MSCs alone ( Figure 7G). Therefore, the immunomodulatory effects of MSCs on ALI were mediated by changes in bacterial community composition in the gut.

| D ISCUSS I ON
There is substantial evidence on the immunomodulatory effects of MSCs in ALI, 40 32 In this study, the therapeutic effects of MSCs were found to be mediated by reduced infiltration of Ly6C + CD8 + T cells, in line with the lower IFNγ level in the LPS/ MSC group, since excessive IFNγ synthesized by large numbers of CD8 + T cells can aggravate tissue injury. 50 Moreover, enhanced macrophage phagocytosis in MSC-treated mice contributed to repair of injured tissue, which is consistent with a prior report. 48 The gastrointestinal mucosal immune system is important for maintaining host immune homeostasis. 51 IELs regulate epithelial cell function; most express the CD8αα homodimer, and 40 to 60% are TCRγδ + T cells. 52 CD8 + T cells of IELs are similar to the effector memory CD8 + T cells that maintain the intestinal mucosal barrier. 53 Inhibition of CD8 + T cells has been shown to regulate immune responses and ameliorate disease in pulmonary inflammation. 54 We analysed the percentage of CD8 + T and CD8ααTCRγδ + T in IELs and PPs. CD8 + T-cell infiltration of the intestinal mucosa was decreased by MSCs, ameliorating injury, which is consistent with the changes in the lung. Therefore, MSCs have an immunosuppressive effect in the lung and gut in ALI.
MSCs may also promote intestinal healing by altering the intestinal bacterial community composition. Their interactions with the microbiota influence the functions of MSCs, including immunomodulation. 55 In this study, the changes in the bacterial community were reversed by MSCs. For example, MSCs increased the Firmicutes and Bacteroidetes abundance. Lachnospiraceae, in the phylum Firmicutes, attenuated colitis in mice. 56 MSCs improved intestinal healing by increasing the gut microbial community diversity.
The complex relationship between intestinal bacterial communities and peripheral T-cell immunity during infection is of interest. 57,58 However, further studies are needed to determine how the intestinal microflora or activated immune response affects ALI in the complex intestinal microecological environment. We found that when antibiotics depleted the bacterial communities in ALI mice, the imbalance in intestinal microbiota aggravated lung and intestinal damage and immune response. Importantly, the damage to the lung and intestine structure was reduced by the MSC treatment. In addition, compared with the LPS group, our study demonstrated that the intestinal microbes contributed to the local host defence against pathogenic stimulus such as LPS. Immunomodulation is an important aspect of MSC therapy, so we analysed the T-cell response of IELs over time. Interestingly, the results in antibiotic-treated mice were similar to those in mice that were not treated with antibiotics.
MSCs reduced the percentage of CD8 + cytotoxic T cells to alleviate inflammation in mice, which is consistent with a previous study. 59 F I G U R E 7 Effects of MSCs on antibiotic-treated ALI mice. (A) Treatment with four broad-spectrum antibiotics (ABX) in drinking water for 3 weeks before LPS injection in C57/BL mice to deplete the gut microbiota. Also shown is the treatment schedule for ABX water feeding, LPS injection and MSCs or PBS intratracheal instillation. (B) Kaplan-Meier survival curves of LPS-induced ALI mice after ABX treatment. Blue line and red line are as shown in Figure 1B; purple line, C57/BL6 mice induced by LPS without MSCs (A-LPS/PBS group, n = 18); orange line, LPS-induced mice treated with MSCs (A-LPS/MSC group, n = 18). *P <.05. (C) Representative images of histological changes in the ileum and lung assessed by H&E staining. Magnifications 20× (scale bar = 100 μm). On days 3 and 7 after LPS treatment, the lung of the A-LPS/PBS group exhibited a more severely distorted structure. A large number of inflammatory cell infiltrations were observed in the pulmonary interstitium, and many red blood cells were observed in the lung vasculature, while leakage occurred in the bronchial lumen. A-LPS/PBS group also showed gut villus separation and gut mucosal damage. (D) Numbers of IELs over time in antibiotic-treated ALI mice with or without MSCs transplantation. Each symbol indicates an individual mouse (mean ± SEM, n = 3). (E) Representative flow cytometry plots of CD8 + T cells in IELs showing the effects of MSCs on antibiotic-treated mice at days 1 and 7. (F) Flow cytometry analyses of T cells and macrophages in IELs of ALI mice in the A-PBS, A-LPS/PBS and A-LPS/MSC groups. Each symbol indicates an individual mouse (mean ± SEM, n = 3). (G) Flow cytometry results of the LPS/MSC and A-LPS/MSC groups at three time points (mean ± SEM, n = 3). *P <.05, **P <.01, ***P <.001 by Student's t-test. ABX/A: mice treated with antibiotics Indeed, the percentages of CD8 + T and CD8TααCRγδ + T cells in our ALI mice treated with MSCs were dramatically decreased. The antiinflammatory activity of MSCs likely promotes recovery of ALI by modulating the synthesis of inflammatory cytokines by T cells and macrophages. 60,61 The dysregulation of the intestinal microflora increased the immune response to LPS, which may aggravate immune damage to the lung, while MSCs have the potential to serve as a therapeutic approach for lung injury due to their immunosuppressive activity.

ACK N OWLED G EM ENTS
We would like to thank the staff of the Laboratory Animal Centre of Zhejiang University, China, for their support with mouse feeding and also thank Dr Yanyuan Li of the Department of Pathology at the First Affiliated Hospital of Zhejiang University for her kind review of the histopathology.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R CO NTR I B UTI O N S
YX and HC performed the experiments. YX, JZ, BF, XL and FL analysed the data. JZ, JL, XS and QP designed the project. YX, JY, HC and YZ analysed the data and wrote the manuscript. HC and LL designed the project and revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All supporting data are included in the article and its additional files.