Placental mesenchymal stem cells boost M2 alveolar over M1 bone marrow macrophages via IL-1β in Klebsiella-mediated acute respiratory distress syndrome

Abstract

Rationale: Acute respiratory distress syndrome (ARDS) is a lethal complication of severe bacterial pneumonia due to the inability to dampen overexuberant immune responses without compromising pathogen clearance. Both of these processes involve tissue-resident and bone marrow (BM)-recruited macrophage (MΦ) populations which can be polarised to have divergent functions. Surprisingly, despite the known immunomodulatory properties of mesenchymal stem cells (MSCs), simultaneous interactions with tissue-resident and recruited BMMΦ populations are largely unexplored.

Objectives: We assessed the therapeutic use of human placental MSCs (PMSCs) in severe bacterial pneumonia with elucidation of the roles of resident alveolar MΦs (AMΦs) and BMMΦs.

Methods: We developed a lethal, murine pneumonia model using intratracheal infection of a clinically relevant Klebsiella pneumoniae (KP) strain with subsequent intravenous human PMSC treatment. Pulmonary AMΦ and recruited BMMΦ analyses, histological evaluation, bacterial clearance and mice survival were assessed. To elucidate the role of resident AMΦs in improving outcome, we performed AMΦ depletion in the KP-pneumonia model with intratracheal clodronate pretreatment.

Measurements and main results: Human PMSC treatment decreased tissue injury and improved survival of severe KP-pneumonia mice by decreasing the presence and function of recruited M1 BMMΦ while preserving M2 AMΦs and enhancing their antibacterial functions. Interestingly, PMSC therapy failed to rescue AMΦ-depleted mice with KP pneumonia, and PMSC-secreted IL-1β was identified as critical in increasing AMΦ antibacterial activities to significantly improve pathogen clearance-especially bacteraemia-and survival.

Conclusions: Human PMSC treatment preferentially rescued resident M2 AMΦs over recruited M1 BMMΦs with overall M2 polarisation to improve KP-related ARDS survival.

Keywords: ARDS; bacterial Infection; macrophage biology; respiratory infection.

Figure 1

Pulmonary infection of a clinically isolated Klebsiella pneumonia (KP)-serotype K2 dramatically decreases resident alveolar macrophages (AMΦs) while increasing recruited bone marrow (BM) MΦs in the lungs. (A) Principal component analysis (PCA) of transcriptomic profiles (National Center for Biotechnology Information-Gene Expression Omnibus or NCBI-GEO database: GSE121970) of KP-infected (n=5) compared to uninfected (n=5) murine lung tissues. The first principal component (PC1) accounts for the largest data variance at 21.3%, while data variance was 18.1% for PC2, and 10.4% for PC3. (B) Metascape pathway analysis for MΦ-related pathways using transcriptomic data of KP-infected murine lung tissue compared with uninfected control, with pathways coloured according to p values. (C) Representative data of t-distributed stochastic neighbor embedding (t-SNE) plots for analyses of changes in MΦs harvested from lung tissues of C57BL/6J mice after intratracheal injection of phosphate buffered saline (PBS; no infection), or infection with sublethal dose (5×10⁶ CFUs) of K2 KP for 2 or 4 hours (h2 or h4). Single-cell suspensions derived from lung lobes were stained with anti-CD45, anti-CD11b and anti-F4/80 for flow cytometric analysis, with gating on CD45⁺F4/80⁺ cells for analysis of MΦ populations using tSNE-based algorithm. G1 and G2 were then manually gated on the t-SNE plot by signal profiles. (D, E) Pooled data for G1 and G2 as defined in (C) (n=3 for each group). (F) Histogram graph for CD11b expression levels in G1 representing CD11b^high MΦs or and G2 representing CD11b^low MΦs as defined in (C). (G) Representative data for frequency analysis of recruited BMMΦs and resident AMΦs in lung lobes taken from uninfected (PBS) and sublethally KP-infected mice sacrificed at h2 and h4 as assessed by flow cytometry. Immune cells harvested from lung tissues were stained with anti-CD45, anti-CD11b and anti-F4/80. Gating for CD45⁺ cells was first performed, with subsequent frequency analysis for CD11b^highF4/80⁺ and CD11b^lowF4/80⁺ to identify for BMMΦs and AMΦs, respectively. (H, I) Pooled data for frequency and absolute numbers, respectively, of BMMΦs (□) and AMΦs (■) in lung lobes as assessed by flow cytometry, with gating for CD45⁺ cells first and then analysis for cell numbers of CD11b^highF4/80⁺ and CD11b^lowF4/80⁺ identified as BMMΦs and AMΦs, respectively (n=6 for each group). Data are shown as mean±SD.

Figure 2

Human placenta mesenchymal stem cells (PMSCs) prevent bone marrow MΦ (BMMΦ) recruitment but preserve alveolar MΦ (AMΦ) population in Klebsiella pneumoniae (KP)-infected lungs. (A) Pathway analyses of transcriptomic profiles obtained from KP-infected versus uninfected lung lobes (NCBI-GEO database: GSE121970) as performed with Molecular Activation Prediction (MAP) tool in Ingenuity Pathway Analysis (IPA), for prediction of mechanisms involved in migration of recruited BMMΦs or quantity of resident AMΦs during pulmonary KP infection. IPA showed regulatory relationships between downregulated (green) and upregulated (red) proteins; the MAP tool showed that ‘migration of BMMΦs’ and ‘quantity of AMΦs’ are upregulated (orange) through positively regulated downstream molecules (orange lines) or inconsistent findings on some mediators (yellow lines) after occurrence of KP pneumonia (red). (B) Enrichment of immune-related pathways as assessed in transcriptomic data of tumour necrosis factor (TNF)-α-treated BMMΦs versus control BMMΦs (NCBI-GEO database: GSE160163) using Metascape analysis and coloured by p values. (C) Assessment of ex vivo lung inflammation with detection of TNF-α in non-infected and KP-infected lung tissues after phosphate buffered saline (PBS) and PMSC treatment. Mice were intratracheally infected with 5×10⁶ CFUs of K2 KP for 2 hours, and then sacrificed for extraction of lung immune cells which were ex vivo co-cultured with human PMSCs at the ratio of 10:1 for 2 hours further and supernatants collected for TNF-α detection (n=10 for each group). (D) Representative data for frequency analysis of recruited BMMΦs and resident AMΦs in lung lobes as assessed with flow cytometry. C57BL/6J mice were intratracheally injected with 5×10⁶ CFUs of K2 KP followed by intravenous administration of PBS or 3×10⁵ PMSCs 2 hours later. Single-cell suspensions harvested from lung tissues of non-infected mice as well as infected mice injected with PBS or PMSCs were stained with anti-CD45, anti-CD11b and anti-F4/80 for flow cytometric analysis. Gating for CD45⁺ cells was first performed, with subsequent frequency analysis for CD11b^highF4/80⁺ BMMΦs and CD11b^lowF4/80⁺ AMΦs. (E, F) Pooled data for analyses of frequency and absolute counts of recruited BMMΦs in lung lobes of each experimental group, respectively (n=8 for each group). (G, representative data; H, pooled data) Relative CD11b expression levels of recruited BMMΦs in lung lobes of each experimental group by flow cytometric analysis (n=8 for each group). (I, J) Pooled data for analyses of frequency and absolute number of resident AMΦs in lung lobes of each experimental group, respectively (n=8 for each group). Data are shown as mean±SD.

Figure 3

Human placental mesenchymal stem cells (PMSCs) decrease pulmonary inflammation and tissue injury, as well as significantly increase survival of mice with Klebsiella pneumoniae (KP) pneumonia. (A, B) Assessment of bacterial load within lung homogenate (A; n=7 for each group) and peripheral blood (B; n=6 for each group). Mice were intratracheally injected with a 5×10⁶ CFUs of K2 KP followed by intravenous administration of phosphate buffered saline (PBS) or 3×10⁵ PMSCs 2 hours later, with lung lobes excised and peripheral blood collected for CFU calculation. (C, representative data; D, pooled data) H&E staining of histological sections of lung tissues from KP-infected mice after PBS or PMSC treatment (n=6 for each group). Upper panel (a–d): scale bar, 200 µm. Middle (e–h) and bottom panels (i–l), magnifications of lung parenchyma and airways, respectively: scale bar, 100 µm. (E) Kaplan-Meier survival analysis of infected mice treated with or without PMSC treatment. Wild-type C57BL/6J mice were intratracheally injected with a lethal dose (5×10⁷ CFUs) of K2 KP followed by intravenous administration of PBS or 3×10⁵ PMSCs 2 hours later. Survival was observed for 12 days (n=7 for each group). Data are shown as mean±SD.

Figure 4

Human PMSCs modulate both bone marrow MΦs (BMMΦs) and alveolar MΦs (AMΦs) to M2 polarisation in mice with Klebsiella pneumoniae (KP) pneumonia. (A, representative data; B, pooled data) Relative inducible nitric oxide synthetase (iNOS) expression levels of CD11b^highF4/80⁺ BMMΦs in lung lobes of each experimental group as assessed by flow cytometry (n=10 for each group). (C, representative data; D, pooled data) Relative arginase-1 (Arg1) expression levels of CD11b^highF4/80⁺ BMMΦs in lung lobes of each experimental group as assessed by flow cytometry (n=10 for each group). (E, representative data; F, pooled data) Relative iNOS expression levels of CD11b^lowF4/80⁺ AMΦs in lung lobes of each experimental group as assessed by flow cytometry (n=10 for each group). (G, representative data; H, pooled data) Relative Arg1 expression levels of CD11b^lowF4/80⁺ AMΦs in lung lobes of each experimental group as assessed by flow cytometry (n=10 for each group). Data are shown as mean±SD.

Figure 5

Human placental mesenchymal stem cells (PMSCs) reduce the population and function of M1 bone marrow MΦs (BMMΦs) while preserving M2 alveolar MΦs (AMΦ) population in mice with Klebsiella pneumoniae (KP) pneumonia. (A, pooled data; B, representative data) Population analyses for M1 and M2 BMMΦs as well as M1 and M2 AMΦs with PhenoGraft on the MΦ-based t-distributed stochastic neighbor embedding (t-SNE) map. Single-cell suspensions harvested from lung tissues of non-infected mice as well as infected mice injected with phosphate buffered saline (PBS) or PMSCs were stained with anti-CD45, anti-CD11b, anti-F4/80 and anti-CD206, assessed by flow cytometry and analysed with t-SNE-based algorithm. Gating for CD45⁺ cells was first performed, and then CD206^-CD11b^highF4/80⁺ M1 BMMΦs and CD206⁺CD11b^highF4/80⁺ M2 BMMΦs as well as CD206^-CD11b^lowF4/80⁺ M1 AMΦs and CD206⁺CD11b^lowF4/80⁺ M2 AMΦs were gated on the two t-SNE dimensions. (C) Representative data for frequency analyses of M1 and M2 populations in BMMΦs or AMΦs from lung lobes as assessed with flow cytometry. Gating for CD45⁺F4/80⁺ cells was first performed, with subsequent analyses for CD11b and CD206 signals. (D, E) Pooled data for analyses of frequency and absolute number, respectively, of CD206^-CD11b^highF4/80⁺ M1 BMMΦs (n=10 for each group). (F, G) Pooled data for analyses of frequency and absolute number, respectively, of CD206⁺CD11b^highF4/80⁺ M2 BMMΦs (n=10 for each group). (H, I) Pooled data for analyses of frequency and absolute number, respectively, of CD206^-CD11b^lowF4/80⁺ M1 AMΦs (n=10 for each group). (J, K) Pooled data for analyses of frequency and absolute number, respectively, of CD206⁺CD11b^lowF4/80⁺ M2 AMΦs (n=10 for each group). (L, representative data; M, pooled data) Relative TNF-α expression levels of M1 BMMΦs as assessed in CD45⁺CD11b^highF4/80⁺CD206^-. Single-cell suspensions harvested from lung tissues, followed by intracellular staining of tumour necrosis factor (TNF)-α (n=10 for each group). (N, representative data; O, pooled data) Relative TNF-α expression levels of M1 AMΦs as assessed in CD45⁺CD11b^lowF4/80⁺CD206^- cells harvested from lung tissues, followed by intracellular staining of TNF-α (n=10 for each group). Data are shown as mean±SD.

Figure 6

Human placental mesenchymal stem cells (PMSCs) enhanced multiple antibacterial functions in alveolar MΦs (AMΦs) but not bone marrow MΦs (BMMΦs) to significantly improve bacterial clearance and survival in mice with Klebsiella pneumoniae (KP) pneumonia which were abrogated after AMΦ depletion. (A, representative data; B, pooled data) The capability of KP phagocytosis in BMMΦs harvested from lung tissues co-cultured in phosphate buffered saline (PBS) or with PMSCs (n=10 for each group) for 2 hours as assessed with subsequent addition of FITC-labelled KP for 30 min at 37°C. Phagocytic capability of BMMΦs was measured by detecting for the frequency of FITC⁺ cells in CD11b^highF4/80⁺. (C, representative data; D, pooled data) The capability of KP phagocytosis in AMΦs harvested from lung tissues co-cultured in PBS or with PMSCs (n=10 for each group) for 2 hours as assessed with subsequent addition of FITC-labelled KP for 30 min at 37°C. Phagocytic capability of AMΦs was measured by detecting for the frequency of FITC⁺ cells in CD11b^lowF4/80⁺. (E, representative data; F, pooled data) Assessment of BMMΦ respiratory burst as measured by reactive oxygen species (ROS) production of H₂O₂ in each experimental group. Isolated cells from lung tissues were incubated with 10 µM of 2′,7′-dichlorofluorescein diacetate (DCFDA) for 30 min, and then analysed by flow cytometry to assess ROS levels in CD45⁺CD11b^highF4/80⁺ BMMΦs (n=8 for each group). (G, representative data; H, pooled data) Assessment of AMΦ respiratory burst as measured by ROS production of H₂O₂ in each experimental group. Isolated cells from lung tissues were incubated with 10 µM DCFDA for 30 min, and then analysed by flow cytometry to assess ROS levels in CD45⁺CD11b^lowF4/80⁺ AMΦs (n=8 for each group). (I) Representative data for frequency analyses of CD11b^highF4/80⁺ BMMΦs and CD11b^lowF4/80⁺ AMΦs in lung lobes treated with PBS or clodronate as assessed with flow cytometry. C57BL/6J mice were intratracheally injected with 100 μL of PBS or 15 mM clodronate solution. After 2 days, single-cell suspensions harvested from lung tissues were stained with anti-CD45, anti-CD11b and anti-F4/80 for flow cytometric analyses. Gating for CD45⁺ cells was first performed, with subsequent frequency analysis for CD11b^highF4/80⁺ BMMΦs and CD11b^lowF4/80⁺ AMΦs. (J, K) Pooled data for analyses of frequency and absolute number of CD11b^highF4/80⁺ BMMΦs, respectively (n=6 for each group). (L, M) Pooled data for analyses of frequency and absolute number of CD11b^lowF4/80⁺ AMΦs, respectively (n=6 for each group). (N) Assessment of bacterial load within lung homogenate in clodronate-pretreated mice infected with 5×10⁶ CFU of K2 KP. Clodronate-pretreated mice were then intratracheally infected with sublethal dose (5×10⁶ CFU) of K2 KP followed by intravenous administration of PBS or 3×10⁵ PMSCs 2 hours later. Lung lobes were collected for detection of bacterial burden (n=6 for each group). (O) Kaplan-Meier survival analysis of clodronate-pretreated/AMΦ-depleted mice with K2 KP infection (5×10⁶ CFU) after PBS or PMSC treatment. Survival was observed for 12 days (n=8 for each group). Data are shown as mean±SD.

Figure 7

Human placental mesenchymal stem cell (PMSC)-secreted interleukin (IL)-1β prominently increase alveolar MΦ (AMΦ) antibacterial functions to significantly improve Klebsiella pneumoniae (KP) pneumonia disease outcome. (A) Pathway analysis on the process of AMΦ activation with IL1B upregulation as performed by Molecular Activation Prediction (MAP) tool in Ingenuity Pathway Analysis (IPA). The MAP tool showed that ‘activation of AMΦs’ is upregulated (orange) through positively regulated downstream molecules (orange lines) after upregulation of IL1B (red). (B, representative data; C, pooed data) Assessment of bacterial phagocytic capacity of AMΦs co-cultured for 2 hours with phosphate buffered saline (PBS) or PMSCs after knockdown with short hairpin RNA to luciferase (shLuc; negative control) or IL1B (shIL1B) (n=6 for each group). (D, representative data; E, pooled data) Assessment of respiratory burst in AMΦ extracted from lungs of KP-infected mice after intravenous injection of PBS, shLuc-PMSCs or shIL1B-PMSCs (n=6 for each group). (F, G) Assessment of bacterial load within lung homogenate (F; n=7 in each group) and peripheral blood (G; n=14 in each group) of KP-infected mice, respectively, of KP-infected mice after intravenous injection of PBS, shLuc-PMSCs or shIL1B-PMSCs. (H) Kaplan-Meier survival analysis of intravenous treatment of PBS, shLuc-PMSCs or shIL1B-PMSCs of mice intratracheally infected with 5×10⁷ CFU of K2 KP. Survival was observed for 12 days (n=13). Data are shown as mean±SD. (F) Summary: Human PMSCs differentially enhance resident AMΦ function via IL-1β while suppressing recruited BMMΦ inflammatory function, leading to decreased local as well as systemic inflammation and significantly improving survival of mice with severe, lethal KP pneumonia.