The influence of asthmatic inflammation and house dust mite (HDM) exposure on abundance, immune-modulatory potential, and differentiation capacity of the lung-resident mesenchymal stem cells (lrMSCs)

Abstract

Background: Tissue-resident mesenchymal stem cells, also known as mesenchymal stromal cells (MSCs), play a crucial role in maintaining tissue homeostasis and repair. However, their function in chronic inflammatory diseases, such as asthma, remains elusive.

Aim: Here, we aimed to assess the influence of house dust mite (HDM)-induced asthmatic inflammation on the numbers and function of lung resident (lr)MSCs.

Methods: Experimental asthma was induced in female C57BL6/cmdb mice via intranasal HDM administration. LrMSCs were isolated, expanded, and characterized by flow cytometry and differentiation assays. Human adipose tissue-derived (hAD)MSCs were isolated and stimulated with HDM, LPS, or cytokines. Co-culture experiments with peripheral blood mononuclear cells (PBMCs) assessed immunomodulatory potential. Gene expression, cytokine levels, and T-cell proliferation were analyzed.

Results: Here, we showed that asthmatic lung inflammation significantly reduces the number of lrMSCs. More importantly, remaining lrMSCs showed impaired differentiation potential and lacked immunomodulatory functions. Furthermore, we found that exposure of hAD-MSCs to HDM and LPS similarly led to marked inhibition of differentiation potential and suppression of immunosuppressive activities. Notably, this inhibitory effect persisted despite the presence of pro-inflammatory cytokines released by PBMCs in response to LPS and HDM. Furthermore, we showed that inflammatory signaling alone, in the absence of direct LPS and HDM exposure, significantly reduces growth factor-induced adipogenesis and osteogenesis.

Conclusions: Taken together, our findings indicate that asthmatic inflammation not only reduces the number of lrMSCs but also impairs their function, potentially exacerbating disease progression by limiting their immunoregulatory role.

Keywords: Asthmatic lung inflammation; Immunomodulation; Lung resident MSC; MSC; MSC differentiation; MSC phenotype; Mesenchymal stem cells.

Fig. 1

The number of lrMSCs is decreased in asthmatic mice. A Schematic representation of the inflammatory phenotype induction in female C57BL6/cmdb mice via intranasal administration of house dust mite (HDM) extract (100 µg of protein) over three weeks. The exposure protocol included a cyclic pattern of five consecutive days of stimulation (black) followed by a two-day break (blue). The control group (Veh) received 0.9% sodium chloride (NaCl) as a vehicle. Mice were euthanized 72 h after the final HDM exposure (green), and lung tissue was collected for analysis: the upper left lobes were processed for histological staining, while the right lobes were used for flow cytometry. B Representative lung tissue Sects. (3 µm) demonstrate increased inflammatory cell infiltration, as visualized by hematoxylin and eosin (H&E) staining, and enhanced mucus production, as indicated by periodic acid-schiff (PAS) staining, in HDM-exposed mice compared to controls. C Gating strategy for lung-resident mesenchymal stromal cells (lrMSCs), identified within the CD45⁻ MHC⁻ population as CD29⁺ and Sca-1⁺ cells. D Summary of quantitative analysis of lrMSCs numbers in HDM-exposed mice compared to controls. E Summary of quantitative analysis of bone marrow (BM)-MSCs numbers in HDM-exposed mice compared to controls. Statistical significance was determined using the Wilcoxon test (*p < 0.05; n = 5)

Fig. 2

Impaired differentiation and immunosuppressive capacity of lrMSCs isolated from asthmatic lungs A Schematic representation experimental design. Lung resident mesenchymal stromal cells (lrMSCs) were isolated from mice, and functional properties were assessed. B Representative confocal stainings of lrMSCs differentiation. Differentiation potential was assessed by inducing adipogenic and osteogenic differentiation, followed by immunofluorescence staining for adipocyte marker, fatty acid binding protein 4 (FABP4, green) and osteoblast marker, Osteopontin (green). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The percentage of FABP4 + and Osteopontin + cells was calculated as the proportion of marker-positive cells relative to the total cell count. A summary of analyses is presented on the left side. Statistical significance was determined using the Wilcoxon test (**p < 0.01, ****p < 0.0001; n = 3, each with 3 technical replicates). C Representative gating strategy for T-cell analysis in a T-cell proliferation assay. D Summary of analysis of the immunoregulatory potential of lrMSCs in T-cell proliferation assay. Statistical significance was determined using the Wilcoxon test (*p < 0.05; n = 3)

Fig. 3

HDM and LPS stimulation of hAD-MSCs impairs their functional properties. A Schematic representation of the experimental design of an in-vitro model of direct 24 h hAD-MSCs stimulation with lipopolysaccharide (LPS, 1 μg/ml), house dust mite (HDM, 14.2 μg/ml) or 0.9% sodium chloride (NaCl) as a control. B Representative confocal pictures at the end of adipogenesis (fatty acid binding protein 4 (FABP4) +, green; after 14 days of differentiation), osteogenesis (Osteocalcin +, green; after 21 days), and chondrogenesis (Aggrecan +, green; after 21 days). And 4’,6-diamidino-2-phenylindole (DAPI) was used for nuclear stainings. The percentage of positive cells (adipocytes, osteoblasts) was determined by dividing the number of positive cells by the total cell count. The percentage of positive chondrocytes was evaluated using the mean fluorescent intensity (MFI) method. Image analysis was performed using ImageJ software. Wilcoxon test (**p < 0.01, ***p < 0.001, ****p < 0.0001; n = 3, each with 3 technical replicates). C Summary of analysis of the effect of hAD-MSCs exposure to HDM, LPS, and NaCl on T-cell proliferation. Wilcoxon test (**p < 0.01; n = 3). Summary of D IL-6 relative gene expression, expressed as 2^−ΔΔ.^CT and E IL-6 levels in cell culture supernatant upon LPS, HDM, or NaCl stimulation. Wilcoxon test (*p < 0.05, **p < 0.01; n = 6)

Fig. 4

Exposure of hAD-MSCs to conditioned media from HDM or LPS stimulation does not induce IL-10 expression. A Schematic representation of the experimental design. Experimental procedure for human adipose tissue-derived mesenchymal stromal cells (hAD-MSCs) priming. Peripheral blood mononuclear cells (PBMCs) isolated from human buffy coat were stimulated with lipopolysaccharide (LPS, 1 μg/ml) or house dust mite (HDM, 14.2 μg/ml) for 24 h or up to 120 h. Next, 0.9% sodium chloride (NaCl)–treated PBMCs served as the control group. hAD-MSCs were primed with a conditioned medium derived from the PBMCs after stimulation. B Representative flow cytometry graphs of cell viability were analyzed with Annexin V (FITC) and propidium iodide (PI). C Summary of PBMCs viability analyses. The mean data were normalized to the control group and presented as a percentage ratio. D Summary of 24 h analyses of IL-6 and IL-10 levels in cell culture supernatants from conditioned media and media upon hAD-MSCs exposure to conditioned media (primed hAD-MSCs, pMSCs). E Summary of hAD-MSCs viability analyses upon stimulation with conditioned PBMCs media. The mean data were normalized to the control group and presented as a percentage ratio. F Analysis of IL-6 and IL-10 levels in supernatants from pMSCs collected after 48-120 h was measured in supernatants using ELISA. Wilcoxon test was used in all analyses (*p < 0.05; n = 6)

Fig. 5

Impaired potential for adipogenesis and osteogenesis of inflammatory cytokine-primed MSCs. A Schematic representation of the experimental design of human adipose tissue-derived mesenchymal stromal cells (hAD-MSCs) after stimulation with IL-1β, IFN-γ, TNF, and cytokine mix was subjected to differentiation and gene expression analysis. B Representative confocal images of hAD-MSCs differentiated into adipocytes (fatty acid binding protein 4 (FABP4 +)), osteoblasts (Osteocalcin +), and chondrocytes (Aggrecan +) after a 24 h priming with IL-1β, IFN-γ, TNF, and cytokine mix. NaCl-primed cells were used as a control (Veh). C The summary of quantification analyzes the proportion of adipocytes, osteoblasts, and chondrocytes. Adipocytes and osteoblasts were analyzed 14 days after adipogenic differentiation and 21 days after osteogenic differentiation. The cell number was calculated by dividing the number of positively stained cells by the total count. The percentage of positive chondrocytes (day 21 of chondrogenesis differentiation) was assessed by analyzing mean fluorescence intensity (MFI). Image analysis was conducted using ImageJ software. Statistical significance was determined using the Wilcoxon test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n = 3, each with 3 technical replicates). D Transcripts Per Million (TPM) differences in transcriptomic factors associated with adipogenesis (CCAAT/Enhancer-Binding Protein Alpha (CEBPA), CCAAT/Enhancer Binding Protein Beta (CEBPB), poly (ADP-ribose) glycohydrolase (PARG)), E osteogenesis (runt-related transcription factor 2 (RUNX2)), and E, F chondrogenesis (RUNX2, SRY-related HMG-box gene (SOX)5,9). Wilcoxon test (*p < 0.05, **p < 0.01; n = 5)