Gastrointestinal microbes interact with canine adipose-derived mesenchymal stem cells in vitro and enhance immunomodulatory functions

Abstract

Mesenchymal stem cells (MSCs) are somatic, multipotent stromal cells with potent immunomodulatory and regenerative properties. Although MSCs have pattern recognition receptors and are modulated by Toll-like receptor ligands, MSC-microbial interactions are poorly defined. The objectives of this study were to determine the effect of bacterial association on MSC function. We hypothesized that gastrointestinal bacteria associate with MSCs and alter their immunomodulatory properties. The effect of MSC-microbial interactions on MSC morphology, viability, proliferation, migration, and immunomodulatory functions was investigated. MSCs associated with a remarkable array of enteric pathogens and commensal bacteria. MSC interactions with two model organisms, the pathogen Salmonella typhimurium and the probiotic Lactobacillus acidophilus, were further investigated. While ST readily invaded MSCs, LB adhered to the MSC plasma membrane. Neither microbe induced MSC death, degeneration, or diminished proliferation. Microbial association did not upregulate MHC-II, CD80/86, or CD1 expression. MSC-microbial interaction significantly increased transcription of key immunomodulatory genes, including COX2, IL6, and IL8, coupled with significantly increased prostaglandin E2 (PGE2), interleukin (IL)6, and IL8 secretion. MSC-ST coincubation resulted in increased MSC expression of CD54, and significant augmentation of MSC inhibition of mitogen-induced T-cell proliferation. T-cell proliferation was partially restored when PGE2 secretion was blocked from ST-primed MSCs. MSC-microbe interactions have a profound effect on MSC function and may be pivotal in a variety of clinical settings where MSCs are being explored as potential therapeutics in the context of microbial communities, such as Crohn's disease, chronic nonhealing wounds, and sepsis.

FIG. 1.

Canine mesenchymal stem cells (MSCs) are susceptible to microbial association in vitro. Adipose-derived MSCs were coincubated with intestinal bacteria and adhesion/invasion was quantified using the gentamicin protection assay. Bacteria used in (A) include Salmonella enterica ssp enterica serotype Typhimurium 14028S, LT2, and a Type III secretion system mutant (ΔInvA); Salmonella enterica ssp enterica serotype Enteritidis (BCW_4673 and BCW_1342); Salmonella enterica ssp enterica serotype Heidelberg (ATCC 8326 and BCW_89); Salmonella enterica ssp enterica serotype Newport (BCW_1378); and Salmonella enterica ssp enterica serotype Saint Paul (BCW_88), Lactobacillus acidophilus NCFM, Lactococcus lactis, Listeria monocytogenes EGDe. Microbe-specific patterns of MSC invasion were observed (A). Selected Salmonella serotypes demonstrated differential ability to associate and localize within MSCs (A). The morphology of ST (B–D) and LB (E–G) MSC interaction was further defined via light (B, E diff-quick,×600) and TEM (C, D and F, G lead citrate). Note massive intracellular proliferation of the ST with no overt ultrastructure evidence of degeneration such as mitochondrial swelling or chromatin degeneration (C, scale bar=5 μm; D, scale bar=2 μm). Note that most LB organisms are extracellularly and are often closely associated with the plasma membrane (G, scale bar=2 μm; F, scale bar=500 nm). Color images available online at www.liebertpub.com/scd

FIG. 2.

Microbe-MSC interaction effects on basic MSC functions. Coincubation of canine MSCs with ST or LB did not affect MSC viability (A) and proliferation rate (B). MSC migration was inhibited by ST (C). MSCs were coincubated with bacteria cultured in complete media for 24 h (A) and 72 h (B) or plated onto Matrigel^®-coated transwell insert and were let to migrate for 22 h toward complete growth media (C). At the appropriate time, cells were harvested and stained with 7-amino-actinomycin D (7-AAD) (viability) or counted with Trucount tubes (proliferation) and read on a flow cytometer. All experiments were repeated with five different MSC lines. Neither bacteria induced cell death or altered MSC proliferation (A, B). ST inhibited migration toward a chemotactic gradient (C) compared with control untreated MSCs (*P<0.01) and compared with LB-treated MSCs (P<0.05). Migration of LB-treated MSCs was not different from control MSCs.

FIG. 3.

MSCs are activated by intestinal bacteria and upregulate transcription, translation, and cell surface expression of key immunomodulatory genes and mediators, respectively. MSCs were coincubated with ST and LB per protocol prior to cell lysis, RNA extraction, and qPCR. For inflammatory mediator determination, MSCs were further cultured for 4 days, media were harvested, and specific cytokine was determined via ELISA. For cell immunophenotyping experiments, MSCs were cultured for 24 h post-microbial coincubation protocol prior to imunophenotype determination via flow cytometry. Initial gene expression screening experiments (A) were done with one MSC line plated in triplicates. Both organisms induced increased expression of peroxisome proliferator activator receptor gamma (PPARγ), interleukin (IL)6, and IL8 while cyclooxygenase 2 (COX2) and hepatocyte growth factor (HGF) transcription were induced by ST, but not by LB. Lipopolysaccharide (LPS) treatment mostly mirrored ST treatment. Nonetheless, while ST induced a marked increase in COX2 expression, LPS treatment did not induce a similar effect. IL1β transcription was not detected in canine MSCs with any of the treatments. We next determined the transcription of selected genes in four more MSC lines (B). Both bacteria induced upregulation of IL6, IL8, and COX2 gene transcription. Moreover, transcription of IL8 and COX2 genes was statistically significantly higher in the ST-treated MSCs compared with the LB-treated MSCs. Secreted factors followed gene transcription trends and IL6, IL8, and PGE₂ concentrations were higher in the ST-primed MSCs than baseline or LB-primed MSCs (C). Cell surface expression of CD54 was significantly induced by ST compared with baseline and LB-treated MSCs (D). LB interaction did not induce any change in CD54 cell surface expression. *P<0.05, **P<0.01.

FIG. 4.

Bacterial interactions augment MSC capacity to inhibit T-cell proliferation. Peripheral blood mononuclear cells (PBMCs) were activated with the T-cell mitogen concanavalin A (Con-A), in the presence or absence of MSCs and microbial-primed MSCs. Cocultures were incubated for 3 days prior to spiking with BrdU. Cells were harvested 24 h post-BrdU spiking; fixed and stained with a viability dye, a T-cell marker (ie, CD3), and anti-BrdU antibody; and read on a flow cytometer. All experiments were repeated with five different MSC lines. Stimulation of PBMCs with Con-A resulted in the maximal BrdU incorporation by viable T-cells. Coincubation of stimulated PBMCs with native MSCs resulted in a marked decrease in T-cell incorporation of BrdU. While ST augmented MSC capacity to inhibit T-cell proliferation, compared with untreated MSCs and LB-treated MSCs, inhibition of T-cell proliferation by LB-treated MSCs was not different from untreated MSCs. When COX function was blocked with indomethacin, T-cell proliferation was significantly, though partially, restored in ST-treated MSCs. ***P<0.001.

FIG. 5.

A proposed model for MSC interaction with intestinal bacteria and subsequent MSC activation in patients with intestinal injury. Exogenously administered MSCs home to the injured gut where they engraft in the lamina propria (A). Given the inflamed and compromised mucosal barrier, microbes and microbial ligands that leak into the lamina propria will interact with newly engrafted MSCs via yet to be undefined receptors, such as membranal [ie, Toll-like receptor (TLR)] or cytoplasmic [NOD-like receptors (NLRs)] pattern recognition receptors (B). Binding of these receptors by microbes may further activate several cellular signaling pathways that culminate in altered gene transcription, MSC activation, and augmented anti-inflammatory or induced proinflammatory capacity based on the overall signals sensed by the MSCs (C). Color images available online at www.liebertpub.com/scd