The influence of hypoxia and IFN-γ on the proteome and metabolome of therapeutic mesenchymal stem cells

Abstract

Over the past 15 years, mesenchymal stem cells (MSCs) have been assessed for their capacity to suppress inflammation and promote tissue repair. Regardless of whether the cells are primed (exposed to instructive cues) before administration, their phenotype will respond to environmental signals present in the pathophysiological setting being treated. Since hypoxia and inflammation coexist in the settings of acute injury and chronic disease we sought to explore how the proteome and metabolome of MSCs changes when cells were exposed to 48 h of 1% oxygen, interferon gamma (IFN-γ), or both cues together. We specifically focused on changes in cell metabolism, immune modulation, extracellular matrix secretion and modification, and survival capacity. IFN-γ promoted expression of anti-pathogenic proteins and induced MSCs to limit inflammation and fibrosis while promoting their own survival. Hypoxia instead led to cell adaptation to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. While dual priming resulted in additive effects, we also found many instances of synergy. These data lend insight to how MSCs may behave after administration to a patient and suggest how priming cells beforehand could improve their therapeutic capacity.

Introduction

Mesenchymal stem cells (MSCs) are multipotent cells that are found in a wide variety of tissues [1,2]. Indeed, it is reasonable to expect that most, if not all, tissues have resident MSC-like cells, which participate in local regeneration upon injury or disease. Because these endogenous cells exist in limited numbers, many investigators have explored administration of large numbers of exogenous MSCs to enhance their therapeutic effect [3]. These cells are most often given by intravenous injection, with the expectation that they can home to affected sites and have long-ranging impact by secreting paracrine factors [4].

The expression profile and phenotype of MSCs residing in healthy tissues or expanded under basal culture conditions are generally different from those expressed by therapeutic MSCs. Upon administration, MSCs enter the diseased microenvironment of the patient and react to the new cues that are present. Two of these cues, which are common to most ailments, are inflammation and hypoxia [5]. Hypoxia specifically connotes an oxygen tension that is lower than physoxia, which is 3–9% oxygen in most tissues [6]. It affects cell behavior because hypoxia inducible factors (HIF) become hydroxylated at higher oxygen levels and face proteasomal degradation, but at lower oxygen tensions, they are more stable (specifically the alpha subunit), and serve as transcription factors. While the signaling pathways for inflammation and hypoxia start off as being distinct, they have many opportunities for cross-talk, as has been reviewed elsewhere [5,7,8].

In acute injury (such as myocardial infarction or stroke), a blood clot leads to a transient decrease in tissue perfusion, and inflammatory cells rush in to tackle foreign invaders and remove damaged tissue. In this situation, the hypoxia and inflammation may be short lived (days to a week), contingent upon successful resolution of the injury [9]. However, in many chronic disease states these two factors co-exist for prolonged periods of time. For example, in rheumatoid arthritis, the synovial joint space is characterized by regions of hypoxia and inflammation, which are thought to influence local cell survival, angiogenesis, and energy metabolism [9]. Another autoimmune disease – inflammatory bowel disease – also exhibits chronic inflammation, with oxygen gradients at the intestinal mucosa that lead to regions of hypoxia. Curiously, these hypoxic regions are relatively protected compared with the rest of the intestinal tract, as the HIF pathway seems to promote epithelial survival while inducing neutrophil apoptosis [9]. Lastly, in solid tumors, hypoxia and inflammation facilitate tumor vascularization and immune escape [7].

In order to understand how MSCs serve as reparative and regenerative cells, we explored how they would respond to a generic pathological microenvironment in which hypoxic and inflammatory cues were provided in vitro. To simulate hypoxia, we chose an incubator setting of 1% O₂, as this is a lower oxygen tension than the physoxic state of most tissues without being anoxic [10], and to simulate inflammation, we used interferon-gamma (IFN-γ). While many pro-inflammatory cytokines and TLR agonists have been shown to affect MSC behavior, the response of MSCs to IFN-γ is considered to be a standard benchmark of cell function by the International Society for Cellular Therapy (ISCT), and IFN-γ is more natural to a generic inflammatory setting than polyI:C, for example [[11], [12], [13], [14], [15]]. We then analyzed the proteome and metabolome of adipose-derived MSCs upon 48hr exposure to control conditions, hypoxia (1% O₂), inflammation (IFN-γ), or both cues together (a scenario we refer to as “dual priming”).