# HIF1α-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions

Rafael Contreras-Lopez, Roberto Elizondo-Vega, Maria Jose Paredes, Noymar Luque-Campos, Maria Jose Torres, Gautier Tejedor, Ana Maria Vega-Letter, Aliosha Figueroa-Valdés, Carolina Pradenas, Karina Oyarce, et al.

► To cite this version:

Rafael Contreras-Lopez, Roberto Elizondo-Vega, Maria Jose Paredes, Noymar Luque-Campos, Maria Jose Torres, et al. HIF1α-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions. FASEB Journal, 2020, 34 (6), pp.8250-8264. 10.1096/fj.201902232R. hal-03616941

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<figure><img src="image_1.png" /></figure>
RESEARCH ARTICLE

THE
FASEB JOURNAL
www.faseb.org

# HIF1α-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions

Rafael Contreras-Lopez<fnref n="1" /> | Roberto Elizondo-Vega<fnref n="1" />,<fnref n="2" /> | Maria Jose Paredes<fnref n="1" /> |

Noymar Luque-Campos<fnref n="1" /> | Maria Jose Torres<fnref n="3" /> | Gautier Tejedor<fnref n="4" /> |

Ana Maria Vega-Letter<fnref n="5" />,<fnref n="6" /> | Aliosha Figueroa-Valdés<fnref n="5" />,<fnref n="6" /> | Carolina Pradenas<fnref n="1" /> |

Karina Oyarce<fnref n="7" /> | Christian Jorgensen<fnref n="4" /> | Maroun Khoury<fnref n="5" />,<fnref n="6" /> |

Maria de los Angeles Garcia-Robles<fnref n="2" /> | Claudia Altamirano<fnref n="3" /> | Farida Djouad<fnref n="4" /> |

Patricia Luz-Crawford<fnref n="1" />

## Correspondence

Patricia Luz-Crawford, Laboratorio de Inmunología Celular y Molecular, Centro de Investigación e Innovación Biomédica, Facultad de Medicina, Universidad de los Andes, Santiago, Chile.

Email: pluz@uandes.cl

Farida Djouad, INSERM U 1183, IRMB, Université de Montpellier, Hôpital Saint-Eloi, 80 avenue Augustin Fliche, 34295 Montpellier cedex 5, France.

Email: farida.djouad@inserm.fr

## Funding information

MINEDUC | Comisión Nacional de Investigación Científica y Tecnológica (CONICYT), Grant/Award Number: FONDECYT INICIACIÓN 11190690,

## Abstract

Hypoxia-inducible factor 1 α (HIF1α), a regulator of metabolic change, is required for the survival and differentiation potential of mesenchymal stem/stromal cells (MSC). Its role in MSC immunoregulatory activity, however, has not been completely elucidated. In the present study, we evaluate the role of HIF1α on MSC immunosuppressive potential. We show that HIF1α silencing in MSC decreases their inhibitory potential on Th1 and Th17 cell generation and limits their capacity to generate regulatory T cells. This reduced immunosuppressive potential of MSC is associated with a metabolic switch from glycolysis to OXPHOS and a reduced capacity to express or produce some immunosuppressive mediators including Intercellular Adhesion Molecule (ICAM), IL-6, and nitric oxide (NO). Moreover, using the Delayed-Type Hypersensitivity murine model (DTH), we confirm, in vivo, the critical role of HIF1α on MSC immunosuppressive effect. Indeed, we show that HIF1α

**Abbreviations:** 2DG, 2-deoxiglucose; BM, bone marrow; CD, cluster of differentiation; DTH, delayed-type hypersensitivity murine model; ECAR, extracellular acidification rate; FCCP, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; GLUT1, glucose transporter 1; HIF1α, hypoxia-inducible factor 1 α; HRE, hypoxia-response element; ICAM, intercellular adhesion molecule; IFNγ, interferon gamma γ; IL, interleukin; MSC, mesenchymal stem/stromal cells; NO, nitric oxide; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation; PDHK1, pyruvate dehydrogenase (acetyl-transferring) kinase isozyme 1; ROS, reactive oxygen species; Th, T helper; TNFα, tumor necrosis factor α.

Contreras Lopez and Elizondo Vega equally contribute to this work. Patricia Luz Crawford and Farida Djouad jointly supervised this work.

[Correction added on May 11, 2020, after first online publication: Contribution to this work has been updated in the footnote.]

<footnote n="1">¹Laboratorio de Inmunología Celular y Molecular, Centro de Investigación e Innovación Biomédica, Facultad de Medicina, Universidad de los Andes, Santiago, Chile</footnote>

<footnote n="2">²Laboratorio de Biología Celular, Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile</footnote>

<footnote n="3">³Escuela de Ingeniería Bioquímica, Pontificia Universidad Catolica de Valparaiso, Valparaiso, Chile</footnote>

<footnote n="4">⁴IRMB, INSERM, Université de Montpellier, Montpellier, France</footnote>

<footnote n="5">⁵Cells for Cells, Consorcio Regenero, Las Condes, Santiago, Chile</footnote>

<footnote n="6">⁶Laboratory of Nano-Regenerative Medicine, Facultad de Medicina, Universidad de los Andes, Santiago, Chile</footnote>

<footnote n="7">⁷Facultad de Ciencias de la Salud, Universidad San Sebastián, Concepción, Chile</footnote>
FONDECYT INICIACION N° 11160929,
BECA DOCTORADO NACIONAL
N°21141173, BECA DOCTORADO
NACIONAL N°2191997, PROYECTO
REDES N°180211, PROYECTO ECOSUD
N°PC18S04-180032, FONDECYT
POSTDOCTORADO N° 3190462 and
FONDECYT REGULAR 1170852;
Fondo de Ayuda a la Investigación (FAI),
Universidad de los Andes, Grant/Award
Number: Venida profesor extranjero, 2018;
Vicerrectoria de Investigacion y desarrollo
(VRID), Grant/Award Number: VRID-
INICIACION 219.031.116-INI; Agence
Nationale de la Recherche (ANR), Grant/
Award Number: ANR-18-CE18-0010-02
and ANR-11-INSB-005; Vicerrectoria
de investigación y desarrollo (VRID),
Universidad de Concepción, Grant/Award
Number: VRID-asociativo n° 218.031.113-1

silencing impairs MSC capacity to reduce inflammation and inhibit the generation of pro-inflammatory T cells. This study reveals the pivotal role of HIF1α on MSC immunosuppressive activity through the regulation of their metabolic status and identifies HIF1α as a novel mediator of MSC immunotherapeutic potential.

KEYWORDS

glycolytic, HIF1α, immunomodulation, immunosuppression, metabolic reprogramming, metabolism, MSCs

# 1 | INTRODUCTION

Mesenchymal stem/stromal cells (MSC) are multipotent cells present in most adult tissues including bone marrow (BM) and adipose tissue. These cells, distinguishable from other stem cells by the expression of mesenchymal markers including CD70, CD90, and CD105 and the absence of hematopoietic markers, can differentiate into chondrocytes, adipocytes, and osteoblasts.¹ Among the diverse functions exhibited by MSC, their capacity to prevent fibrosis, apoptosis and hypertrophy as well as their potential to support hematopoiesis, regulate immune responses, and stimulate regeneration have been intensively described (For review 1-3). The potential of MSC to differentiate into various cell lineages and to empower endogenous stem cells, have been of high interest for tissue engineering for decades. The more recent but not the least documented anti-inflammatory and immunoregulatory functions of MSC represent a huge interest in clinic for the treatment of inflammatory and autoimmune diseases.¹,⁴,⁵ Indeed, MSC have been described to repress the function of most, if not all, pro-inflammatory immune cells including Th1 and Th17 lymphocytes while inducing the generation of regulatory T cells in vitro and in vivo (for review see 6). Although considered as candidate of choice for diverse therapeutic applications, MSC are highly plastic and consequently, their phenotype and functions are molded by factors present in their microenvironment including the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and Interferon gamma γ (IFNγ). This versatility of MSC needs to be considered for their optimal use in clinic. Moreover, MSC are present in most adult tissues, but at low frequencies. Thus, MSC use for fundamental research and clinical applications requires a high number of cells, and thus, an intensive amplification that might modify MSC functions.

Actually, the expansion of MSC on plastic adherence cell culture dishes, in a media that contains serum, significantly

impairs their proliferation rate and biology due to the constant exposure to high oxygen rates compared to their niche. Indeed, the level of oxygen plays a pivotal role on MSC functions. In physiological conditions, MSC reside in hypoxic niches and are exposed to oxygen level ranging from 2% to 7%.⁷ Thus, many studies have investigated the role of hypoxia on MSC functions focusing mainly on their proliferation and differentiation potential.⁸ Hypoxic culture conditions enhance the survival and proliferation rate of MSC and stabilize their karyotype. In contrast, in normoxia, MSC undergo a loss of their stem cell features as well as enhanced oxidative phosphorylation (OXPHOS), chromosomal alterations, DNA damage and premature senescence.⁹⁻¹² Regarding, MSC functions, hypoxia improves their angiogenic properties, their capacity to migrate toward the site of injury and to release immunoregulatory factors.¹³⁻¹⁵ Hypoxia is also described for its role on MSC morphology, cytoskeletal tension, chondrogenesis,¹⁶ and osteogenesis.¹⁷ While the role of hypoxia has been intensively studied on MSC proliferation and differentiation potential, its role on MSC anti-inflammatory immunosuppressive properties has been poorly investigated.

In response to hypoxia, MSC undergo molecular and functional changes via the hypoxia inducible factor (HIF) pathways. HIF1 is a heterodimeric transcription factor consisting of two subunits; HIF1α regulated by oxygen and HIF1β constitutively expressed. Under hypoxia, HIF1α translocates to the nucleus to form a heterodimer with HIF1β that will bind to hypoxia-response element (HRE) sequence. HIF1α translocation plays a pivotal role during MSC chondrogenesis. Indeed, HIF1α binds to the promoter region of Sox9, a master transcriptional regulator of chondrogenesis, and thereby activate its downstream targets and promote eventually chondrogenesis.¹⁸ Moreover, MSC transduced with HIF1α gene exhibit increased expression levels of angiogenic and osteogenic markers, in vitro under normoxic conditions.¹⁹ While
hypoxia enhances MSC osteogenesis, it represses adipogenesis in a HIF1α-dependent and competitive manner. Thus, during certain pathological processes such as fracture hema-toma, HIF1α, the master regulator of the hypoxia, will guide MSC differentiation toward osteogenesis to the detriment of adipogenesis.²⁰ Thus, while the role of HIF1α has been in-tensively studied on MSC differentiation potential, its role on the capacity of MSC to regulate the immune response needs to be fully addressed.

The role of energy metabolism in MSC functions is pivotal. Indeed, undifferentiated MSC rely on glycolysis for energy while they activate the mitochondrial process of OXPHOS during their differentiation.²¹ Under hypoxic conditions, HIF1α expression level in MSC is increased as well as their glycolytic activity. During osteogenesis, HIF1α expression level progressively decreased in MSC to activate mitochon-drial OXPHOS.²² Regarding MSC immunosuppressive prop-erties, MSC metabolic activity has been recently proposed to be correlated with their suppressive capacity.²³,²⁴ However, the role of the metabolic reprogramming mediated by HIF1α on MSC immunoregulatory function remains to be better defined.

A comprehensive characterization of the role of HIF1α on MSC metabolism and immunosuppressive functions both in vitro and in vivo will have critical therapeutic significance for the treatment of anti-immune and inflammatory diseases and will help for the development of novel MSC-based therapeutic approaches. Thus, in the present study, we explored the rele-vance of Hypoxia-inducible factor 1 α (HIF1α) expression in the immunosuppressive properties of MSC. We investigated how HIF1α inhibition may affect the immunosuppressive function of MSC focusing on Th1 and Th17 cell phenotype and generation and the generation of regulatory T cells both in vitro and in vivo.

2 | MATERIAL AND METHODS

2.1 | MSC culture and characterization

Murine MSC were isolated from BW of wild type C57BL/6 mice, and characterized as previously described.²⁵ The cells were culture in complete DMEM (Dulbecco's Modified Eagle Medium) containing 10% of heat-inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and incubated at 37°C with 5% CO₂ under normoxic conditions.

2.2 | HIF1α knock-down

MSC deficient for HIF1α were obtained by transfec-
tion of a specific small interfering RNA (sequence:
UACUGUUCCUGAGGAAGAA). Transfection was per-
formed Oligofectamine reagent (Life Technology, USA) ac-
cording to the manufacturer's guidelines.

2.3 | T cell isolation, differentiation, and coculture with MSC

Freshly isolated T-CD4 cells were obtained from C57BL/6 mice using the Dynal CD4 negative (Life Technology, USA) isolation kit according to the manufacturer's instructions. Then, T-CD4 cells were stained with cell trace violet (CTV), activated with anti-mouse CD3/CD28 Dynabeads (Life Technology, USA) and cultured in complete Iscove's Modified Dulbecco's Media (IMDM) containing 10% of heat-inactivated FBS, 2 mM of L-glutamine, 100 U/mL of penicillin, 100 µg/mL of streptomycin, 0.1 mM of nonessential amino acids, 1 mM of sodium pyruvate, 20 mM of HEPES, and 50 µM of beta-mercaptoethanol (all from Gibco, USA). To differentiate Th1 cells, CD4⁺ T cells were stimulated with 20 ng/mL of IL12 (R&D Systems, USA) and 2.5 µg/ mL of anti-IL4 (BD Pharmingen, BD Biosciences, USA). Th17 differentiation was obtained by adding 50 ng/mL of IL6 (R&D Systems, USA), 2.5 ng/mL of TGFβ1 (R&D Systems, USA), 2.5 µg/mL of anti-IFNγ (BD Pharmingen, USA), and 2.5 µg/mL of anti-IL4 (BD Pharmingen, USA). To evaluate the role of HIF1α and reactive oxygen species (ROS) in the suppressive activity of MSCs, CD4⁺ T cells were cultured alone or in the presence of MSC in different states: (1) naïve MSC, (2) MSC knock-down for HIF1α, (3) MSC knock-down for HIF1α and pretreated with 1 µg/ mL of the ATP synthase inhibitor oligomycin for 24 hours, and (4) MSC pretreated with 1 mg/mL of the ROS scavenger N-acetyl-L-cysteine (NAC) for 24 hours at a MSC:T cell ratio of 1:10 in complete IMDM media. After 72 hours, proliferation and CD4⁺ T cell differentiation was quantified by flow cytometry.

2.4 | Flow cytometry analysis

Proliferation and differentiation of lymphocytes were quantified by intracellular staining in order to detect the production of specific cytokines and transcription factors. For that purpose, 3 days after the coculture, T cells were stimulated with phorbolmyristate acetate (PMA) (50 ng/mL; Merck, Germany) and ionomycin (1 mg/mL; Merck, Germany), in the presence of brefeldin A (10 mg/mL; Sigma, USA) for 4 hours. Then, surface staining against CD25 (BD, pharmingen) was performed together with LIVE/DEAD Fixable near-IR stain (Invitrogen, Thermo Fisher, USA) in order to achieve the analysis only in live cells. Then, cells were fixed at 4°C with the FoxP3 Cytofix/Cytoperm buffer (eBioscience, USA) and subsequently stained with intracellular fluorochrome-conjugated antibodies against, IFNγ (BD pharmingen), IL17 (BD pharmingen), and FoxP3 (eBioscience) diluted in Perm/Wash buffer (eBioscience, USA) following manufacturing instruction.
To evaluate Intercellular Adhesion Molecule (ICAM) and HIF1α expression, MSCs were either stained with an antibody against ICAM (R&D, USA) or permeabilized and stained using a specific antibody against HIF1α (R&D, USA) after 24 hours of TNFα and IFNγ (R&D, USA) activation.

## 2.5 | Immunofluorescence

MSC pretreated or not with TNFα and IFNγ were fixed in 100% of methanol for 20 minutes at 4°C. Then, permeabilized with PBS containing 0.25% of Triton X-100 for 10 minutes and blocked with 10% of goat serum in PBS during 1 hour at room temperature. HIF1α expression was measured with a rabbit monoclonal antibody against HIF1α (1:100; Cell Signaling Technology, USA) overnight at 4°C. The cells were washed three times with PBS followed by incubation with secondary antibody Alexa Fluor 594 goat anti-rabbit IgG (Abcam, USA) for 1 hour at room temperature. Laminin staining was used to outline the nuclei (Cell Signaling Technology, USA). Finally, cells were observed by confocal laser scanning microscopy TCS SP5 (Leica Myrosystems, Germany). Total nuclear translocation was quantified with the open-source software for biological-age analysis Fiji.

## 2.6 | Seahorse assay

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the XF96 analyzer (Seahorse Biosciences, North Billerica, MA, USA). Pretreated murine MSCs were plated on 96 well plates and analyzed according to manufacturer's recommended protocol. Three independent readings were taken after each sequential injection. Instrumental background was measured in separate control wells using the same conditions without biologic material.

Basal glycolytic rate was calculated after glucose injection. Maximal glycolytic rate was measured after oligomycin injection and glycolytic capacity as the difference of oligomycin-induced ECAR and 2-Deoxiglucose (2DG)-induced ECAR. OCR was measured in XF media (non-buffered DMEM medium, containing 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate), under basal conditions and in response to 1 µM of oligomycin, 1 µM of carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), and 1 µM of antimycin A and rotenone (all chemicals from Sigma Aldrich). Basal OCR was calculated as the difference between baseline measurements and antimycin A/rotenone-induced OCR; maximal OCR was measured as the difference between FCCP-induced OCR and antimycin A/rotenone-induced OCR.

## 2.7 | Reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR)

Total RNA from cell cultures was isolated using TRIzol reagent (Invitrogen, USA), and then, treated with DNase I (Life Technology, USA) before reverse transcription processing following manufacturing instructions. The RNA concentration was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Reverse transcription and qRT-PCR were performed as we previously described using an Mx3000P QPCR System (Agilent Technologies, USA).²⁶ The following sets of oligonucleotides were used: HIF 5'-TG TGACCATGAGGAAATGAGAG-3' (sense) and 5'-GGTT CACAAATCAGCACCAAG-3' (antisense), Glucose Transporter 1 (GLUT1) 5'-TCTGTCGGCCTCTTTGTTAA TC-3' (sense) and 5'-TGACACCTCTCCCACATACA-3' and 18S 5'-GCCCCGAAGCGTTTACTTTGA-3' (sense) and 5'-TTGCGCCGGTCCAAGAATTT-3' (antisense).

## 2.8 | Quantification of immunosuppressive molecules and HIFα target produced by MSC

In order to study the expression levels of the immunosuppressive molecules supernatants from MSC or MSC<sub>siHIF1α</sub> activated or not with TNFα plus IFNγ for 24 hours were collected. Then, IL6 production was measured by ELISA following manufactured instructions (R&D, USA), while NO<sub>2</sub> production was determined by using a modified Griess reagent reaction (Thermo Fisher, USA).

## 2.9 | Mice and ethical statement

C57BL/6 and BALB/c mice, 10-12 weeks old were purchased from the Central Animal Facility JANVIER Labs (Grenoble, France). Animals were housed under standard laboratory conditions and maintained with food and water ad libitum. Experimental procedures and protocols were performed according to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of the Universidad de los Andes, Santiago, Chile (Bioethics grant certificate of Universidad de los Andes N°201630) and in accordance with the Ethical Committee for animal experimentation of the Languedoc-Roussillon (Approval CEEA-LR-2016050918509993).

## 2.10 | DTH model

To assess the effect of HIF1α in the capacity of MSC to achieve inflammation in vivo and the generation of different
subpopulation of CD4⁺ lymphocytes, we rely on the Delayed Type Hypersensitivity inflammation protocol (Delayed-Type Hypersensitivity murine model, DTH) murine model. DTH was induced by injection of administration of chick ovalbumin emulsified in complete Freund's adjuvant at the base of the tail. Five days postimmunization, the boost was performed with 30 µg chick ovalbumin resuspended in a saline solution (control DTH) or together with MSC. The thickness of the footpads was measured at the boost and compared with the measures performed 24 hours after prior the euthanasia of the mice. Different subpopulation of pro-inflammatory and anti-inflammatory T cells were studied in the blood and LN by flow cytometry.

**2.11 | Immunoblotting**

Total protein extracts were obtained from MSC or MSC<sub>siHIF1α</sub> cultures. Briefly, samples were lysed in RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma) and homogenized by pass-through a 25-gauge needle at least 10 times, and then, incubated for 1 hour on ice. Proteins were resolved by SDS-PAGE (50 µg/lane) in a 10% (w/v) polyacrylamide gel, transferred to PVDF membranes (0.45 µm pore, Amersham Pharmacia Biotech., Piscataway, NJ, USA), and probed for 16 hours at 4°C with antibodies against HIF1α or PDHK1 (both from cell Signaling). After extensive washing, PVDF membranes were incubated for 2 hours at room temperature with Alexa-fluor 680 labeled goat anti-rabbit IgG (1:20.000 Invitrogen by Thermo Fisher USA) or Alexa Fluor 750 labeled goat anti-mouse IgG (1:20.000; Life Technologies by Thermo Fisher USA). The reaction was developed using LI-COR OdysseyCLx Imaging System. Images shown are representative of at least four analyses performed on samples from at least four separate experiments. β-actin expression levels were used as a loading control.

**2.12 | Statistical analysis**

Data are presented as mean ± SD. All in vitro experiments were performed using at least four different independent experiments. For the in vivo studies 6 to 8 animals were used for each experimental or control groups and repeated at least three independent times. The *P* values were generated using nonparametric analysis using the Kruskal-Wallis or the Mann-Whitney U test to either compare between more than two or between two experimental groups, respectively. *P* < .05 (*), *P* < .01 (**), or *P* < .001 (***) were considered statistically significant. All the analyses were performed using the GraphPad Prism TM 6 software (GraphPad Software, San Diego, California, USA).

**3 | RESULTS**

**3.1 | Pro-inflammatory cytokines induce HIF1α expression and ROS production in MSC independent on hypoxic conditions**

In cells such as pterygium fibroblasts, inflammation, well described to prime MSC immunoregulatory properties, has been shown to upregulate HIF1α expression level even under normoxic conditions.²⁷ To study the role of HIF1α in the immunosuppressive and therapeutic activity of MSC, we first evaluated whether pro-inflammatory cytokines such as TNFα and IFNγ that prime MSC immunosuppressive functions, were able to induce HIF1α expression. Western blot and FACS analysis showed that the activation of MSC with TNFα and IFNγ activation significantly increased the protein expression levels of HIF1α under normoxic conditions (Figure 1A-D). Moreover, confocal microscopy showed that upon TNFα and IFNγ activation, HIF1α expression is translocated to MSC nucleus as revealed by its increased colocalization with nuclear laminin (Figure 1E,F). A similar HIF1α nuclear translocation was observed upon cobalt chloride (CoCl₂)-mediated hypoxia (Figure 1E,F, white bar). Additionally, we showed that MSC activation with TNFα and IFNγ significantly increased the production of ROS (Figure 1G,H) known to be pivotal for HIF1α induction and stabilization through NF-κβ molecular pathway.²⁸ Thus, our results suggest that MSC activation with pro-inflammatory cytokines, described to prime their immunosuppressive potential, enhanced the amount of ROS produce inducing and maintaining HIF1α expression level under normoxic conditions.

**3.2 | HIF1α controls the immunosuppressive activity of MSC in vitro**

Going further, we investigated whether HIF1α expression level in MSC was associated with their immunosuppressive potential. Since the inhibitory effects of MSC on Th1 and Th17 cell proliferation and differentiation is well described,²⁹ we performed coculture experiments with naïve or HIF1α silenced MSC and freshly isolated naïve T CD4 cells, induced to differentiate into Th1 or Th17 cells. To knock-down HIF1α, we transfected MSC with specific small interfering RNA (siRNA) against HIF1α (siHIF1α) and compared them with MSC transfected with a control siRNA (MSC<sub>siControl</sub>). HIF1α knock-down in MSC (MSC<sub>siHIF1α</sub>), significantly reduced HIF1α levels (70%) as compared to MSC<sub>siControl</sub> (Figure S1A). Also, our in vitro results showed that, the suppressive potential of MSC on Th1 and Th17 proliferation was significantly impaired when HIF1α was silenced (Figure 2A,D). Moreover, while IFNγ production was significantly decreased only when Th1 cells were cocultured
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

**FIGURE 1** Pro-inflammatory cytokines induce HIF1α expression in MSC. A-B, Expression levels of HIF1α was determined by western blot (A-B) and by FACS (C-D) 24 hours after MSC activation with IFNγ and TNFα (E-F) Immunodetection of HIF1α (red) localization and laminin (green) to stain cellular and nuclear membrane in MSC were determined by confocal microscopy. Fluorescence intensity reflecting HIF1α expression profile (either total or nuclear) was quantified in different fields of each image for each condition including the CoCl₂ as a positive staining control group using the ImageJ program. Cells were visualized and photographed using a confocal microscope. G-H, Expression levels of mitochondrial ROS upon TNFα and IFNγ activation in MSCs, was determined by FACS using the Mitosox reagent. Results represent the mean ± SD of three independent experiments. Statistics: non-paired Mann-Whitney test. \*P < .05; \*\*P < .01; \*\*\*P < .001. Unless otherwise indicated, *P* values refer to values obtained for MSCs under basal conditions

with MSC<sub>siControl</sub> (Figure 2B, black bar), a similar decrease in IL17 production by Th17 cells was observed when the cells were cultured either with MSC<sub>siControl</sub> or MSC<sub>siHIF1α</sub> (Figure 2E, black and blue bars). Regarding, the generation of T regulatory (Treg) cells, it was significantly impaired when Th1 and Th17 cells were cocultured with MSC<sub>siHIF1α</sub> as compared to MSC<sub>siControl</sub> (Figure 2C,F, blue bars).

It has been well described that the immunosuppressive abilities of MSCs, depends on the expression of several suppressive mediators including ICAM, IL6, and NO₂ among others. These factors are produced by MSC upon pro-inflammatory cytokine stimulation with TNFα and IFNγ.³⁰ Consequently, we investigated the effects of HIF1α knock-down in MSC on their capacity to produce the main mediators
<figure><img src="image_10.png" /><figcaption>(B)
% of Proliferating Cells
CD4-Th1</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>CD4 (Th1 or Th17)
+MSC<sub>siControl</sub>
+MSC<sub>siHIF1α</sub></figcaption></figure>

<figure><img src="image_12.png" /><figcaption>(C)</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>(D)</figcaption></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>(E)</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>(F)</figcaption></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>(G)</figcaption></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>(H)</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>(I)</figcaption></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

**FIGURE 2** HIF1α expression drives the immunosuppressive properties of MSCs in vitro. HIF1α expression regulates the suppressive effect of MSCs on pro-inflammatory Th1 and Th17 cells in vitro. A-F, Naïve T CD-4 cells from Bl6 mice were labeled with cell trace violet (CTV) stain and induced to differentiate into Th1 (A,C) or Th17 (D,F) cells and cultured in the presence or absence (white bar-T-CD4 alone) of murine MSC with (blue bar) or without (black bar) siRNA against HIF1α. Proliferation, pro-inflammatory phenotype (IFNγ and IL17 production for Th1 and Th17, respectively), and Treg generation was evaluated by FACS. Results represent the mean ± SD of three independent experiments with four different mice each time. Statistics: non-paired Kruskal-Wallis test. \*P < .05; \*\*P < .01; \*\*\*P < .001. Unless otherwise indicated, *P* values refer to values obtained for either CD4-Th1 or CD4-Th17 when cultured alone. G-I, HIF1α inhibition on MSC significantly impairs the expression and production of classical immunosuppressive molecules in vitro. MSC and MSC<sub>siHIF1α</sub> were cultured in the presence or absence of TNFα and IFNγ, and the expression levels of ICAM (G) was evaluated by FACS using the geometric mean fluorescence intensity (MFI) quantification. IL-6 (H) and NO<sub>2</sub> (I) production were evaluated in the supernatants of either MSC or MSC<sub>siHIF1α</sub> by either ELISA or modifying colorimetric Griess detection, respectively. Results represent the mean ± SD of four independent experiments. Statistics: non-paired Kruskal-Wallis test. \*P < .05; \*\*P < .01; \*\*\*P < .001
<figure><img src="image_24.png" /><figcaption>**FIGURE 3** HIF1α expression controls the therapeutic abilities of MSCs on a DTH-induced arthritis model. A, Scheme that shows the DTH murine model. B, Paw swelling analysis was determined in the hindlimb of the mice 24 hours after boost and MSCs injection. Pro-inflammatory and anti-inflammatory T cells were evaluated in the blood (L,N) and drained lymph nodes (O,Q) of DTH mice (white bar) and DTH mice treated with either MSC (black bar) or MSC<sub>siHIF1α</sub> (blue). Results represent the mean ± SD of three independent experiments of at least 15 animals per experimental group. Statistics: Non-paired Kruskal Wallis test. \*P < .05; \*\*P < .01; \*\*\*P < .001. Unless otherwise indicated, *P* values refer to values obtained for DTH experimental groups</figcaption></figure>

involved in their immunosuppressive properties. Compared to MSC<sub>siControl</sub>, we showed that the reduced immunoregulatory properties of MSC<sub>siHIF1α</sub> on Th1 and Th17 cells upon TNFα and IFNγ was associated with a significant decrease of ICAM expression (Figure 2G, blue bar), IL6 (Figure 2H, blue bar), and NO<sub>2</sub> production (Figure 2G, blue bar). These results suggest that HIF1α might regulate the production of MSC immunosuppressive mediators increased upon stimulation with pro-inflammatory cytokines.

## 3.3 | HIF1α drives the therapeutic potential of MSC in the murine DTH model

In order to determine the impact of HIF1α silencing on MSC therapeutic potential in vivo, we relied on the delayed-type hypersensitivity (DTH) murine model (Figure 3A). Compared to untreated DTH mice, mice treated with

MSC<sub>siControl</sub> exhibited a reduced paw swelling (Figure 3B, dashed bar) while the injection of MSC<sub>siHIF1α</sub> significantly increased the mice paw swelling (Figure 3B, blue line bar). This beneficial effect mediated by MSC<sub>siControl</sub> was correlated with a reduced percentage of Th1 cells in the peripheral blood (Figure 3C, blue bar). No statistically significant differences were observed on the frequency of both Th17 (Figure 3D, blue bar) and Treg cells (Figure 3E, blue bar) between the different groups (Figure 3C-E). In the popliteal lymph nodes (LN) of mice treated with MSC<sub>siControl</sub>, we observed a significant decrease of the percentages of both pro-inflammatory T cell populations (Figure 3F,G, blue bars) and a significant increase of Treg cells (Figure 3H, black bar). Altogether, these data suggest that HIF1α expression is critical for the therapeutic potential of MSC in the DTH experimental model by reducing the frequency of pro-inflammatory Th1 and Th17 cells and inducing an anti-inflammatory environment.
<figure><img src="image_25.png" /><figcaption>**FIGURE 4** HIF1α inhibition impairs the immunosuppressive potential of MSC by prompting an OXPHOS dependent metabolism. A-C, The metabolic status of murine MSC (Black line or bar) or MSC<sub>siHIF1α</sub> (blue line or bar) was determined by evaluating the oxygen consumption rates (OCR) (A), the extracellular acidification rate of the media (ECAR) (B), and the ratio among those values (C) using the Agilent Seahorse XF technology. The expression levels of PDHK1 (D and E) and GLUT1 (F) were determined by western blot or qPCR, respectively. Results are represented as the mean ± SD of three independent experiments. Statistics: non-paired Mann-Whitney test. \*P < .05; \*\*P < .01; \*\*\*P < .001</figcaption></figure>

## 3.4 | The inhibition of HIF1α expression is associated with an increased OXPHOS metabolism in MSC

Since it is well known that HIF1α is a master regulator of glycolysis, we thus studied the metabolic status of MSC<sub>siHIF1α</sub> compared to MSC<sub>siControl</sub> in real-time using the Seahorse XF Extracellular Flux Analyzer. HIF1α knock-down in MSC significantly increased OCR (Figure 4A, blue dashed line) without modifying ECAR (Figure 4B). Consequently, the ECAR/OCR ratio measurement revealed that HIF1α silencing enhanced the OXPHOS metabolism of MSC without changing their glycolytic metabolism in the experimental time window (Figure 4C, blue bar). Going further, we assessed the impact of HIF1α knock-down in MSC on the expression levels of its targets such as pyruvate dehydrogenase kinase 1 (PDHK1), an important enzyme that regulates the metabolic switch from OXPHOS toward glycolysis,³¹ and GLUT1, one of the main glucose transporter.³² Compared to MSC<sub>siControl</sub>, MSC<sub>siHIF1α</sub> expressed lower levels of PDHK1 (Figure 4D,E, blue bar) and GLUT1 (Figure 4F, blue bar).

Thus, these results unveiled that HIF1α is a key regulator of MSC metabolic status.

## 3.5 | HIF1α-dependent metabolic switch is crucial for MSC immunosuppressive functions

Due to the pivotal role of HIF1α on MSC metabolic status and functions, we wondered whether we could restore MSC immunoregulatory properties lost in response to HIF1α silencing by promoting glycolysis. For that purpose, we treated MSC<sub>siHIF1α</sub> with oligomycin (a known inhibitor of ATPase and OXPHOS metabolism) during 24 hours and studied their bioenergetic status.

We assessed the bioenergetic capacities of MSC<sub>siControl</sub> and MSC<sub>siHIF1α</sub> by measuring the OCR and the ECAR after a 24 hours treatment with oligomycin and found that, oligomycin-treated MSC<sub>siControl</sub> exhibited a lower OCR and higher glycolytic capacity compared to non-treated cells (Figure 5A,B, black dashed lines). Moreover, the treatment of MSC<sub>siHIF1α</sub> with oligomycin, significantly decreased their
OCR to a similar extent as in MSC<sub>siControl</sub> exhibiting a glycolytic status (Figure 5A,B, blue dashed lines). Furthermore, the ratio of ECAR to OCR of oligomycin-treated MSC<sub>siHIF1α</sub> was about three times lower, than the ratio measured for MSC<sub>siControl</sub> (Figure 5C, blue bar), but did not reach the ECAR to OCR ratio level of MSC<sub>siControl</sub> treated with oligomycin

(Figure 5C, grey dashed bar). Furthermore, the establishment of an energetic graph (Figure 5D) indicated that HIF1α silencing significantly increased the oxygen consumption of MSC, indicating that MSC<sub>siHIF1α</sub> underwent mainly an aerobic respiration. The treatment of MSC<sub>siHIF1α</sub> with oligomycin, significantly decreased their OCR while increasing their

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**FIGURE 5** HIF1α regulates the immunosuppressive activity of MSC through a metabolic dependent manner. A-D, The metabolic status of MSC (black circles or bar) and MSC<sub>siHIF1α</sub> (blue circles or bar) pretreated or not with oligomycin (crosswise black lines for each condition) was determined by analyzing the oxygen consumption rates (OCR) (A) or the extracellular acidification rate of the media (ECAR) (B) and the ratio among those values (C) using the Agilent Seahorse XF technology. D, Energetic graph showed the metabolic status of MSC and MSC<sub>siHIF1α</sub> in the presence or absence of oligomycin. E-K, Naïve T-CD4 murine cells induced to differentiate into Th1 (E-G) or Th17 (H-J) cells were labeled with CTV and cultured in the presence or absence (white bars) of either MSC (Black bars) or MSC<sub>siHIF1α</sub> (blue bars) pretreated or not with oligomycin (crosswise black lines for each condition). Proliferation, pro-inflammatory phenotype (IFNγ, IFNγ, and IL17 production by Th1 and Th17, respectively), and Treg generation was evaluated by FACS. Results represent the mean ± SD of three independent experiments with three different mice each time. Unless otherwise indicated, *P* values refer to differences obtained for either CD4-Th1 or CD4-Th17 cells when cultured alone. Statistics: non-paired Kruskal-Wallis test. \* *P* < .05; \*\* *P* < .01; \*\*\* *P* < .001. Oligomycin pretreatment partially recovers the expression and production of classical immunosuppressive molecules in MSCs deficient for HIF1α in vitro. K,L, MSC<sub>siHIF1α</sub> were pretreated or not with oligomycin and the expression levels of ICAM (K) and IL-6 production (L) were measured by FACS and ELISA, respectively. M, MSC<sub>siHIF1α</sub> were pretreated or not with oligomycin and cultured in the presence or absence of TNFα and IFNγ prior to measure NO₂ production in the supernatants of these cells using a modifying colorimetric Griess detection. Results represent the mean ± SD of four independent experiments. Statistics: non-paired Kruskal-Wallis test. \* *P* < .05; \*\* *P* < .01; \*\*\* *P* < .001

ECAR, revealing that MSC<sub>siHIF1α</sub> are more energetic when switched toward a glycolytic status.

Thus, we investigated whether the immunosuppressive activity of MSC<sub>siHIF1α</sub> could be restored by switching their metabolism. To that end, we performed coculture experiments with CD4<sup>+</sup> T cells induced to differentiate into Th1 and Th17 lymphocytes and untreated or oligomycin-treated MSC. We demonstrated that the oligomycin-induced metabolic switch of MSC<sub>siHIF1α</sub> to glycolysis rescued their capacity to inhibit the proliferation and pro-inflammatory phenotypes of both Th1 (Figure 5E,F, blue dashed bars) and Th17 cells (Figure 5H,I, blue dashed bars), to a similar extent as MSC<sub>siControl</sub>. Additionally, we showed that in contrast to untreated MSC<sub>siHIF1α</sub>, oligomycin-treated MSC<sub>siHIF1α</sub> were able to polarize Th1 cells into Treg cell (Figure 5G blue dashed bar). However, no Treg generation was observed in any coculture conditions of Th17 cells (Figure 5J). Moreover, when MSC<sub>siHIF1α</sub>, where pretreated with oligomycin, ICAM expression was significantly restored (Figure 5K). Conversely, IL6 (Figure 5L, blue dashed bar) and NO<sub>2</sub> production (Figure 5M) were slightly increased in oligomycin-treated MSC<sub>siHIF1α</sub> (blue dashed bar) as compared to MSC<sub>siHIF1α</sub>, (blue bars) although the difference was not significant. Altogether, these results show that HIF1α regulates the immunosuppressive activity of MSC through a glycolytic dependent manner.

## 3.6 | ROS production regulates the immunosuppressive activity of MSC in vitro

Since ROS control HIF1α activity,³³ we decided to evaluate their role in the immunosuppressive activity of MSC in vitro. For that purpose, we performed coculture experiments with freshly isolated T-CD4 cells induced to differentiate into Th1 or Th17 cells with untreated MSC or MSC pretreated with 1 mg/mL of the ROS scavenger N-acetyl-L-cysteine (NAC).

Compared to untreated MSC, MSC pretreated with NAC were significantly less efficient to inhibit the proliferation of both Th1 (Figure 6B, calypso bar) and Th17 (Figure 6D, calypso bar) cells. Additionally, the immunosuppressive activity of MSC (Figure 6B, black bar) on IFNγ production by Th1 cells was significantly decreased when MSC were blocked to produce ROS (Figure 6B, calypso bar). Conversely, MSC and MSC pretreated with NAC similarly inhibited the capacity of Th17 to produce IL17 (Figure 6E black and calypso bar, respectively). Moreover, Treg generation was also reduced when Th1 and Th17 cells where cultured with MSC pretreated with NAC as compared to non-treated MSC (Figure 6C,F). Altogether, these data indicate that the ROS produced by MSC play an important role on MSC immunoregulatory potential presumably through HIF1α regulation (Figure 6K).

## 4 | DISCUSSION

Analysis of the role of HIF1α expressed by MSC revealed that HIF1α is a main regulator of MSC immunoregulatory and therapeutic potential. We provide the unique demonstration of a direct association between HIF1α expression in MSC, their metabolism and anti-inflammatory and therapeutic potential. First, we showed that the priming of MSC with pro-inflammatory cytokines, described to enhance their immunosuppressive potential, increased the amount of ROS produced by MSC, leading to the induction and maintenance of HIF1α expression level under normoxic conditions. Then, in coculture experiments with activated pro-inflammatory T cell subsets and MSC, we demonstrated that HIF1α is an upstream regulator of the factors associated with MSC immunoregulatory properties, critical for this latter MSC function. Indeed, MSC deficient for HIF1α neither alters the proliferation of Th1 and Th17 cells nor impact on their pro-inflammatory phenotype, in contrast to naïve MSC as
<figure><img src="image_40.png" /><figcaption>**FIGURE 6** ROS control the immunosuppressive activity of MSCs in vitro. A-F, Naïve T CD-4 cells from Bl6 mice were labeled with cell trace violet (CTV) stain and prompts to differentiate into Th1 (A,C) or Th17 (D,F) cells and cultured in the presence or absence (white bar-T-CD4 alone) of murine MSC that where pretreated (calypso bars) or not (black bars) for 24 hours with 1 mg/mL of the ROS scavenger N-acetyl-L-cysteine (NAC. Proliferation, pro-inflammatory phenotype (IFNγ and IL17 production for Th1 and Th17, respectively), and Treg generation was assessed by FACS. Results represent the mean ± SD of two independent experiments with three different mice each time. Statistics: non-paired Kruskal-Wallis test. \*P < .05; \*\*P < .01; \*\*\*P < .001. Unless otherwise indicated, *P* values refer to values obtained for either CD4-Th1 or CD4-Th17 when cultured alone. K, Scheme that summarized the role of HIF1α and ROS in the suppressive activity of MSC</figcaption></figure>
we have previously shown.³⁰ Additionally, we provided evidence that HIF1α expression is required for MSC therapeutic potential in the DTH experimental model to reduce, in the lymph nodes, the frequency of pro-inflammatory Th1 and Th17 cells and inducing an anti-inflammatory environment in treated mice. This pivotal role of HIF1α on MSC immunosuppressive functions is correlated with its capacity to control the metabolic status of MSC, already described to be essential for the differentiation potential of MSC.²¹,²²

Recently, using dental tissue-derived MSC, Martinez and colleagues have shown that HIF1α overexpression slightly enhances the capacity of MSC to inhibit dendritic cell differentiation without affecting their inhibitory potential on activated T cells.³⁴ However, it is important to mention that this slight or absence of effect induced by HIF1α overexpression in MSC might be due to the overexpression of endogenous HIF1α, which is highly unstable under normoxic conditions.³⁵ Therefore, the overexpression of a stable version of HIF1α would provide a better insight on its role on MSC functionality. Our study revealed that HIF1α expression in MSC, is increased in response to activation with IFNγ and TNFα, priming also described to enhance MSC immunoregulatory functions. This suggests that the well documented improved immunoregulatory potential of MSC induced with pro-inflammatory cytokines,³⁶⁻³⁸ promising to maximize their therapeutic effects in inflammatory and autoimmune disease, is mediated by HIF1α. Moreover, we showed that the stimulation of MSC with IFNγ and TNFα also enhanced the production of ROS. ROS are potent inducers of HIF1α protein accumulation in cancer cells, that acts by reducing HIF1α degradation and also, by controlling its transcriptional activity in an NF-κB dependent manner.²⁸,³⁹ Our results suggest that, a similar mechanism would take place in MSC in which IFNγ and TNFα activation under normoxic conditions will result in HIF1α stabilization and accumulation. The levels of ROS in cells is influenced by NF-κB activity, which is in turn also controlled the ROS levels (for review see 40). This observation with the description of the role of NF-κβ on MSC immunosuppressive abilities,⁴¹,⁴² indicate that IFNγ and TNFα priming of MSC enhances their immunosuppressive properties via a tight control of ROS production and NF-κβ activity.

Since HIF1α expression was significantly increased in MSC primed with pro-inflammatory cytokines, we investigated the effect of its silencing on MSC immunosuppressive properties. It has been shown that HIF1α overexpression slightly increased the capacity of MSCs to inhibit dendritic cell differentiation without affecting the suppressive abilities of MSCs to diminish the activation and proliferation of T cells.³⁴ In this context, it has been well documented that HIF1α protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions.³⁵ However, ROS can stabilize HIF1α by impeding the activity of HIF-prolyl hydroxylases

(PHDs) oxygen-dependently hydroxylate HIF1α, which label it for proteasomal degradation.⁴³ Thus, the strategy of increasing the expression of HIF1α without controlling the activity of PHDs might not be the best approximation to determine the role of HIF1α. Thus, our strategy to evaluate the potential role of HIF1α on MSCs immunosuppressive capacity was through the inhibition of its expression. Our results showed that MSC knocked-down for HIF1α are less effective in inhibiting the proliferation and differentiation of activated T-CD4 cells into Th1 lymphocytes while a moderate effect of HIF1α knocked-down on MSC immunosuppressive effect on Th17 cells was observed. These results are in line with our previous data showing that the immunosuppressive activities of MSC on Th1 and Th17 not only rely on common, but also different mechanisms.³⁰ Similarly, in vivo, using the DTH murine model, we showed that HIF1α expression drives MSC capacities to decrease inflammation by reducing the percentage of pro-inflammatory Th1 and Th17 cells and increasing the frequency of regulatory T cells. These results are in line with a recent study showing that short-term hypoxia might enhance MSC anti-inflammatory properties by reducing the levels of pro-inflammatory cytokine such as IL-8, while increasing the levels of the anti-inflammatories, IL1RA and GM-CSF, produced by MSC.¹³ IL1RA secreted by MSC, plays a key role on MSC immunosuppressive properties by controlling the polarization of macrophages toward an anti-inflammatory phenotype and inhibiting the differentiation of B cell in vivo.⁴⁴ Thus, HIF1α could be an upstream regulator of the mediators involved in MSC immunosuppressive potential such as IL1RA. Going further, we showed in the present study that, HIF1α also regulate the expression of other factors implicated in MSC immunoregulatory function, including ICAM expression, IL6, and NO₂ production. Altogether, these findings suggest that HIF1α is a novel upstream regulator that controls the expression levels of the key mediators involved on MSC immunosuppressive properties and consequently a key player of MSC immunosuppression.

Additionally, we showed that HIF1α expression by MSC play a key role on MSC functions by governing their glycolytic status. Indeed, HIF1α silencing in MSC induced a metabolic switch of MSC from glycolysis toward OXPHOS. This metabolic switch observed in HIF1α deficient MSC, was associated with a significant down regulation of PDHK1 and GLUT1, and was reversed upon a treatment with oligomycin. In line with this result, we showed that the reduced immunosuppressive activity of MSC deficient for HIF1α, was significantly increased by switching their metabolism toward glycolysis. Indeed, we demonstrated that the oligomycin-induced metabolic switch of MSC<sub>siHIF1α</sub> to glycolysis, rescued their capacity to inhibit the proliferation and pro-inflammatory phenotypes of both Th1 and Th17 to a similar level as control MSC.
The present study identifies for the first time that HIF1α as a
main regulator of MSC metabolic status and immunoregulatory
potential and bring some insights into a better clinical utilization
of MSC that relies on HIF1α expression level. Specific strate-
gies are required to limit the parameters that make unpredictable
the clinical outcome of administrated MSC presumably through
a deregulation of HIF1α expression. MSC amplification, prior
their administration in experimental disease models or in clin-
ical trials, is usually performed in standard atmospheric oxy-
gen level culture. Therefore, our results suggest that it is crucial
that stem cells designated for human application should be ex-
panded in low oxygen condition or modified for the expression
of hypoxia inducible factors to augment their biologic potential.
The hypoxic preconditioning of MSC during the manufactur-
ing process, to enhance HIF1α expression might increase MSC
therapeutic functions and open novel avenues for an enhanced
MSC-based therapy.

ACKNOWLEDGMENTS

This work was supported by grants from the Chilean “Agencia Nacional de Investigación y Desarrollo”:ANID: “Fondecyt Iniciación” N°11160929, “Fondecyt Iniciación” N°11190690, “Fondecyt Regular” N°1170852, “Programa de apoyo a la formación de redes internacionales” N°180211; “Programa de Cooperación Científica ECOS-CONICYT” N° PC18S04-ECOS180032; Beca Doctorado Nacional 2014 RC-L N° 21141173; “Beca Doctorado Nacional” 2019 NL-C N° 2191997; “Fondecyt Postdoctorado” N° 3190462, “Proyecto FAI: ‘Venida profesor extranjero,’ 2018,” Universidad de los Andes, Santiago, Chile. This project was also funded by the “Vicerrectoria de Investigación y Desarrollo,” Proyecto VRID-iniciación 219.031.116-INI and Proyecto VRID-asociativo n° 218.031.113-1 Universidad de Concepción, Concepción, Chile. We also acknowledge the Agence Nationale pour la Recherche (ANR) for the financial support with the project “PPAROA” (ANR-18-CE18-0010-02), the national infrastructure: “ECELLFRANCE: Development of a national adult mesenchymal stem cell-based therapy platform” (ANR-11-INSB-005), Inserm, the University of Montpellier, the Société Française de Rhumatologie (SFR). The authors would like to specially thank the staff of the platform SMARTY (part of “Réseau des Animaleries de Montpellier, France”) for expert care of the mice colonies.

CONFLICT OF INTEREST

The authors declare no competing financial interests.

AUTHOR CONTRIBUTIONS

R. Contreras-Lopez, R. Elizondo-Vega, P. Luz-Crawford,
and F. Djouad designed the all project and the experiments
with the input of M. Garcia-Robles, C. Altamirano, and
M. Khoury. R. Contreras-Lopez, R. Elizondo-Vega, N.
Luque-Campos, G. Tejedor, C. Pradenas, M.J. Paredes,

A.M. Vega-Letter, K. Oyarce, M.J. Torres, and P. Luz-Crawford performed the experiments and analyzed the results. R. Contreras-Lopez, R. Elizondo-Vega, P. Luz-Crawford, and F. Djouad wrote the manuscript with the input of M. Khoury, C. Jorgensen, C. Altamirano, and M. Garcia-Robles.

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## SUPPORTING INFORMATION

Additional Supporting Information may be found online in the Supporting Information section.

**How to cite this article:** Contreras-Lopez R, Elizondo-Vega R, Paredes MJ, et al. HIF1α-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions. *The FASEB Journal.* 2020;34:8250–8264. https://doi.org/10.1096/fj.201902232R