SerpinB1 expression in Th17 cells depends on hypoxia-inducible factor 1-alpha

Highlights

• SerpinB1 is highly expressed in Th17 cells.

• SerpinB1 is pivotal for Th17 cell to acquire pathogenic signature upon IL-23 priming.

• SerpinB1 expression is mediated by mTOR/HIF1α/glycolysis in Th17 cells.

Abstract

SerpinB1, previously known as MNEI (monocyte/neutrophil elastase inhibitor), has been well established to maintain the survival of neutrophils. Our recent studies showed that SerpinB1 is also the signature gene of IL-17-producing γδT cells and Th17 cells, and its expression is maintained by IL-23 signaling. Deficiency of SerpinB1 largely ameliorates the experimental autoimmune encephalomyelitis (EAE) with enhanced granule protease-mediated mitochondrial damage leading to suicidal cell death of pathogenic CD4 T cells. However, the mechanism that induces SerpinB1 expression in Th17 cells still remains elusive. Here, we showed that SerpinB1 was induced in Th17 cells, and plays a pivotal role to maintain the pathogenic signature of IL-23-primed Th17 cells in vitro. Its expression in Th17 cells was independent of Th17-lineage specific transcript factor retinoic acid-related orphan receptor γ t (RORγt), but was controlled by glycolysis and the mammalian target of rapamycin (mTOR) signaling. Finally, by using two specific pharmacological inhibitors, our study further deciphered that hypoxia-inducible factor 1α (HIF-1α) specifically controlled the SerpinB1 expression in Th17 cells. On the other side, when HIF-1α stabilizer Dimethyloxalylglycine (DMOG) was applied, SerpinB1 expression was significantly increased in Th17 cells. Taken together, this study is the first to report that SerpinB1 expression in Th17 cells is mediated by glycolysis/mTOR/HIF-1α pathway.

Introduction

SerpinB1, previously called MNEI (monocyte/neutrophil elastase inhibitor), is an ancestral member of the superfamily of Serpins (SERine Protease Inhibitors) [1], [2]. Early studies showed high levels of SerpinB1 in myeloid cells, especially neutrophils, the short-lived cells recruited immediately to infection sites to destroy pathogenic microbes. Three related neutrophil serine proteases (NSPs), elastase, proteinase-3 and cathepsin G that can be inhibited by SerpinB1, are stored in azurophil granules of neutrophils [3], [4], [5]. In bacterial lung infection, SerpinB1 protects against inflammatory tissue injury and neutrophil death [6], [7], and in naïve mice, SerpinB1 preserves the bone marrow reserve of mature neutrophils by restricting spontaneous cell death mediated by the granule serine proteases cathepsin G and proteinase-3 [8], [9]. Most recently, SerpinB1 was discovered to be the signature gene expressed in interleukin (IL)-17-producing γδ T cells [10], IL-17-producing natural killer (NK) T cells [11], and IL-17-producing CD4 (Th17) cells [12], and plays key role in maintaining the Th17 cell pathogenicity in murine model of experimental autoimmune encephalomyelitis [13].

CD4 helper T cells are important immunoprotectants with specialized functions; Th1 protect against intracellular pathogens, Th2 against helminths, and Th17 against fungi and extracellular bacteria [14], [15]. Th17 cells produce IL17A and IL17F, which activate IL17R-expressing cells to produce inflammatory chemokines and cytokines including granulocyte-colony stimulating factor (G-CSF), facilitating granulopoiesis and neutrophil recruitment [16], [17], [18]. Although the recognition of Th17 cells as a unique lineage among CD4 T cells is relatively recent, their developmental pathway has been characterized in substantial details. Polarization of naive CD4 cells to the Th17 lineage requires transforming growth factor (TGF)-β and IL-6 [19], [20]. Retinoic acid receptor-related orphan receptor gamma t (RORγt) is the key transcription factor (‘master regulator’) for IL-17 production [21], as IL-17 expression is blocked by RORγt antagonists (digoxin [22], SR1001 [23], and TMP778 [24]). Central events of Th17 differentiation are mammalian target of rapamycin (mTOR) activation and increased glycolysis orchestrated by transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) [25], [26]. Th17 cells themselves are not pathogenic, but can be converted into pathogenic CD4 cells under the priming of myeloid cell-derived IL-23 [27]. These pathogenic CD4 cells lose IL-17-producing ability and produce IFNγ and GM-CSF instead [28], [29], [30], [31]. Altogether, the known factors that determine the Th17 cell generation fall into three categories: (i) cytokines (TGF-β, IL-6 and IL-23), (ii) transcription factors (RORγt) and (iii) metabolic regulators (mTOR, HIF-1α). In recent studies we discovered a novel SerpinB1-dependent proteolysis regulation of Th17 cell differentiation program [12], [13].

The involvement of SerpinB1 in Th17 regulatory mechanism was first hinted by the study of SerpinB1 KO (serpinb1a^−/−) mice in influenza-infection [7]. Soon we found that SerpinB1 deficiency leads to selective expansion of IL-17⁺ γδ T cells in influenza infection and at steady stage [10]. Subsequently we found that the gene (serpinb1a) is selectively expressed late during Th17 differentiation through systematic evaluation of SerpinB1 expression in CD4 cells [12]. Most recently, our study showed that SerpinB1 expression is downstream of IL-23 signaling, and its expression maintains the survival of pathogenic Th17 cells in vivo. Deficiency of SerpinB1 largely protects mice from experimental autoimmune encephalomyelitis [13].

In the current study, we demonstrated that SerpinB1 plays a key role in conferring Th17 cells the pathogenic signature upon IL-23 priming; we also investigated the events that regulate the SerpinB1 expression in Th17 cells, and discovered that SerpinB1 expression in Th17 cells is RORγt independent, however is HIF-1α/mTOR/glycolysis dependent.