Hypoxia-Inducible Factor-2α Is an Essential Catabolic Regulator of Inflammatory Rheumatoid Arthritis

Hypoxia-inducible factor-2α (HIF-2α) is sufficient to cause experimental rheumatoid arthritis and acts to regulate the functions of fibroblast-like cells from tissue surrounding joints, independent of HIF-1α.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that mainly targets the synovial membrane, resulting in destruction of the joint architecture. The pathophysiology of RA involves numerous cell types, including T cells, B cells, macrophages, synoviocytes, chondrocytes, and osteoclasts, all of which contribute to the process of RA pathogenesis [1]. T-cell-mediated autoimmune responses play an important role in RA pathogenesis, in which interleukin (IL)-17-producing T-helper cells (T H 17) act as crucial effectors [1,2]. RA is characterized by synovial hyperplasia and synovitis with infiltration of immune cells. Synovial tissues express numerous cytokines that have been directly implicated in many immune processes of RA pathogenesis [1,3]. Additionally, an aggressive front of hyperplastic synovium, called the pannus, invades and destroys mineralized cartilage and bone through the action of osteoclasts [1,3]. Synovial hyperplasia results from a marked increase in macrophage-like and fibroblast-like synoviocytes (FLS). Accumulating evidence indicates that activated FLS are among the key players in RA joint destruction [4]. FLS actively contribute to the initiation, propagation, and maintenance of synovial inflammation through secretion of factors and direct cellcell interactions. For instance, cytokines and chemokines produced by FLS attract T cells to RA synovium, and the interaction of FLS with T cells results in activation of both cell types. FLS in the inflamed synovium also contribute to RA pathogenesis by producing matrix-degrading enzymes involved in cartilage destruction; RANKL (receptor activator of nuclear factor-kB ligand), which regulates osteoclast differentiation, leading to bone erosion; and angiogenic factors associated with blood vessel formation [4].
Despite therapeutic advances, the etiology of RA pathogenesis has not yet been entirely elucidated, and effective treatment of RA remains a significant unmet medical need.
A prominent feature of the inflamed RA synovium is hypoxia [5][6][7], suggesting a possible role for hypoxia-inducible factors (HIFs) in RA pathogenesis. HIFs are members of a transcription factor family that act as ''master regulators'' of the adaptive response to hypoxia [8,9]. Of the three isoforms, HIF-1a (encoded by HIF1A) and HIF-2a (encoded by EPAS1) are the most extensively studied. HIF-1a is up-regulated in RA synovium [10][11][12], where it appears to be associated with angiogenesis [5][6][7]. HIF-1a is also expressed in T H 17 cells, where it serves to regulate T H 17/T reg balance; a lack of HIF-1a in T H 17 cells impairs their differentiation [13,14]. Additionally, loss of HIF-1a in myeloid cells reduces the RA pathogenesis caused by K/BxN serum transfer [15]. Although these results suggest that HIF-1a is an important mediator of RA pathogenesis, whether HIF-1a is sufficient to cause RA pathogenesis in vivo has not been previously demonstrated. Most strikingly, HIF-2a, which is closely related to HIF-1a, has not yet been investigated for a role in RA pathogenesis. Indeed, despite many similarities between HIF-1a and HIF-2a, these two isoforms show different sensitivity to oxygen tension and display distinct, and sometimes opposing, cellular activities [8,9]. Here, we present an extensive study of the function of HIF-2a in experimental inflammatory arthritis in mice. We also investigated whether the role of HIF-2a is independent of, complementary to, or redundant with that of HIF-1a in the development and pathogenesis of experimental RA. We report here that HIF-2a is an essential catabolic regulator of RA pathogenesis, independent of the action of HIF-1a.

HIF-1a and HIF-2a Are Differentially Up-Regulated in RA Synovium
To explore possible functions of HIFs in RA pathogenesis, we first examined the expression pattern of HIFs by immunostaining human RA joint sections. HIF-2a was highly expressed in the intimal lining of human RA synovium, where other markers of inflamed RA synovium were expressed, including IL-6, matrix metalloproteinase (MMP)3, and MMP13 ( Figure 1A). Indeed, double immunostaining for HIF-2a and these markers revealed their co-localization in human RA synovium ( Figure 1B). HIF-2a was also up-regulated in tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in bone tissue and chondrocytes in damaged cartilage, but not in the intact, undamaged part of human RA cartilage ( Figure S1A). In contrast, HIF-1a was detected only in a few cells in the sublining and deep layer of human RA synovium ( Figure 1A). However, neither HIF-1a nor HIF-2a was detected in human osteoarthritis (n = 10), psoriatic arthritis (n = 2), or gouty arthritis (n = 2) synovium ( Figure S1B). These results indicate RA-specific differential up-regulation of HIF-1a and HIF-2a in synovial tissues.
We extended these results using the collagen-induced arthritis (CIA) model of RA in DBA/1J mice. This is a commonly used experimental model of inflammatory joint arthritis caused by a Tcell-dependent, antibody-mediated autoimmune response directed against cartilage type II collagen [16]. Compared with nonimmunized (NI) control joints, joints in CIA mice exhibited destruction typical of RA (Figure S1C-E). HIF-2a was highly upregulated in the region lining the CIA synovium ( Figure 1C), where it was co-localized with the RA-synovium markers, IL6, MMP3, and MMP13 ( Figure 1B). Unlike HIF-2a expression, HIF-1a was rarely detected in the intimal lining, but was detected in cells of the sublining and deep layer ( Figure 1C). Similar to human RA joint tissues, HIF-2a was also detected in pannus and damaged cartilage ( Figure S1F). Quantitation of relative HIF expression levels further confirmed the marked up-regulation of HIF-2a compared with HIF-1a in human RA and mouse CIA synovia ( Figure 1D). HIF-2a-positive cells were much more abundant in synovial lining cells (fibroblast-like and macrophage-like synoviocytes) compared with sublining macrophages and endothelial cells in blood vessels of RA synovium ( Figure 1D).

Overexpression of HIF-2a, But Not HIF-1a, in Joint Tissues Causes Experimental RA
The expression patterns of HIF-1a and HIF-2a in RA synovium suggested differential roles of HIF isoforms. To explore the possible in vivo functions of HIFs, we overexpressed HIF-1a or HIF-2a in the knee joint tissues of DBA/1J mice via intra-articular (IA) injection of Ad-Hif1a or Ad-Epas1 adenoviruses (1610 9 plaque-forming units [PFUs]), respectively. Immunostaining of joint tissue sections 3 wk after IA injection revealed that the respective adenoviruses caused marked overexpression of HIF-1a and HIF-2a in the synovium, cartilage, and meniscus of joint tissues (Figure 2A and B). HIF-2a expression in joint tissues caused typical RA-like phenotypic manifestations, including synovial hyperplasia and severe synovitis, determined by hematoxylin and eosin (H&E) staining and scoring of inflammation ( Figure 2C and D); marked cartilage destruction, determined by safranin-O staining and scored by Mankin's method ( Figure 2E); pannus formation and invasion into calcified cartilage and bone, determined by hematoxylin/safranin-O staining and scoring ( Figure 2E); and angiogenesis in the synovium, determined by immunostaining for CD31 and counting blood vessels in synovia of knee and ankle joints ( Figure 2E). Overexpressed HIF-2a in the synovium of Ad-Epas1-injected mice was co-localized with the RAsynovium marker IL6, as determined by double-immunofluorescence microscopy ( Figure S1G). In contrast to HIF-2a, HIF-1a overexpression did not cause any changes in joint architecture, including hallmarks of RA such as synovitis, pannus formation, angiogenesis, and cartilage destruction ( Figure 2C-E). Collectively,

Author Summary
Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic inflammation in joint tissues leading to destruction of cartilage and bone. Despite some therapeutic advances, the etiology of RA pathogenesis is not yet clear, and effective treatment of RA remains a significant, unmet medical need. Hypoxia is a prominent feature of inflamed tissue within RA-affected joints, and earlier work has implicated limited involvement of hypoxia-inducible factor (HIF)-1 a. We explored the role of a second HIF family member, HIF-2a, in RA pathogenesis. We showed that HIF-2a is markedly increased in the tissue lining the RA-affected joints. Notably and in contrast to HIF-1a, when overexpressed in normal mouse joint tissues, HIF-2a is sufficient to cause RA-like symptoms. Conversely, an HIF-2a deficiency blocks the development of experimental arthritis in mice. We discovered further that HIF-2a regulates RA pathogenesis by modulating various RA-associated functions of joint-specific fibroblastlike cells, including proliferation, expression of cytokines, chemokines, and matrix-degrading enzymes, and boneremodeling potential. HIF-2a also increases the ability of these cells to promote interleukin-6-dependent differentiation of T H 17 cells, a known effector of RA pathogenesis. We thus show that HIF-1a and HIF-2a have distinct roles and act via different mechanisms in RA pathogenesis.
these results indicate that ectopic expression of HIF-2a, but not HIF-1a, causes typical RA-like joint destruction in mice, suggesting distinct functions of HIF-1a and HIF-2a in RA pathogenesis.
We further validated HIF-2a functions in CIA by locally deleting Epas1 in joint tissues via IA injection of Ad-Cre (1610 9 PFU) in Epas1 fl/fl mice. Immunostaining of joint sections revealed that Ad-Cre injection effectively reduced the elevated levels of HIF-2a induced by CIA in joint tissues, including synovium, cartilage, and pannus ( Figure 4A). Moreover, local deletion of Epas1 in joint tissues by Ad-Cre injection significantly inhibited RA pathogenesis by blocking synovitis and synovial hyperplasia, pannus formation and invasion into calcified cartilage and bone, angiogenesis in inflamed synovium, and cartilage destruction ( Figure 4B and C). These results collectively indicate that Epas1 knockdown (Epas1 +/2 ) or local deletion (Ad-Cre) inhibits experimental RA in mice.

HIF-2a Modulates Immune Responses Without Affecting Immune System Development
Next, we investigated the inhibitory mechanisms of RA pathogenesis in Epas1 +/2 DBA/1J mice by examining immune responses. Epas1 +/2 mice showed normal populations of CD4 + and CD8 + T cells in lymph nodes, as determined by flow cytometry ( Figure 5A). Flow cytometry also revealed no differences in immune cell populations between WT and Epas1 +/2 DBA/1J mice, including CD4 + and CD8 + T cells in spleen and thymus; Foxp3-expressing regulatory T cells (T reg ) in lymph node, spleen, and thymus; naïve (CD44 low CD62L high ) and memory (CD44 high CD62L low ) CD4 + T cells in lymph node and spleen; and B220 + B cells and CD11c + dendritic cells in lymph node and spleen ( Figure S2A-D). Proliferation of CD4 + T cells and B220 + B cells isolated from lymph nodes and spleens was similar between WT and Epas1 +/2 DBA/1J mice ( Figure S2E and F). Additionally, CD4 + T cells isolated from lymph nodes and spleens of Epas1 +/2 mice showed a normal capacity to differentiate into T H 1, T H 2, and T H 17 cells, as determined by the detection of specific cytokines ( Figure 5B and Figure S2G and H).
Although immune system development was not affected in Epas1 +/2 mice, HIF-2a knockdown in these mice modulated immune responses under CIA conditions. The population of IL17A-producing cells in lymph nodes and spleens as well as the levels of secreted IL17A, which plays a key role in T H 17 cell differentiation and autoimmune responses, were significantly down-regulated under CIA conditions in Epas1 +/2 mice ( Figure 5C). We further validated the effects of Epas1 knockdown on pathogenic cytokine expression in synovial cells using a total mixed-cell population isolated from synovial tissues of WT and Epas1 +/2 mice. mRNA levels of the pathogenic cytokines IL1b, IL6, IL12, IL17A, IL17F, TNFa, and interferon (IFN)-c under CIA conditions were significantly down-regulated in the total synovial cell population isolated from Epas1 +/2 mice compared with WT littermates ( Figure 5D). Conversely, IA injection of Ad-Epas1 (1610 9 PFU) significantly increased mRNA levels of IL6, IL17A, and IL17F in the total synovial cell population compared with those in Ad-C-injected mice ( Figure 5D). Collectively, our results indicate that Epas1 knockdown in Epas1 +/2 DBA/1J mice does not alter the development pattern of the immune system, but does significantly reduce the production of pathogenic cytokines under CIA conditions.

HIF-2a Is Overexpressed in FLS of RA Synovium
HIF-2a is up-regulated mainly in the intimal lining of RA synovium, which primarily consists of FLS and macrophage-like synoviocytes [4]. We therefore examined which cell types overexpress HIF-2a in inflamed RA synovium. Double-immunofluorescence microscopy of human RA ( Figure 6A) and mouse CIA ( Figure 6B) synovia revealed co-localization of HIF-2a with FLS markers (vimentin or CD55), whereas only a subset of CD68positive macrophages expressed HIF-2a. We further examined HIF-2a expression in primary cultures of the total synovial cell  Figure 6D). These results collectively suggest that HIF-2a is mainly up-regulated in FLS of RA synovium, where it may play a major role in RA pathogenesis.
Next, we investigated the mechanisms regulating HIF-2a expression using primary cultures of mouse FLS. The proinflammatory cytokines IL1b and TNFa induced up-regulation of HIF-2a in FLS, whereas IL6 and IL17 did not affect HIF-2a expression ( Figure 6E). A pharmacological analysis using inhibitors of nuclear factor-kappaB (NF-kB) and mitogen-activated protein (MAP) kinase subtypes indicated that IL1b-and TNFa-induced HIF-2a expression in FLS is mediated by the NF-kB pathway, but not by the MAP kinase pathway ( Figure 6F). Because hypoxia is a prominent feature of the inflamed RA synovium [5][6][7], we additionally examined the role of hypoxia in HIF-2a expression in FLS. Mouse CIA synovium was hypoxic, as determined by pimonidazole staining ( Figure 6G). However, unlike HIF-1a protein levels, which were markedly elevated in FLS under hypoxic conditions, HIF-2a protein showed only minimal accumulation under the same conditions; however, Ad-Epas1 infection under normoxic conditions caused marked expression of HIF-2a protein ( Figure 6G). Collectively, these findings suggest that pro-inflammatory cytokines, rather than hypoxia, are the leading cause of HIF-2a expression in FLS under CIA conditions.

HIF-2a Regulates RA-Associated FLS Functions
FLS play a crucial role in RA pathogenesis by producing various regulatory factors [4]. We therefore explored whether upregulated HIF-2a in FLS modulates FLS functions and thereby RA pathogenesis. Because increased survival and/or proliferation of FLS contribute to synovial hyperplasia [4], we first examined HIF-2a regulation of apoptosis and proliferation in these cells. Ad-Epas1-mediated HIF-2a overexpression in primary cultured FLS did not cause apoptosis or modulate apoptosis induced by an anti-Fas antibody (unpublished data) known to cause FLS apoptosis [4]. However, HIF-2a overexpression significantly increased proliferation of FLS, and IL1b-induced proliferation was inhibited in Epas1 +/2 FLS ( Figure 7A). Moreover, staining for the cell proliferation marker Ki67 revealed the presence of proliferating cells in the intimal lining of both CIA and Ad-Epas1-infected synovia; notably, this staining was markedly reduced in Epas1 +/2 DBA/1J mice ( Figure 7B). Double immunostaining for HIF-2a and Ki67 indicated that 16% and 24% of HIF-2a-positive cells were proliferative in inflamed synovia caused by CIA and Ad-Epas1 injection, respectively ( Figure 7C).
Pannus formation and invasion into adjacent cartilage and bone are important regulatory steps in cartilage and bone erosion, which is mediated by the actions of osteoclasts [1,3,4]. Osteoclastogenesis is regulated by RANKL, which is produced by FLS and T cells, and requires physical contact of precursor cells with RANKL-expressing FLS or T cells in RA synovium [3,19]. We therefore examined a possible role for HIF-2a in FLS regulation of RANKL expression, osteoclastogenesis, and pannus formation. HIF-2a overexpression or IL1b treatment of FLS caused significant up-regulation of RANKL mRNA levels ( Figure 7D). Additionally, immunostaining indicated markedly increased levels of RANKL protein in CIA synovium of WT mice, an effect that was reduced in Epas1 +/2 mice ( Figure 7E). HIF-2a and RANKL were co-localized in CIA synovium, as determined by double immunostaining ( Figure 7E). Consistent with this, TRAP staining revealed an increase in the number of multinucleated osteoclasts in the pannus of the bone-cartilage interface of CIA and Ad-Epas1infected joints of WT mice; this too was also significantly reduced in Epas1 +/2 mice ( Figure 7F). The role of HIF-2a in osteoclastogenesis was further determined using Epas1 +/2 precursor cells, with and without HIF-2a overexpression. Osteoclastogenesis of Epas1 +/2 precursor cells was significantly reduced compared with that of WT cells ( Figure 7G). Moreover, overexpression of HIF-2a in precursor cells by Ad-Epas1 infection enhanced osteoclastogenesis ( Figure 7G). These results collectively support the idea that HIF-2a-mediated production of RANKL in FLS and osteoclastogenesis of precursor cells contribute to cartilage and bone erosion during HIF-2a-induced RA pathogenesis.

HIF-2a Up-Regulates Catabolic Factor Expression in FLS
FLS regulate RA pathogenesis by producing various cytokines, chemokines, and matrix-degrading enzymes involved in inflammation, chemotaxis, cartilage destruction, and bone erosion [4]. This led us to explore a possible role for HIF-2a in the expression of these regulatory factors. Ad-Epas1-infected FLS exhibited   (IL1b, IL6, IL11,  IL12, IL17, IL21, LIF, and TNFa), both mRNA and protein levels of IL6 and TNFa were increased in response to HIF-2a overexpression ( Figure 8B and C). Moreover, IL1b-induced upregulation of catabolic factors (matrix-degrading enzymes, cytokines, and chemokines) was abolished by the knockdown of Epas1 with two independent small interfering RNAs (siRNAs) ( Figure  S3). In contrast to the effects of HIF-2a, overexpression of HIF-1a in FLS caused up-regulation of MMP9, IL6, COX2, and VEGF, but not that of other factors regulated by HIF-2a ( Figure 8C).  [2,3], and HIF-1a has been previously shown to regulate T H 17 cell differentiation [13,14]. For instance, enhanced HIF-1a expression in T H 17 cells positively regulates T H 17 differentiation by upregulating RORct, an isoform of RAR-related orphan receptor gamma [13]. We therefore examined possible functions of HIF-2a in T H 17 cell differentiation, and thereby RA pathogenesis. We first examined mRNA levels of HIF-1a and HIF-2a during T H 17 cell differentiation. Compared with precursor CD4 + T cells, differentiated T H 17 cells exhibited significant down-regulation of HIF-2a expression and significant up-regulation of HIF-1a expression ( Figure 9A). Unlike the case with HIF-1a, which enhances T H 17 cell differentiation [13], overexpression of HIF-2a in precursor CD + T cells did not affect T H 17 cell differentiation ( Figure 9B), suggesting that HIF-2a in CD4 + T cells does not directly modulate T H 17 cell differentiation.
It is well established that IL6 plays a key role in T H 17 cell differentiation [2,3]. Consistent with this, in vitro T H 17 cell differentiation was dependent on the addition of exogenous IL6 protein ( Figure 9C). Given the marked overexpression of IL6 in FLS induced by HIF-2a, we explored possible functions of HIF-2a-regulated, FLS-derived IL6 in T H 17 cell differentiation by treating CD4 + precursor T cells with conditioned medium (CM) prepared from Ad-C (control)-or Ad-Epas1-infected FLS. T H 17 cell differentiation was evaluated by monitoring IL17A production using an enzyme-linked immunosorbent assay (ELISA). As shown in Figure 9C, addition of CM from Ad-Epas1-infected FLS from WT mice induced T H 17 cell differentiation, even in the absence of exogenous IL6 protein. The specific role of IL6 in CM was confirmed by preparing CM from FLS of Il6 2/2 mice or by adding IL6 neutralizing antibody to the CM. Compared with CM from WT FLS, CM of Ad-Epas1-infected FLS from Il6 2/2 mice did not affect in vitro T H 17 cell differentiation ( Figure 9C). Furthermore, addition of a neutralizing antibody against IL6, but not TNFa, blocked stimulation of T H 17 cell differentiation by the CM of Ad-Epas1-infected FLS ( Figure 9D). We additionally confirmed T H 17 cell differentiation by monitoring mRNA levels of IL17A and IL17F using quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis ( Figure S4). Immunostaining of synovial sections also revealed the presence  of IL17A-producing cells in mouse synovium infected with Ad-Epas1 or under CIA conditions, whereas no positive immunostaining was observed in Ad-C-infected or NI synovium ( Figure  S5A-C). Indeed, IL17A-positive cells were located in close proximity to HIF-2a-positive cells in human RA and mouse CIA synovia, as determined by double immunostaining ( Figure  S5D).

HIF-2a Does Not Cause a RA Phenotype in Il6 2/2 Mice
The above results suggest that FLS-derived IL6 plays an important role in HIF-2a regulation of experimental RA by regulating T H 17 cell differentiation. To confirm this, we investigated IL6 functions in HIF-2a-induced experimental RA using Il6 2/2 mice. Consistent with the inhibition of CIA by Il6 knockout [20,21], we also observed significantly greater inhibition of synovitis, pannus formation and invasion, cartilage destruction, and angiogenesis in inflamed synovium under CIA conditions in Il6 2/2 DBA/1J mice compared with WT littermates (Figure 9E and F). More importantly, the development of RA-like phenotypic manifestations, including synovitis, pannus formation and invasion, cartilage destruction, and angiogenesis, induced in inflamed synovium by IA injection of Ad-Epas1 was markedly diminished in Il6 2/2 DBA/IJ mice compared with WT mice ( Figure 9E and F). Our results collectively suggest that FLS-derived IL6 plays an important role in T H 17 cell differentiation and thereby contributes to HIF-2a regulation of experimental RA.

Discussion
Our current findings provide two novel insights into the regulation of RA pathogenesis by HIF pathways: the catabolic role of HIF-2a in RA pathogenesis and the differential actions of HIF-1a and HIF-2a in this disease.
In the first case, we demonstrate an essential role for HIF-2a in the pathogenesis of RA. Despite circumstantial evidence for the hypoxic status of RA synovium [5][6][7] and increased expression of HIF-2a in the synovial lining of human RA patients [9], little is currently known about the role of HIF-2a in RA pathogenesis. The results of our loss-of-function studies utilizing Epas1 knockdown in mice (Epas1 +/2 ) or local deletion in Epas1 fl/fl mice by Ad-Cre injection strongly support our conclusion that HIF-2a is necessary for RA pathogenesis. This conclusion is reinforced by the marked up-regulation of HIF-2a observed in RA synovia of humans and mouse models of RA as well as the RA-like phenotype revealed in gain-of-function studies involving IA injection of Ad-Epas1. In RA joint tissues, HIF-2a is up-regulated in various tissues, including synovium, pannus, cartilage, meniscus, and TRAP-positive osteoclasts. IA injection of Ad-Epas1 also caused up-regulation of HIF-2a in these tissues. Because numerous cell types in joint tissues contribute to the process of RA pathogenesis [1], up-regulated HIF-2a in any of these tissues could contribute to RA pathogenesis. However, because HIF-2a levels were most markedly increased in synovial cells, which are also the primary targets of adenovirus infection, we characterized HIF-2a functions in synovial tissue in the regulation of RA development. In RA synovial tissue, HIF-2a was up-regulated in most FLS in the synovium lining compartment, although some other cell types, such as macrophages, also exhibited HIF-2a up-regulation. Although we cannot rule out a contribution of these other cell types, we were able to demonstrate that HIF-2a regulates RAassociated FLS functions in experimental RA pathogenesis. These include proliferation; expression of cytokines, chemokines, and matrix-degrading enzymes; RANKL expression and osteoclastogenesis; IL6 production; and IL6-dependent T H 17 cell differentiation. Among these, IL6-dependent T H 17 cell differentiation is a crucial effector of RA pathogenesis. In this context, we demonstrated that IL6 present in CM prepared from FLS caused T H 17 cell differentiation. Moreover, IL17A-positive cells were located in close proximity to HIF-2a-positive cells, suggesting that IL6 production mediated by HIF-2a in the inflamed RA synovium affects differentiation of neighboring T H 17 cells. Additional support for this relationship is provided by our demonstration that global deletion of Il6 abolished HIF-2a-induced RA pathogenesis. Although it remains possible that production of IL6 by cell types in synovial tissue besides FLS could also contribute to the regulation of T H 17 cell differentiation, establishing this definitively would likely require a conditional FLSspecific Il6-knockout model, which, to our knowledge, has not yet been developed.
The second novel finding of this study is that HIF-1a and HIF-2a have distinct roles and act via different mechanisms in RA pathogenesis. HIF-1a is up-regulated in RA synovium [10][11][12], where it is associated with angiogenesis [5][6][7]. It has previously been demonstrated that HIF-1a regulates RA pathogenesis by directly modulating T H 17 cell functions [13,14]. In the current study, HIF-1a expression, in contrast to that of HIF-2a, was detected in a small number of cells in the sublining and deep layer of RA synovium in both humans and experimental mouse models of RA, a result consistent with other reports [10,11]. We did not extensively explore the underlying mechanisms of this differential expression of HIF isoforms in the current study. However, HIF-1a and HIF-2a show different sensitivity to oxygen tension and display distinct, and sometimes opposing, cellular activities [8,9]. Indeed, we found in this study that sensitivities to hypoxia and to pro-inflammatory cytokines differed between HIF-1a and HIF-2a in FLS. These differences may reflect the differential expression pattern of HIF-2a and HIF-2a in RA synovium. Nevertheless, ectopic expression of HIF-1a in joint tissues by IA injection of Ad-Hif1a did not cause an RA-like phenotype, suggesting that HIF-1a overexpression is not sufficient to induce RA pathogenesis. In striking contrast, HIF-2a overexpression was sufficient to activate RA pathogenesis and did so by regulating FLS functions. Collectively, our results suggest that HIF-2a regulates RA pathogenesis by acting globally to modulate the RA pathogenesis program, including angiogenesis and FLS functions, whereas HIF-1a contributes to RA pathogenesis by modulating the effector functions of myeloid and T cells. Moreover, the observation that HIF-2a deficiency, which does not affect HIF-1a expression, is sufficient to inhibit experimental RA underscores the specific roles played by HIF-2a.
RA and OA are the most common types of joint arthritis. We have previously shown that HIF-2a is a catabolic regulator of OA cartilage destruction [18,[22][23][24], demonstrating that HIF-2a causes OA pathogenesis by up-regulating catabolic enzymes such as MMP3 and MMP13 in chondrocytes, and further showing that chondrocyte-specific Col2a1-Epas1 TG mice exhibit spontaneous cartilage destruction with no evidence of synovitis [18]. Although RA and OA phenotypes share certain features, such as cartilage destruction, their etiology and pathogenesis are completely different. RA and OA also differ with respect to outcomes, cell types associated with the pathogenesis, and therapeutic approaches. For instance, OA is a degenerative joint disease (''wear and tear'' arthritis) that begins with the destruction of surface articular cartilage, subchondral bone sclerosis, and osteophyte formation in a single joint. In this type of arthritis, mechanical stresses, including joint instability and injury, and factors that predispose toward OA, such as aging, are important causes of pathogenesis [25,26]. In contrast to OA, RA is a systemic autoimmune disorder, which manifests as chronic inflammation that results in destruction of cartilage and bone tissues [2][3][4]. The inflammatory process initially affects a single joint, but the disease usually progresses to affect nearly all joints [27]. Thus, our results indicate that, despite their different etiologies and pathogenesis, both RA and OA are regulated by HIF-2a via completely different mechanisms: HIF-2a regulates OA pathogenesis by up-regulating matrix-degrading catabolic enzymes in articular chondrocytes, whereas it appears to regulate RA pathogenesis by regulating angiogenesis, various functions of FLS, and IL6-dependent T H 17 cell differentiation.
In summary, our current studies suggest that HIF-2a is an essential catabolic regulator of RA pathogenesis that acts by modulating various RA-associated FLS functions. Because the etiology of RA pathogenesis has not yet been entirely elucidated and effective treatment of RA remains a significant unmet medical need, HIF-2a may serve as an effective therapeutic target in RA treatment. In this context, an important question that remains to be evaluated is whether recently developed small-molecule inhibitors of HIF-2a [28,29] inhibit RA pathogenesis in vitro and in vivo. Additionally, because HIF-1a and HIF-2a appear to regulate RA pathogenesis through different mechanisms, both HIF isoforms could be alternative therapeutic targets in the treatment of RA disease.

Ethics Statement
The use of human materials was approved by the Institutional Review Board of Chonnam National University Hospital and Wonkwang University Hospital, and written informed consent was obtained from all individuals before the operative procedure. Mice were housed in specific pathogen-free barrier facilities and were used in accordance with protocols approved by the Animal Care and Ethics Committees of the Gwangju Institute of Science and Technology.

Human Arthritic Joint Tissues
Human RA, psoriatic arthritis, gouty arthritis, and OA joint tissues were collected from patients undergoing knee arthroplasty (Tables S1, S2, S3) and then embedded in paraffin. All RA patients had a median disease duration of ,6 y, high disease activity (i.e., median DAS of 5.61), and received medications, including a variety of disease-modifying antirheumatic drugs (Table S1). Because joint tissues were obtained from patients undergoing knee arthroplasty, our samples represent relatively late-stage RA.

Mice and Experimental Arthritis
Male DBA/1J, C57BL/6, Epas1 +/2 , Epas1 fl/fl , and Il6 2/2 mice were used for experimental RA studies. The C57BL/6 strains of Epas1 +/2 , Epas1 fl/fl , and Il6 2/2 mice were described previously [18,22]. Epas1 +/2 , Epas1 fl/fl , and Il6 2/2 (C57BL/6) mice were backcrossed against the DBA/1J strain for eight generations to generate Epas1 +/2 DBA/1J, Epas1 fl/fl DBA/1J, and Il6 2/2 DBA/ 1J mice, respectively. CIA was produced in WT and Epas1 +/2 DBA/1J mice using a standard protocol [16]. Briefly, mice were intradermally injected at the base of the tail with incomplete Freund's adjuvant alone (control) or Freund's adjuvant containing 100 mg of collagen type II; a booster injection was given 21 d later. Epas1 fl/fl DBA/1J mice were IA-injected with Ad-C or Ad-Cre (1610 9 PFU) on days 0, 3, and 6, followed by a booster injection with collagen type II. Mice were maintained for an additional 2 wk. The incidence and severity of RA were evaluated on the indicated days after the first immunization. Severity was evaluated using a clinical score (grade 0-4) of paw swelling based on the level of inflammation in each of the four paws [16]. Joint tissues from mice were fixed, decalcified with 0.5 M EDTA (pH 8.0), embedded in paraffin, and sectioned at 5-mm thickness. Synovitis was evaluated by H&E staining of joint sections, and synovial inflammation (grade 0-4) was scored as described by Tang et al. [30]. The pannus in joint tissues adjacent to cartilage and bone was visualized by H&E staining with or without safranin-O staining of cartilage, and pannus formation was scored (grade 0-4) as described by Tang et al. [30]. Cartilage destruction was examined by safranin-O staining and scored using Mankin's method, as previously described [18,31].

FLS Culture, CM Preparation, and Proliferation Assays
Total synovial cells were isolated from knee joint synovium of CIA mice. Synovial tissues were minced and digested in collagenase for 4 h at 37uC. The cells were plated on coverslips in RPMI-1640 medium and incubated for 4 d. FLS were isolated from NI and CIA joint tissues of WT and Il6 2/2 mice [33]. FLS between passage 4 and 8 were used for further analysis. Pure FLS (.90% CD90 + /,1% CD14 + ) were identified by flow cytometry using antibodies against the fibroblast marker CD90 and the macrophage marker CD14 (Abcam). For the preparation of CM, FLS were infected with Ad-C or Ad-Epas1 at a multiplicity of infection (MOI) of 800 for 2 h and incubated on 35-mm culture dishes containing 1 ml of RPMI-1640 medium. CM was used to treat CD4 + precursor T cells during differentiation into T H 17 cells. FLS proliferation in culture was quantified by measuring BrdU incorporation during DNA synthesis. Proliferating cells in synovial sections were identified by detecting Ki67 using an antibody obtained from Novus Biologicals.

T-Cell Differentiation and Proliferation
CD4 + T cells from WT and Epas1 +/2 mice were purified from lymph nodes and spleens. T H cell differentiation was induced by plating cells (2610 6 cells/ml) on culture dishes coated with anti-CD3 antibody (1 mg/ml) in the presence of soluble anti-CD28 antibody (2 mg/ml) under the following T H cell-skewing conditions: T H 1 cells, IL12 (10 ng/ml) and anti-IL4 antibody (10 mg/ ml); T H 2 cells, IL4 (20 ng/ml) and 10 mg/ml of antibodies against IFNc and IL12; T H 17 cells, tumor growth factor (TGF)-b (3 ng/ ml), IL6 (30 ng/ml), and 10 mg/ml of antibodies against IL4, IFNc, and IL12. IL2 (100 U/ml) was added after 24 h, and cells were cultured for 6 d. Antibodies and cytokines were purchased from BD Biosciences or PeproTech. The cells were stimulated with PMA (50 ng/ml), ionomycin (1 mM), and brefeldin A (1 mg/ ml; eBioscience). T H cell differentiation was evaluated by flow cytometry after staining for intracellular cytokines and by detecting cytokines by ELISA and qRT-PCR. Where indicated, activated CD4 + T cells were transfected by electroporation with empty vector or vector carrying Epas1. The cells were cultured under neutralizing conditions (10 mg/ml of antibodies against IL4 and IFNc) or T H 17 cell-skewing conditions for 4 d. The effects of HIF-2a overexpression on skewed CD4 + T cells were evaluated by detecting IL17A production by ELISA. For cell proliferation assays, CD4 + T and B220 + B cells were isolated from lymph nodes and spleens of WT and Epas1 +/2 DBA/1J mice. T-cell proliferation was induced by stimulating cells with anti-CD3 antibody (10 mg/ml), and B-cell proliferation was induced by stimulating cells with LPS (10 mg/ml), LPS plus IL4 (5 ng/ml), or antibodies against IgM (20 mg/ml; Jackson ImmunoResearch) and CD40 (10 mg/ml; BioLegend). Proliferation was assessed by measuring [ 3 H]thymidine (0.5 mCi/well) incorporation during the last 18 h of a 72-h culture period.

Hypoxia in FLS and Joint Tissues
For detection of the hypoxic status of mouse CIA synovium, mice immunized with type II collagen were intraperitoneally injected with hypoxyprobe-1 (pimonidazole HCl; Hypoxyprobe Inc.) at a dosage of 60 mg/kg body weight and sacrificed 6 h after injection. Paraffin-embedded joint tissues were sectioned at 5-mm thickness, and pimonidazole was detected by immunofluorescence microscopy, according to the manufacturer's instructions. For hypoxic culture of mouse FLS, cells were exposed to hypoxia for 12, 18, or 24 h in a GasPak anaerobic chamber (BBL GasPak Pouch; Becton Dickinson) at 37uC, as described previously [18]. The proportion of oxygen in each chamber was #1%.

Flow Cytometric Analysis
Leukocytes were prepared from lymph nodes draining the inflamed joint, spleen, and thymus of NI and CIA mice. Synovial cells were harvested by digesting synovial tissues with collagenase. The cells were incubated with primary antibodies for 15 min at 4uC. Antibodies against CD4, CD8, CD44, Foxp3, B220, and CD11c were purchased from eBioscience; the anti-CD62L antibody was from BD Pharmingen. Nonspecific staining was ascertained using isotype-matched control antibodies. T H cells were fixed in fixation/permeabilization buffer for 30 min; resuspended in 100 ml of permeabilization buffer; incubated for 30 min at 4uC with Alexa 488-or phycoerythrin (PE)-conjugated anti-IFNc (eBioscience), fluorescein isothiocyanate (FITC)-conjugated anti-IL4, PE-conjugated anti-IL17A or isotype control antibodies (eBioscience); and analyzed by flow cytometry using EPICS XL and EXPO32 software (Beckman Coulter).

ELISA of Cytokines and Autoantibody Production
Representative cytokines involved in RA pathogenesis (IFNc, TNFa, IL4, and IL17A) and produced by T H subsets were detected using ELISA kits (eBioscience), according to the manufacturer's protocol. IL6 and TNFa secreted into serum-free culture media by FLS infected with Ad-C or Ad-Epas1 were quantified by ELISA. Collagen type II-specific antibodies were measured by ELISA. Sera from NI and CIA mice were added into 96-well plates coated with type II collagen (5 mg/ml), incubated overnight at 4uC, washed, and incubated for 1 h with alkaline phosphatase-labeled monoclonal antibodies against mouse IgG1, IgG2a, or IgG2b (Immunology Consultants Lab). Wells were developed using p-nitrophenyl phosphate as a substrate, and the resulting color reaction was quantified using an ELISA plate reader.

Bone Marrow Culture, Osteoclastogenesis, and TRAP Staining
Bone marrow cell culture, osteoclastogenesis, and TRAP staining were performed as described previously [34]. Briefly, bone-marrow-derived macrophages were isolated from WT or Epas1 +/2 mice, seeded in 48-well plates (4610 4 cells/well), and cultured for 4 d (Ad-C and Ad-Epas1 infection in WT cells) or 5 d (WT and Epas1 +/2 precursor cells) with M-CSF (macrophage colony stimulating factor; 30 ng/ml) and RANKL (100 ng/ml) to induce osteoclastogenesis. The surface area of TRAP-stained multinuclear osteoclasts containing three or more nuclei was measured using an Osteomeasure system (Osteometrics). TRAP activity was also determined in paraffin sections of joint tissues from NI and CIA mice or mice IA-injected with 1610 9 PFU of Ad-C or Ad-Epas1. The numbers of TRAP-positive osteoclasts and their precursor cells were counted in a blinded fashion in all regions of pannus-formed bone-cartilage interface and synovium for each knee joint.

Statistical Analysis
The nonparametric Mann-Whitney U test was used for the analysis of data based on an ordinal grading system, such as synovitis, pannus, and Mankin scores. For results obtained in qRT-PCR assays, ELISAs, and analyses of blood vessel numbers, joint thickness, TRAP-positive cells, BrdU incorporation, thymidine incorporation, and apoptotic cell numbers, data were first tested for conformation to a normal distribution using the Shapiro-Wilk test and then were analyzed by Student's t test (pair-wise comparisons) or analysis of variance (ANOVA) with post hoc tests (multicomparison), as appropriate. Significance was accepted at the 0.05 level of probability (p,0.05).  Figure S2 Epas1 knockdown in mice (Epas1 +/2 ) does not affect immune system development. (A-D) Leukocytes were isolated from the indicated tissues of naïve WT and Epas1 +/2 DBA/1J mice. Cells were stained for markers of various immune cell types, and immune cell populations were determined by flow cytometry and quantified (n.10 mice). CD4 + helper T cells and CD8 + cytotoxic T cells (A); Foxp3-expressing regulatory T cells (T reg ) (B); naïve (CD44 low CD62L high ) and memory (CD44 high CD62L low ) CD4 + T cells (C); B220 + B cells and CD11c + dendritic cells (D). (E and F) CD4 + T cells (E) and B220 + B cells (F) were isolated from lymph nodes and spleens from WT and Epas1 +/2 DBA/1J mice (n.8 mice). Proliferation of cells cultured for 3 d in the absence or presence of the appropriate T-or B-cell stimulants was assessed by [ 3 H]thymidine incorporation assays. Results are expressed as counts per minute (CPM). (G and H) CD4 + T cells were purified from the lymph nodes and spleens of WT and Epas1 +/2 DBA/1J mice. T H cell differentiation was induced under T H 1-, T H 2-, or T H 17-skewing conditions. Recombinant IL2 (100 U/ml) was added after 24 h, and cells were cultured in complete medium for 6 d. Cells were restimulated with PMA, ionomycin and brefeldin A, or left untreated, and then stained for intracellular cytokines. The indicated cytokines were detected by ELISA in untreated cells (G) and restimulated cells (H) (n = 6). Values are presented as means 6 SEM. (TIF) Figure S3 Knockdown of Epas1 by siRNA inhibits IL1b-induced catabolic factor expression in primary culture FLS. (A and B) FLS were left untreated (None) or were treated with 100 nM control siRNA (C-siRNA) or the indicated amounts of two different Epas1specific siRNAs-siRNA-1 (A) or siRNA-2 (B)-and then were exposed to IL1b for an additional 24 h. mRNA levels of the indicated catabolic factors were quantified by qRT-PCR (n = 10). Values are means 6 SEM (*p,0.05, **p,0.01, ***p,0.001 compared with C-siRNA treatment). (TIF) Figure S4 IL6 produced by FLS regulates T H 17 cell differentiation. T H 17 cell differentiation was evaluated by detecting mRNA levels of IL17A (A) and IL17F (B) (n = 6). Precursor CD4 + T cells were left untreated or were treated with antibodies against CD3 and CD28 (Abs), Mix (i.e., TGFb, IL2, and antibodies against IL4, IFNc, and IL12), the indicated amount of IL6, or CM from WT FLS infected with Ad-C or Ad-Epas1 (800 MOI), or WT and Il6 2/2 FLS infected with Ad-Epas1 (800 MOI). T H 17 cell differentiation was evaluated by monitoring IL17A expression (left panels). T H 17 cell differentiation in the presence of CM from WT FLS infected with Ad-Epas1 and/or the indicated amounts of neutralizing antibodies against IL6 or TNFa was evaluated by monitoring IL17A (right panels). Values are means 6 SEM (*p, 0.05, **p,0.005, ***p,0.0005). (TIF) Figure S5 Epas1 knockdown in mice (Epas1 +/2 ) inhibits IL17 expression. (A) CIA was produced in WT and Epas1 +/2 DBA/1J mice, and IL17 protein was detected in NI and CIA synovial sections after 6 wk by immunohistochemistry. (B) The knee joints of WT and Epas1 +/2 DBA/1J mice were injected with Ad-C or Ad-Epas1 (1610 9 PFU). After 3 wk, synovial sections were immunostained for IL17 and counterstained with hematoxylin. (C) IgG was used as a negative immunostaining control. Representative images are shown (n = 6). (D) Typical images of triple-stained (HIF-2a, IL17A, and DAPI) human RA synovium and mouse CIA synovium (n = 6). Scale bar, 50 mm. (TIF)