IL-27 receptor signaling regulated stress myelopoiesis drives Abdominal Aortic Aneurysm development

Abdominal Aortic Aneurysm (AAA) is a vascular disease, where aortic wall degradation is mediated by accumulated immune cells. Though cytokines regulate the inflammatory milieu within the aortic wall, their contribution to AAA through distant alterations, particularly in the control of hematopoietic stem cells proliferation and myeloid cell differentiation remains poorly defined. Here we report an unexpected pathogenic role for interleukin-27 receptor (IL-27R) in AAA development as genetic inactivation of IL-27R protected mice from AAA induced by Angiotensin (Ang) II. The mitigation of AAA in IL-27R deficient mice is associated with a blunted accumulation of myeloid cells in suprarenal aortas due to the surprising attenuation of Ang II-induced expansion of HSCs. The loss of IL-27R engages transcriptional programs that promote HSCs quiescence and suppresses myeloid lineage differentiation, decreasing mature cell production and myeloid cell accumulation in the aorta. We, therefore, illuminate how a prominent vascular disease can be distantly driven by cytokine dependent regulation of the bone marrow precursors.


Introduction
Abdominal Aortic Aneurysm (AAA) is a cardiovascular disease, which due to limited therapeutic options is a significant cause of death in the elderly population 1 . AAA is characterized by immune cell infiltration into the aortic wall and progressive degradation of the medial layer, resulting in the dilatation and rupture of the aorta, ultimately leading to fatal bleeding 2,3,4 . Since current standard of care for AAA is still limited to surgical interventions at the late stages, a better understanding of its mechanisms, particularly the inflammatory nature of this disease, is urgently needed. Various risk factors are associated with AAA pathogenesis, including elevated blood pressure that is mediated by the activation of the renin-angiotensin system (RAS) and an increase in Angiotensin II (Ang II). Long-term infusion of Ang II in mice is able to recapitulate many aspects of AAA in humans, as it induces AAA formation in the ascending aorta 5,6 . In addition to regulating blood pressure, Ang II is known to control the function of various vascular wall cells predominantly through AT1a receptor (AT1aR) signaling 7, 8, 9, 10, 11, 12 . Chronic inflammation caused by the infiltration of various immune cells is a key driver of AAA etiology and pathogenesis 2,3,4 . In particular, neutrophils and monocytes, which are bone marrow (BM) derived myeloid cells, play an important role in the initiation of aortic wall destruction 13,14,15,16,17 . Their output by hematopoietic stem and progenitors cells (HSPCs) in the BM can be influenced by environmental stimuli and stresses. During infections or inflammatory insult, a variety of cytokines and danger associated molecular patterns (DAMPs) stimulate HSPCs to rapidly increase the production of innate immune cells 18,19,20 . Although alterations in myelopoiesis have been reported to play a role in AAA progression 4,16 , the key cytokine signaling factors that control HSPCs fate and myelopoiesis in AAA have yet to be determined. Hematopoietic stem cells (HSCs) were shown to express the Angiotensin II receptor, AT1aR and respond to elevated Ang II levels during cancer development 21 .
Furthermore, Ang II infusion promotes the expansion of murine HSPCs, suggesting that HSPCs may be affected during AAA pathogenesis characterized by elevated levels of Ang II 22 .
Interleukin (IL)-27 is a member of IL-6/IL-12 cytokine superfamily that regulates various hematopoietic and non-hematopoietic cells in infectious diseases and autoimmunity 23,24,25,26,27,28 . Nevertheless, knowledge regarding the role of IL-27 in regulation of hematopoiesis in chronic inflammatory diseases remains limited. Moreover, the role of IL-27R signaling in AAA pathogenesis, inflammation and HSPCs homeostasis has yet to be investigated.
Here we found that inactivation of IL-27R protects mice from Ang II-induced AAA.
Mechanistically, we showed that IL-27R signaling is essential to drive Ang II-mediated HSPCs proliferation and myeloid differentiation. Our unexpected findings illustrate how IL-27 signaling acts distantly to control AAA development by cooperating with stress-induced factors in the bone marrow to accelerate stress myelopoiesis, promote the production of myeloid cells that are subsequently recruited to the aortic wall mediating its destruction.

IL-27R signaling promotes Ang II-induced abdominal aortic aneurysm development
Inactivation of IL-27R exacerbates atherosclerosis 26,27,28 and leads to the development of abdominal aortic lesions which typically are rare in atherosclerotic mice. As atherosclerosis and AAA may share some underlying chronic inflammatory mechanisms 29 , we anticipated that IL-27R-deficiency would increase inflammation and promote AAA.
To evaluate the potential role of IL-27R signaling in AAA, we employed a well characterized mouse model of AAA driven by chronic Ang II infusion through a surgically implanted osmotic mini-pump (800 ng · kg −1 min −1 ) into mice on Apoe -/background 5,30 . To exclude any differences in genetics or microbiota, we used cage-mate and littermate controls. Male and female Apoe -/-, Apoe -/-Il27ra +/or Apoe -/-Il27ra -/mice were fed with a western diet (WD) for 8 weeks followed by Ang II pump implantation. Four weeks later, mice were assessed for bulging of abdominal aorta and AAA development ( Figure 1A). We found that Ang II infusion induces AAA formation in IL-27R proficient Apoe -/and Apoe -/-Il27ra +/control mice, while unexpectedly the incidence of AAA was markedly reduced in Apoe -/-Il27ra -/mice ( Figure 1B-F). Both male and female Apoe -/and Apoe -/-Il27ra +/mice developed larger AAAs with visual hemorrhages in the artery walls compared to their Apoe -/-Il27ra -/counterparts ( Figure 1B, C). Blood pressure was elevated in response to Ang II infusion; but IL-27R regulated AAA independent of effects on blood pressure, body weight or plasma lipids (Supplemental Figure 1A,B, not shown). Verhoffvan Gieson staining revealed extensive disruption and degradation of elastic lamina in the aortas of both Apoe -/and Apoe -/-Il27ra +/mice, while no significant elastin degradation was found in Apoe -/-Il27ra -/mice ( Figure 1D). Female Apoe -/and Apoe -/-Il27ra +/mice ( Figure 1E) developed slightly lower rates of AAA than did their male counterparts ( Figure 1F); however, the incidence of AAA was reduced by IL-27R-deficiency in Apoe -/-Il27ra -/mice of both genders ( Figure 1E,F), indicating that IL-27R mediated promotion of AAA in both genders. While both Apoe -/and Apoe -/-Il27ra +/mice experienced significant AAA-related mortality, Apoe -/-Il27ra -/mice exhibited a 100% ( Figure 1G, H). Pathological severity index based on the level of aortic wall degradation and immune infiltrate as previously described 31 , revealed that both female and male Apoe -/and Apoe -/-Il27ra +/mice had more advanced AAA (IV stage), than was observed in IL-27R deficient Apoe -/mice, where AAA progression was restricted to the early stages (I-II) (Supplemental Figure 2). Therefore, our data demonstrate that IL-27R signaling unexpectedly promotes AAA.
Thus, IL-27R deficiency leads to the reduced accumulation of myeloid cells, especially monocytes and neutrophils in suprarenal aortas. This further results in a decreased expression of myeloid-derived chemokines, cytokines and enzymes in the area of AAA formation.

IL-27R signaling is required for the regulation of BM HSPCs during AAA development
Inflammation associated with infection and injury is also known to modulate output and mobilization of myeloid cells from the bone marrow 20, 32 . Next we sought to determine if reduced myeloid cell accumulation in AAA sites of suprarenal aortas and AAA development in the absence of IL-27R is associated with alterations in hematopoiesis. We analyzed the cellular composition of BM isolated from Apoe -/-Il27ra -/and control Apoe -/-Il27ra +/mice. In steady-state (PBS-infusion), IL-27R-deficiency did not cause dramatic changes in LSK compartment (Lineage -, Sca-1 + , c-Kit + ) or in myeloid progenitors (Lineage -, c-Kit + , Sca-1 -) ( Figure 3A, C, D), including common myeloid progenitors (CMP, Lineage -, Sca-1 -, c-Kit + , FcgII/III -, CD34 + ) and granulocyte-monocyte progenitors (GMP, Lineage -, Sca-1 -, c-Kit + , FcgII/III + , CD34 + ) ( Figure 3D and data not shown). The percentage of long-term progenitors (LT-HSC, Lineage -, Sca-1 + , c-Kit + , CD150 + , CD48 -) was slightly elevated, while the percentage of LSK CD48 + CD150population was slightly reduced in IL-27R deficient mice ( Figure 3B,C). In agreement with previous observations 22 , Ang II infusion significantly increased the percentage of LSK and total myeloid progenitors in Apoe -/-Il27ra +/mice ( Figure 3A, C, D) compared to PBS treated "non-stressed" controls, indicating that Ang II-induced AAA development is indeed accompanied by significant changes in myeloid cell development. Surprisingly, Ang II driven expansion of LSK or myeloid precursors was blunted in the absence of IL-27R ( Figure 3A, C, D). The percentage of LT-HSC did not change in response to Ang II in Apoe -/-Il27ra +/but remained elevated in Apoe -/-Il27ra -/mice (Fig 3B, C). A mild increase in CD48 + CD150population in response to Ang II was found in controls, while Ang II-treated IL-27R deficient mice still displayed a reduction of these cells ( Figure 3B, C). Ang II infusion also decreased CMP and GMP in Apoe -/-Il27ra -/-( Figure 3D).
Only a slight reduction of myeloid progenitors in the absence of IL-27R signaling was found in the spleen (data not shown).
The Ang II receptor, AT1aR is expressed on HSPCs 21 and Ang II infusion promotes both proliferation and myeloid-biased differentiation of HSCs 22 . To test the ability of HSPCs to proliferate in response to Ang II, we assessed BM cell proliferation by in vivo BrDU incorporation. We found that BrDU incorporation was significantly lower among various precursor cells in the BM of Ang II-infused Apoe -/-Il27ra -/mice compared to Apoe -/-Il27ra +/controls ( Figure 3E, F), while no difference in proliferation level was observed in PBS-infused mice (Supplemental Figure 3). These data suggest that IL-27R signaling potentiates the proliferative effect of Ang II on HSPCs.
To evaluate the effect of IL-27R signaling on the clonogenic and differentiation capacities of HSPCs, we performed ex vivo colony formation assay. Lineage-depleted (Lin-) BM HSPCs were isolated from Apoe -/-Il27ra -/and Apoe -/-Il27ra +/mice after 4 weeks of Ang II infusion and cultured in M3434 media under myeloid differentiation conditions. We found that IL-27R deficient HSPCs showed a marked decrease in total numbers of colonies compared to IL-27R sufficient cells ( Figure 3G). Our data suggest that IL-27R signaling potentiates the Ang IIinduced "stress" myelopoiesis during AAA development.

AAA
To evaluate if a cell-intrinsic lack of IL-27R signals render HSPCs unable to expand and contribute to AAA in an overall IL-27R sufficient environment, we conducted competitive bone marrow transfer (BMT) analysis. BM isolated from Apoe -/-CD45.1 + (further referred as WT) or Apoe -/-Il27ra -/-CD45.2 + (further referred as Il27ra -/-) congenic mice were mixed in different ratios including 90%:10%, 50%:50% and 10%:90%, respectively, and transplanted into lethally irradiated Apoe -/-CD45.1 IL-27R sufficient recipients. Mice were allowed to reconstitute the BM for 4 weeks followed by 10 week WD feeding. The efficiency of BM reconstitution was confirmed by FACS for CD45.1/CD45.2 ratio in the blood of naive unchallenged mice 4 weeks after BMT (Supplemental Figure 4). After 8 weeks of WD feeding mice were implanted with Ang II pump to induce AAA and assess the effect of Ang II on the BM cells and their role in AAA ( Figure 4A).
Interestingly, all mice transplanted with a 90%wt:10%Il27ra -/mix of BM developed late stages of AAA and some died due to AAA rupture ( Figure   Of note, IL-27R deficiency was previously reported to promote atherosclerosis in Apoe -/mice 28 . Here we found that Ang II infusion into mice with already developed atherosclerosis (8 weeks of WD) strongly accelerated the disease in control, but not in IL-27R deficient mice (Supplemental  Figure 5D). These results provide an explanation for equal atherosclerotic lesion sizes between Apoe -/-Il27ra +/and Apoe -/-Il27ra -/-Ang II infused mice.
Overall, our data suggest that Ang II-driven expansion of HSCs and myelopoiesis facilitates myeloid cell accumulation and AAA and this process is dependent on IL-27R signaling, which provides HSCs and myeloid progenitors with competitive fitness required for the expansion.

IL-27R signaling regulates HSCs quiescence transcriptional programs in Ang II-induced myelopoiesis
To gain insights into the mechanisms by which IL-27R signaling influences HSCs function and "stress-induced" myelopoiesis in AAA, we performed whole transcriptome RNA sequencing analysis of FACS-sorted CD150 + CD48 -LT-HSCs isolated from the BM of Apoe -/-Il27ra -/-, Apoe -/-Il27ra +/or Apoe -/fed with WD and infused with PBS or Ang II for the last 2 weeks of WD feeding. Consistent with our functional data in vivo and ex vivo, we found that the lack of IL-27R signaling does not significantly affect transcriptional profile of LT-HSCs in "steady state" (PBSinfused mice), where only 16 genes were differentially expressed (FDR<5%) between Apoe -/-Il27ra -/and Apoe -/-LT-HSCs; however, Ang II infusion dramatically changed transcriptional profile and led to significant differential expression of 587 genes (FDR<5%) between Apoe -/-Il27ra -/and Apoe -/-LT-HSCs ( Figure 5A). Interestingly, Agr2, an inhibitor of p53 pathway 33 that is downregulated in Apoe -/-Il27ra -/-LT-HSCs, was the only gene that was differentially expressed in both PBS and Ang II-treated IL-27R-deficient HSCs ( Figure 5A). Gene set enrichment analysis using Ingenuity Pathway Analysis (IPA) of genes specific to Apoe -/-Il27ra -/-LT-HSCs revealed several significantly de-regulated pathways that had a common effect among all member genes (Supplemental Table 1). Using IPA upstream regulator analysis, we found 24 regulators with a significant number of their known targets (at least 5 targets, p<0.05) overrepresented in the list of significantly affected by IL-27R deficiency genes whose combined behavior showed change in activation status of upstream regulator ( Figure 5B). The majority of the regulators that we found to be significantly activated or inhibited have been implicated into These results suggest that IL-27R signaling maintains the balance between quiescence, proliferation and differentiation in LT-HSCs and show that the responsiveness of HSPCs to Ang II is blunted in the absence of IL-27R, suggesting that the lack of IL-27R signaling is associated with a previously described more quiescent state 45 . These data are consistent with our finding showing reduced BrDU incorporation in IL-27R deficient LT-HSCs. We next validated some of the genes from RNA sequencing analysis on isolated Lin -HSPCs. Q-RT-PCR analysis revealed in IL-27R deficient cells a reduction of the expression of genes previously involved into regulation of proliferation and myeloid lineages differentiation 34, 38, 46, 47 (Supplemental Figure   S6) Along with the quiescence gene signature, IL-27R deficient HSPCs from Ang II treated mice were characterized by elevated p21 ( Figure 6A). Importantly, infusion of Ang II into Apoe -/-Il27ra +/controls triggered p21 downregulation, whereas p21 was still expressed in Apoe -/-Il27ra -/-HSPCs ( Figure 6A). To assess whether the observed effect on p21 expression was dependent upon Ang II and not another aspect of the BM microenvironment, we isolated HSPCs from Apoe -/-Il27ra +/or Apoe -/-Il27r -/mice and stimulated them in vitro with Ang II. We found that direct Ang II stimulation strongly reduces p21 expression in control Apoe -/-Il27ra +/cells, but not in Apoe -/-Il27ra -/-HSPCs ( Figure 6B).
Several signaling pathways could participate in the regulation of p21 expression, including interferon and Myc pathways, both of which are enriched in Ang II-treated wild type cells ( Figure 5B, C). In addition, p53 is one of the key upstream bona fide regulators of p21 expression. p53 displayed the most number of overrepresented targets among the gene list (29 targets, p=2x10 -6 ; z-score=1.74). We also found that p53 protein expression was reduced by in vitro stimulation with Ang II in Apoe -/-Il27ra +/-HSPCs, while the downregulation was incomplete in IL-27R deficient cells ( Figure 6B).
Collectively, these data suggest that HSPCs depends on IL-27R signaling to regulate the expression of p21 and other quiescence/proliferation regulators. Therefore, IL-27R signaling is required to control the balance between quiescence and cell-cycle entry in response to AngIIinduced myelopoiesis during AAA progression ( Figure 7).

Discussion
The function of IL-27R signaling was extensively investigated in various infectious models 23,24,25 , and an anti-inflammatory role IL-27R has been demonstrated in atherosclerosis 26,27,28 . Moreover, some of the IL-27R deficient mice employed in the atherosclerosis studies developed frank abdominal aorta lesions, similar to incipient AAA sites, raising the possibility that IL-27R might also restrict spontaneous AAA. The role of IL-27R in AAA, another vascular pathology with clear role of yet unidentified immune cytokine-driven mechanisms, however has never been assessed. Here we made the unanticipated observation that Apoe -/-Il27ra -/mice were largely protected from Ang II-induced AAA. This correlated with diminished accumulation of monocytes and neutrophils in suprarenal aortas, which is a key early event in AAA 13,14,15,16,17,41 . The expression of myeloid cell derived cytokines and chemokines was also decreased in the AAA lesions of Apoe -/-Il27ra -/mice infused with Ang II. Although homing mechanisms play an important role in the regulation of immune cell accumulation at the site of inflammation (AAA) 17,48 , the recruitment of a large amount of immune cells into rapidly developing AAA lesions should also require an increased BM output in production of these cells, known as "stressinduced" BM myelopoiesis. The unifying regulators, required for the AAA development, vessel inflammation and 'stress myelopoiesis", however remained unknown. Ang II was shown to induce the mobilization of splenic monocytes, while splenectomy and subsequent reduction of circulating monocytes suppresses AAA development 16 . Moreover, Ang II was shown to act directly on HSCs in BM and induce their self-renewal and rapid amplification of myeloid progenitors with subsequent production of mature myeloid cells 22 . Here we found that in our model Ang II causes the expansion of various HSCs and progenitor cells, including LT-HSCs and CD48 + CD150population as well as more mature myeloid precursors in the BM of control Apoe -/or Apoe -/-Il27ra +/mice. A surprising observation of our studies was, however, that inactivation of IL-27R signaling significantly blunted Ang II-driven expansion of HSCs and progenitor cells, indicating a novel role of IL-27R signaling potentiating Ang II-induced myelopoiesis, which in turn promotes AAA.
Several cytokines have been implicated in the regulation of immune cells in AAA site 3, 29, 49, 50, 51, 52, 53, 54, 55 ; however, the possible contribution of cytokine signaling in controlling of AAAdriven "stress induced" HSCs expansion and differentiation toward myeloid lineages has never been reported. Here, we for the first time establish IL-27R signaling as a critical regulator of Ang II-driven proliferation and differentiation of HSC, essential for Ang II-induced stress myelopoiesis in a non-infectious, chronic vascular injury model. Given the role of myeloid cells in host defense and tissue repair 56, 57, 58 this cytokine-driven induction of HSCs may be a common mechanism regulating the rapid need for increased BM output during various pathophysiological processes.
Quiescence is one of the key characteristics of HSCs at the steady state, when a significant BM output of myeloid cells is not needed. HSCs quiescence is tightly regulated by many factors including IFN signaling and miRNAs (mir-21, mir-223, let-7) 36, 59, 60, 61 .
Maintenance of quiescence is crucial for prevention of both stem cell pool exhaustion and development of hematopoietic malignancies 62,63 . In chronic diseases, like AAA, prolonged exposure to "stress" factors ultimately affecting BM cells, such as high fat diet and Ang II 22 By performing RNAseq analysis of purified LT-HSCs we found that IL-27R deficiency rendered LT-HSCs unable to upregulate the expression of genes involved in cell proliferation and myeloid cell differentiation normally induced by Ang II. Moreover, IL-27R deficient LT-HSCs were characterized by a transcriptional profile that supports a quiescent state even following exposure to Ang II. Specifically, we found that ablation of IL-27R causes the upregulation of mir-21, mir-223, let-7 and p53 and the downregulation of E2F, CCND1, IRF3, IFN-"gene signature" and Myc, which are have been linked to a less proliferative and more quiescent state. In agreement, we also detected reduced BrDU incorporation in Apoe -/-Il27ra -/-HSCs in vivo. Many of these regulators have also been previously suggested into the control HSCs fate. Indeed, mir-21 was previously implicated in hematopoietic suppression via activation of TGFβ signaling 39 . Mir-223 has been reported to attenuate hematopoietic cell proliferation 40 , while let-7 was shown to regulate HSCs fate by controlling self-renewal, proliferation, quiescence and differentiation via inhibition of TGFβ pathway 67,68 . Moreover, let-7 family members were shown to repress cell cycle regulators (Cyclin D1) and negatively regulate Myc expression 42, 43, 44 . Our RNA seq data also revealed downregulation of Cyclin D1, E2F, Myc and "IFN-gene signature".
TGFβ, p53 and IFN pathways may converge at the level of p21 expression, a key negative regulator of cell proliferation, whose levels are decreased by Ang II stimulation in an IL-27R dependent manner. Taken together, the gene expression profile of IL-27R-deficient LT-HSCs is characteristic of quiescent state and remains as such even when AAA-inducing AngII driven stress is applied.

Implantation of Angiotensin II pumps
Ang II containing osmotic mini-pumps were prepared and implanted as previously described 5 .
Briefly, mice were anesthetized and osmotic mini-pumps (Alzet 2004) loaded with Angiotensin II (800ng/kg/min; Calbiochem) were surgically subcutaneously implanted in the mid-scapular area over the shoulder blade followed by closing the wound with clips. Abdominal aortic aneurysm formation was analyzed after 14 or 28 days of Angiotensin II infusion.

Blood pressure measurements
Systolic blood pressure was measured on conscious mice after 4 weeks after infusion of Ang II Immunofluorescence staining was performed as previously describe 27 . Briefly, 5-µm frozen sections of suprarenal aortas, containing abdominal aortic aneurysm, were fixed in cold acetone for 10 min at room temperature followed by fixation in 1% paraformaldehyde in 100mmol/L dibasic sodium phosphate containing 60 mmol/L lysine and 7 mmol/L sodium periodate at pH 7.4 on ice. After sections were blocked with avidin/biotin (Vector Laboratories, Burlingame, CA) for 10 min each, followed by blocking with 5% normal goat serum in 1% BSA in PBS for 15 min.

Verhoeff-Van Gieson staining of elastic fibers
5-µm sections of suprarenal aortas (with or without AAA) were cut and staining of elastic fibers was performed. Frozen sections were hydrated followed by staining in Verhoeff's solution for 1h.
After slides were differentiated in 2% ferric chloride for 2 min and treated with 5% sodium thiosulfate for 1 min. Sections were counterstained in Van Gieson's solution for 5 min and dehydrated (all reagents were purchased from Electron Microscopy Sciences, Hatfield, PA). All images were acquired with microscope Eclipse 80i.

Flow cytometry analysis of isolated cells from AAA
Cells were isolated from suprarenal aortas (with or without AAA) of Apoe -/-Il27ra +/and Apoe -/-Il27ra -/mice infused with Ang II and analyzed by flow cytometry. Briefly, mice were sacrificed by CO 2 inhalation, and aortas were perfused with PBS containing 2% heparin to remove all traces of blood. Suprarenal aortas were isolated, cut into small pieces and incubated in a cocktail of

In vivo BrDU incorporation
Mice were injected i.p. with a single dose of BrDU (1mg/mouse) (BD Pharmigen) in sterile PBS.

Immunomagnetic isolation of HSPCs
HSPCs were isolated using Easy Sep Mouse Hematopoietic Progenitor Cell Isolation Kit

Western Blot
Cell lysates of sorted HSPCs were separated by 4-12% Bis-Tris Protein Gels (Invitrogen) and transferred to PVDF transfer membranes (Invitrogen). Each membrane was washed with TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20) and blocked with 5% skimmed milk for 1 h prior to incubation with a 1:1,000 dilution of the appropriate primary antibody. Each membrane was washed, and primary antibodies were detected with a 1:5000 dilution of HRPconjugated rabbit anti-mouse IgG or mouse anti-rabbit IgG (Cell Signaling). The reactive bands were visualized with enhanced KODAK chemiluminescence BioMax film (Carestream Health Inc.).

RNA-sequencing and data processing and analysis
A total of 5,000 LT-HSCs (Sca-1 + c-kit + CD150 + CD48 -) were FACS-sorted from the BM of Apoe -/ , Apoe -/-Il27ra +/or Apoe -/-Il27ra -/mice infused with Ang II or PBS for 2 weeks. RNA was extracted using Quigen RNA isolation kit according to manufacture protocol. Total RNA libraries were prepared by using Pico Input SMARTer Stranded Total RNA-Seq Kit (Takara). 250pg-10ng total RNA from each sample was reverse-transcribed via random priming and reverse transcriptase. Full-length cDNA was obtained with SMART (Switching Mechanism At 5' end of RNA Template) technology. The template-switching reaction keeps the strand orientation of the RNA. The ribosomal cDNA is hybridized to mammalian-specific R-Probes and then cleaved by ZapR. Libraries containing Illumina adapter with TruSeq HT indexes were subsequently pooled and loaded to the Hiseq 2500. Paired end reads at 75bp with 30 million reads per sample were generated for the bioinformatic analysis. RNA-seq data was aligned using STAR 69