Transcriptomic analysis identifies Toll‐like and Nod‐like pathways and necroptosis in pulmonary arterial hypertension

Abstract Inflammation and immunity play a causal role in the pathogenesis of pulmonary vascular remodelling and pulmonary arterial hypertension (PAH). However, the pathways and mechanisms by which inflammation and immunity contribute to pulmonary vascular remodelling remain unknown. RNA sequencing was used to analyse the transcriptome in control and rats injected with monocrotaline (MCT) for various weeks. Using the transcriptional profiling of MCT‐induced PAH coupled with bioinformatics analysis, we clustered the differentially expressed genes (DEGs) and chose the increased expression patterns associated with inflammatory and immune response. We found the enrichment of Toll‐like receptor (TLR) and Nod‐like receptor (NLR) pathways and identified NF‐κB‐mediated inflammatory and immune profiling in MCT‐induced PAH. Pathway‐based data integration and visualization showed the dysregulated TLR and NLR pathways, including increased expression of TLR2 and NLRP3, and their downstream molecules. Further analysis revealed that the activation of TLR and NLR pathways was associated with up‐regulation of damage‐associated molecular patterns (DAMPs) and RIPK3‐mediated necroptosis was involved in the generation of DAMPs in MCT‐induced PAH. Collectively, we identify RIPK3‐mediated necroptosis and its triggered TLR and NLR pathways in the progression of pulmonary vascular remodelling, thus providing novel insights into the mechanisms underlying inflammation and immunity in the pathogenesis of PAH.


| INTRODUC TI ON
Pulmonary arterial hypertension (PAH) is a devastating disease characterized by perivascular infiltration of inflammatory cells and pulmonary vascular remodelling, ultimately resulting in the right heart failure and premature death. Patient survival of advanced PAH remains poor, 1 and the pathogenic mechanisms contributed to the progression of pulmonary vascular remodelling in PAH are not well understood.
It is widely accepted that inflammation and immunity are linked to pulmonary vascular remodelling in PAH. 2 The infiltration of inflammatory cells, such as mast cells, macrophages, dendritic cells and lymphocytes, was identified in the PAH lung, and an array of inflammatory mediators, including TNFα, IL-1β, IL-6, IL-8, IL-12, MCP-1 and RANTES, was abnormally elevated in peripheral blood. 2 In addition, inflammatory infiltration was positively correlated with pulmonary arterial remodelling parameters. 3 Although it is well established inflammation and immunity are involved in pulmonary vascular remodelling and pulmonary hypertension, the pathways and mechanisms by which inflammation and immunity contributed to pulmonary vascular remodelling remain unknown.
The monocrotaline (MCT) model of PAH was widely used for over 50 years. 4 After administration of MCT, significant changes in pulmonary artery pressure, pulmonary arterioles remodelling and right ventricular hypertrophy occur. 5 MCT was thought to induce a syndrome, composed of acute lung injury, necrotizing pulmonary arteritis in about one third of the animals and pulmonary hypertension, etc. 6 The development of MCT-induced PAH was associated with dysregulated inflammation/immunity, because inflammatory cells, mainly neutrophils, macrophages, dendritic cells and lymphocytes infiltrated the lung, mainly in perivascular areas. 4 Consistently, our previous study has showed an elevated marker of macrophage infiltration. In addition to inflammatory cell infiltration, the inflammatory mediators, such as TNFα and IL-6, were elevated, with concomitantly increased pulmonary arterial remodelling parameters WT% and WA% in the progression of MCT-induced PAH. 7 The transcriptomic change during the PAH progression was investigated by using microarray. 8 Recently, high-throughput RNA sequencing (RNA-seq) has emerged as a more powerful alternative to microarray. 9 We have performed RNA-seq analysis of rat lungs isolated from control and monocrotaline (MCT)-treated rats that had been treated with MCT for a variety of weeks, and this study showed that inflammatory and immune response was occurred at the early time-point of PAH development and dysregulated inflammation/immunity were involved in the onset and progression of PAH. 10 The changes of inflammation and immunity and pulmonary vascular remodelling that occur during the PAH progression largely result from the changes in the transcriptome. Therefore, in the current study, we use the RNA-seq data set and bioinformatics approach to carry out a further analysis of the transcriptome in MCT-induced PAH, aiming to have a deeper understanding of inflammatory and immune mechanisms in pulmonary vascular remodelling.

| Animal and treatment
All procedures have been conducted in accordance with the ARRIVE guidelines and were approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University (Approval No. 2017-070, Fuzhou, China). Sprague-Dawley rats (4 -5 weeks male and female rats, 200-250g) were purchased from Shanghai SLACCAS Laboratory Animal Co., Ltd. (Certificate No. SCXK 2012-0002). The rats were raised and housed in the animal room and received food and water ad libitum. PAH model in rats was induced by a single intraperitoneal injection of 40 mg/ kg MCT (Sigma-Aldrich) as described previously. 7 A total of 17 rats were used in this study: 12 rats were randomly assigned into 4 groups and treated with MCT (n = 3, each group) and five remaining rats served as control and treated with saline. Before killing, an effort was made to diminish suffering by intraperitoneal injection of 30 mg/kg sodium pentobarbital. The MCT-treated rats were killed at the end of weeks 1, 2, 3 and 4, and control rats were killed at week 0, and as with the corresponding MCT-treated rats, at the end of weeks 1, 2, 3 and 4. Rat lungs were immediately isolated and frozen in liquid nitrogen and then stored at −80°C.

| RNA extraction, cDNA library preparation and RNA-seq
Total RNA was isolated from 50 mg lung tissues using 1 mL TRIzol reagent (Life Technology) following the manufacturer's instructions.
RNA integrity and quality were assessed by gel electrophoresis, and its concentration and purity were determined by the Thermo Scientific NanoDropTM instruments. Total RNA with high quality was used for cDNA library preparation. Library preparation and RNA-seq were performed on an Illumina HiSeq 2000 platform by Genergy Biotechnology (Shanghai) Co., Ltd. The generated raw sequences were processed through a series of steps: (a) removing the low quality reads and adapter sequences, (b) quality control using the FastQC software, (c) mapping the clean reads to rat reference genome using STAR software, (d) assembling transcripts using the software of StringTie and Cufflinks-Cuffmerge, (e) calculation of transcripts abundance using FPKM, and (f) identification of DEGs using DESeq2 software.

| Bioinformatics analysis
Bioinformatic analysis tools, including DAVID, 11

| Real-time PCR analysis
Total RNA was isolated from lung tissues of control and rats injected with MCT for 4 weeks. First-strand cDNA was synthesized by using the Transcriptor First Strand cDNA Synthesis Kit, according to the manufacturer's protocol. Real-time PCR was performed in accordance with the manufacturer's instructions, as previously described. 16 The forward and reverse primers were synthesized by Sangon Biotechnology Co.,

| Statistical analysis
Morpheus software and R package software were used for RNAseq data set analysis. The DEGs between control and MCT treatment were identified by DESeq2 package in R using a threshold of fold-change ≥2 and P ≤ .05. Data were shown as mean ± SEM, and comparison of two conditions in Pathview was performed by using Morpheus software. More details of the statistical analysis were provided in the figure legends.

| Identification of pathways related to inflammation and immunity
Our previous study has showed the elevated markers of macrophage infiltration and inflammatory mediators, such as TNFα and IL-6, with concomitantly increased pulmonary arterial remodelling parameters WT% and WA% in the progression of MCT-induced PAH. 7 RNAseq analysis of rat lungs isolated from control and MCT-treated rats identified a total of 23 200 transcripts, of which 280, 1342, 908 and 3155 were differentially expressed at the end of weeks 1, 2, 3 and 4, respectively. 10 Further hierarchical clustering analysis of the differentially expressed genes (DEGs) revealed 10 clusters of expression pattern. Cluster 1, cluster 3 and cluster 4 showed an increased pattern. In contrast, cluster 2, cluster 5 and cluster 10 showed the opposite pattern ( Figure 1). GO enrichment analysis of all the 10 clusters using DAVID showed that only cluster 1 and cluster 3 whose expression pattern resembled the changes of pulmonary arterial remodelling parameters, WT% and WA%, were associated with inflammatory and immune response ( Figure 2A; Figure S1). KEGG pathway enrichment of cluster 1 and cluster 3 using KOBAS revealed 28 significantly enriched pathway terms in cluster 1 and 10 significantly enriched pathway terms in cluster 3. The majority of the pathway terms were linked to inflammation and immunity, including Nod-like receptor (NLR) signalling pathway, Toll-like receptor (TLR) signalling pathway and NF-κB pathway in cluster 1, as well as cytokinecytokine receptor interaction and chemokine signalling pathway in cluster 3 ( Figure 2B).

| Identification of inflammatory and immune profiling
Further analysis of enriched inflammatory and immune genes in cluster 1 and cluster 3 using Venny 2.1 showed that a total of 70 and 41 genes were linked to inflammation and immunity, of which 23 and 15 were overlapped ( Figure 3A). Hierarchical clustering of the overlapped genes using Morpheus showed most of the genes were increased in a time-dependent manner ( Figure 3B). GO enrichment analysis using DAVID showed that the overlapped genes were associated with chemokine and cytokine activity ( Figure 3C). Analysis of the interaction among overlapped genes using GeneMANIA revealed that the majority of genes were co-expressed and shared C-C/C-X-C chemokine domain ( Figure 3D). Enrichment analysis using Enrichr revealed that Rela, the p65 subunit of NF-kB, was the most significantly enriched transcription factor in the overlapped genes ( Figure 3E). Collectively, the overlapped genes were co-expressed, associated with chemokine and cytokine activity and predominantly regulated by NF-κB pathway, thus maybe representing the inflammatory and immune profiling that lead to pulmonary vascular remoulding in MCT-induced PAH.

| The change of TLR and NLR pathways in MCTinduced PAH
Due to having enrichment of TLR and NLR pathways in cluster 1 by KEGG pathway enrichment analysis, we then characterized the changes of TLR and NLR pathways in response to MCT treatment.
The TLRs are specific families of pattern recognition receptors capable of detecting microbes and generating innate immunity. Upon recognizing specific structures of microorganisms, TLRs activate NF-κB pathway, resulting in the alteration of effector mechanisms, including up-regulation of TNFα, IL-1β, IL-6 and IL-12. 17 In addition to showing up-regulation of previously well-known effector genes in PAH, such as TNFα, IL-6 and IL-12, 2,18 pathway-based data integration and visualization using the Pathview and hierarchical clustering analysis of DEGs using Morpheus also revealed up-regulation of less well-appreciated genes in TLR pathways, such as up-regulated genes including TLR2, MyD88, CD14, LBP and TAB1, as well as down-regulated genes including TAB2, NFKBIA (IκBα) and TRAF6 ( Figure 4A,B). Visualization of global pathway change using Pathview showed that the gene expression changes in TLR and NLR pathways were not always synergistic. As an example, the reduced expression of IκBα, TRAF6 and TAB2 was identified in TLR pathway ( Figure 4B).
The reduced IκBα, a NF-κB inhibitor, was associated with activation of NF-κB pathway. However, the reduced TRAF6 and TAB2 expression may restrict activation of NF-κB pathway. It was likely that the presence of negative feedback mechanisms prevented the overactivated or prolonged TLR and NLR pathways in the progression of MCT-induced PAH.

| The activation of TLR and NLR pathways by DAMPs
It is well established that TLRs and NLRs could be activated by endogenous molecules termed damage-associated molecular patterns (DAMPs), and a series of DAMPs and related receptors have been identified by previous studies (Tables S1 and S2). In our RNA-seq data set, some of the intracellular and extracellular DAMPs were found to be differentially expressed. To characterize these dif- , S100 proteins (S100A3, S100A4, S100A8 and S100A9  -log 10 (p-value) -log 10 (p-value) -log 10 (p-value) -log 10 (p-value) and COL18A1), fibronectin (FN1) and laminin (LAMA1) ( Figure 5B).

| D ISCUSS I ON
In the present study, RNA-seq and bioinformatics methods were used to identify the pathways related to inflammation and immunity in pulmonary vascular remodelling in PAH. We found

F I G U R E 6
The change of necroptosis pathways in response to monocrotaline (MCT) treatment. A, The integration and visualization of gene expression change in necroptosis pathways using modified Pathview. Each coloured box represents the comparison of MCT treatment 1 wk with control, MCT treatment 2 wk with control, MCT treatment 3 wk with control and MCT treatment 4 wk with control. Colour represents P-value for each comparison of MCT treatments with control by using Morpheus software (unpaired t test); genes with relatively increased and reduced expression were shown in red and green, respectively, while white represents P ≥ .  CTW0  CTW1  CTW2  CTW3  CTW4  MCTW11  MCTW12  MCTW13  MCTW21  MCTW22  MCTW23  MCTW31  MCTW32  MCTW33  MCTW41  MCTW42  MCTW43   id   CFLAR  RIPK1  CASP8  CYLD  DNM1L  TRPM7  XIAP  SPATA2  TNFAIP3  BIRC2  MLKL  BIRC3  RIPK3  PARP1  RBCK1  PGAM5  SHARPIN  TRADD  TNFRSF1A  TRAF2  The expression of IL-1β, IL-18, GSDMD and NLRP3 was elevated in NLR pathway, which was consistent with previously reported activation of NLRP3 inflammasome in PAH. 20 Lysosomal destabilization and CTSB release were capable of activating NLRP3 inflammasome following DAMP phagocytosis. 40 The up-regulation of CTSB suggested lysosomal destabilization after DAMP phagocytosis was one of the mechanisms that result in the activation of NLRP3 inflammasome in PAH.
Increased deposition of ECMs in pulmonary arterioles contributes to the progression of PAH, and both inhibition of synthesis and genetic deletion of ECMs reduce pulmonary arterial remodelling. 41,42 The elevated expression of ECMs, including elastin, biglycan, collagens, fibronectin and laminin, was identified in the present study.  58 and S100 proteins (S100A3 and S100A4) 48 60 Necroptosis is a regulated form of necrosis, which is associated with generation or exposure of DAMPs. 21,22 Necroptosis was originally defined as being dependent on the kinase RIPK1; subsequently, RIPK3 was also established to be required for necroptosis. 23 But now, RIPK1 is known to inhibit necroptosis as a negative regulator. 24,25 In this study, the reduced expression pattern of RIPK1 was observed, which was completely opposite to the expression pattern of RIPK3 in MCT-induced PAH. Apart from the RIPK1, XIAP 26 and CFLAR/CASP8 complex 28 were also known as the negative regulators of necroptosis. In the present study, the ex- To our knowledge, this is the first time to reveal a role of RIPK3mediated necroptosis in the pathogenesis of PAH through bioinformatics analysis. Thus, it is likely that PAH also belongs to the necroptosis diseases. Accordingly, a model illustrating the role of necroptosis and its triggered TLR and NLR pathways in PAH is proposed in Figure 7. This study has some limitations. Firstly, only lung tissues were chosen for gene expression analysis rather than specific cell types. Secondly, the biological replicates in MCT treatment groups may be limited. Finally, this study was based on transcriptomic data, the protein levels, for instance IκBα, IL1β, IL18 and TLR2, and the phosphorylation states of RIPK3 and MLKL proteins were not examined. Consequently, further studies in the future may be needed to verify the role of necroptosis in PAH.
In summary, we identify dysregulated TLR and NLR pathways in the progression of pulmonary vascular remodelling. RIPK3-mediated necroptosis may be associated with exposure of DAMPs and consequent activation of TLR and NLR pathways. Thus, these results provide novel insights into the mechanisms underlying immunity and inflammation in PAH.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81873537 and 81570446 to Liangdi Xie).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The raw RNA-seq data that support the findings were deposited in the gene expression omnibus (GEO) repository with an accession number GSE14 9713.