Identication of the difference of neutrophils in different locations in RA patients

Rheumatoid Arthritis (RA) is a complex systemic disease in which numerous cell types are involved. Neutrophils play an important role in the onset and development of RA. In our study, we aim to identify different functions of neutrophils in different conditions (blood and synovium) of RA patients by using a bioinformatics method to clarify their potential pathogenesis. The gene expression proles of the GSE154474 dataset were originally produced by using the high-throughput Illumina HiSeq 2000 (Homo sapiens). The biological categories and biochemical pathways were identied and analyzed by the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Gene Ontology (GO), and Reactom enrichment. KEGG and GO results showed the biological pathways related to the immune and cellular structure were mainly different. Moreover, we identied several genes including GNB4, RHOA, and TECB2 were involved in the regulation of inammation. Therefore, this study provides different insights into the pathogenesis of RA.


Introduction
Rheumatoid arthritis (RA) is a systemic disease that can cause the disability of the immue system without proper treatment. It is unclear that the precise pathogenetic mechanisms underlying the development of RA 1 . At the onset of RA, patients indicate increased articular in ammation usually in the form of a symmetric polyarthritis 2 . Leukocyte migrates and aggregates in the joint area with early in ltration of neutrophils, T cells, B cells, and plasma cells that produced autoantibodies in the synovium tissue 3 . Not only the neutrophils, other innate immune cells such as macrophages and natural killer cells are also recruited to the synovium 4,5 . Finally, the local in ammatory condition contributes to fundamental changes and promotes the formation of hyperplastic, in ammatory synovium 6,7 .
Neutrophils are the rst cell type that respond to acute in ammation and are endowed with the ability of antimicrobial mechanisms 8 . Neutrophils are from bone marrow and respond to granulocyte colonystimulating factors. It is believed that integrins and selectins are critical in the process of neutrophil egression 9 . Neutrophils also express the C-X-C chemokine receptors. The balance between CXCR2 and CXCR4 and their respective chemokines appears to play a basic role in neutrophil mobilization 10 . In the absence of in ammatory stimuli, mature neutrophils locate in the bloodstream and remain in the circulation for a short period 8 . Thus, we consider the neutrophils in the blood as the less active neutrophils than those in the synovium in RA patients.
Here, we studied the relative neutrophil changes between blood and synovium. We identi ed and analyzed a series of DEGs, the relevant biological processes, and biological functions of neutrophils in the synovium in comparison to those in the blood by using comprehensive bioinformatic analysis 11 . We performed the signal pathway analysis, the functional enrichment, and protein-protein interaction (PPI) for discovering the features of the neutrophils in the synovium from RA patients. The identi ed genes and pathways could be critical to favor future clinical and therapeutic studies.

Data resources
The dataset GSE154474 was obtained from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). The data was produced by Illumina HiSeq 2000 (Homo sapiens), Institute of Life Course and Medical Sciences, University of Liverpool, United Kingdom. Bulk RNA-Seq analysis was performed using Neutrophils (purity > 97%) from paired peripheral blood and synovial uid (SF) from n = 3 patients with severe rheumatoid arthritis.

Data acquisition and preprocessing
The dataset GSE154474 that includes Neutrophils from paired peripheral blood and synovial uid was analyzed and conducted by R script 12,13 . We performed a classical t test to identify DEGs with P < .01 and fold change ≥ 1.5 as being statistically signi cant.

Gene functional analysis
The Gene Ontology (GO) is a functional genomics research that develop a comprehensive model through knowledge-informed computational analysis of biological data. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database is widely used for identifying the high-level functions and utilities of the biological system. The GO analysis and KEGG pathway enrichment analysis were performed by using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (http://david.ncifcrf.gov/). P < .05 and gene counts > 10 were considered statistically signi cant.

Module analysis
The Molecular Complex Detection (MCODE) of Cytoscape software was used to analyze the densely connected regions in protein-protein interaction (PPI) networks. The signi cant modules were from the constructed PPI network using MCODE. The function and pathway enrichment analyses were performed by using DAVID, and P < .05 was used as the cutoff criterion.

Reactome pathway analysis
We used the Reactom pathway to obtain the visualisation, interpretation and analysis of potential pathways (https://reactome.org/). P < .05 was considered statistically signi cant.

Results
Identi cation of DEGs of neutrophils from peripheral blood in comparison to synovial uid in RA patients The neutrophils were collected from the paired peripheral blood and synovial uid from patients with severe rheumatoid arthritis. To gain the insights on the different genes, the neutrophils from blood were compared to those from synovial uid. A total of 416 genes were identi ed to be differentially expressed with the threshold of P<0.005. The top 10 up-and down-regulated genes are list in table 1.
KEGG analysis of DEGs of neutrophils from peripheral blood in comparison to synovial uid in RA patients To further identify the biological roles and potential mechanisms of the DEGs of neutrophils from blood and synovial uid, we performed KEGG pathway and GO categories enrichment analysis (Supplemental Table S1) 12 . The KEGG pathway (http://www.genome.jp/kegg/) includes curated sets of genes that are to understand the molecular interaction, reaction and relation networks. Our study indicated top ve enriched KEGG pathways including "Serotonergic synapse", "Regulation of actin cytoskeleton", "Alzheimer's disease", "Proteoglycans in cancer" and "Pathways in cancer" (Figure 1).

PPI (protein-protein interactions) network and Module analysis
The PPI networks were constructed to analyze the relationships of DGEs at the protein level. The criterion of combined score >0.7 was set and the PPI network was created by using the 224 nodes and 320 interactions. Among these nodes, the top ten of most signi cant genes with highest scores are shown in Table 2. The top two signi cant modules versus blood samples were selected to indicate the functional annotation ( Figure 2).

Reactome Pathway of neutrophils from peripheral blood in comparison to synovial uid in RA patients
We identi ed a series of signaling pathways by using Reactome Pathway Database (https://reactome.org/). We identi ed top ten signaling pathways including: "Defective Base Excision Repair Associated with MUTYH", "Defective MUTYH substrate processing", "VEGF ligand-receptor interactions", "VEGF binds to VEGFR leading to receptor dimerization", "Activation of RAS in B cells", "RUNX3 regulates RUNX1-mediated transcription", "Polymerase switching", "Leading Strand Synthesis", "Activated NTRK2 signals through FRS2 and FRS3", and "PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases" (Supplemental Table S2). We then constructed the reaction map according to the signaling pathways (Figure 3).

Discussion
Neutrophils are the common cell type in RA synovial uid and are also detected in RA synovial tissues 14 .
Various proteases express in neutrophils play crucial roles in joint damage and in ammation 8 . Moreover, these proteases can activate the proin ammatory cytokines, modulate chemokine function and trigger different pathways [15][16][17][18] . Thus, the molecules present in neutrophil granules lead to in ammation and tissue damage 19 .
To better understand the different roles of neutrophils in blood and synovium in RA, we analyzed the RNAseq of peripheral blood and synovial uid neutrophils from rheumatoid arthritis patients. Thus, by using this, we could learn more about the functions of neutrophils in different conditions and environments. By analyzing the DEGs, we selected 10 proteins that may be critical according to the PPI network analysis. G proteins and Regulator of G protein signaling proteins are widely expressed in various tissues and involved in the immune process 5,20,21 . In our study, the G Protein Subunit Beta 4 was involved in calcium signaling during infection or in ammation 22 . CXCL12/CXCR4 can activate the RhoA to further promote the in ammation-driven colorectal cancer progression 23 . The absence of OA inhibits Th17 cell differentiation and allergic airway in ammation 24 . TCEB2 is involved in the regulation of apoptosis.
TCEB2 is increased in response to the treatment with proteasome inhibitor bortezomib for 24 hours 25 .
Laminin beta 1(LAMB1) is highly expressed in lung tissue and it is critical for both lung morphogenesis and physiological function 26 . KRAS is the most mutated oncogene in cancer and its receptor AMG 510 contributes the regression of KRAS mutant tumors and improves the anti-tumor e cacy of chemotherapy via formation a pro-in ammatory tumor microenvironment 27 . F2 accounts for the fate of at least 90% of the prothrombin in plasma, which may affect the blood clotting in RA 28 . DNMT3a can regulate the transcriptional inhibition on opiate-induced synaptic and behavioral plasticity via UBE2B 29 . ITGB3 was reported as a hub regulator in the tumor microenvironment, which may regulate the immune system during the tumor genesis 30 . As a mitochondrial protein, ATP5 proteins are important for the construction of complex V. The loss of ATP5F1D can lead to a metabolic disorder 31 . enhanced ITGA2B was discovered in bone marrow megakaryocytes of sepsis onset. Subsequent upregulation of ITGA2B were seen in circulating platelets 32 . Circadian clocks play important roles in physiological and pathophysiological processes such as aging 33 , bone 7, 34 , metabolism diseases [35][36][37][38] . Most interestingly, circadian control of neutrophil responsiveness contributes to changes in different location in in ammation condition, which may in uence the development of RA 39 . Interestingly, the genes such as RHOA, and KRAS are critically regulated by circadian clocks 40,41 . Thus, these PPI proteins are majorly involved in the in ammation and circulation environment during RA. It is suggested that neutrophils in different places and environments may activate different proteins and play different roles during RA.
KEGG and GO analysis showed that cancer related protein, immune and cell skeletal protein play critical roles by comparing the neutrophils from blood and synovium. The KEGG analysis showed the "Cancer pathways" and "Regulation of actin cytoskeleton" were the major different pathways in neutrophils from blood and synovium. It is suggested that the function of neutrophils from different locations is based on the environment. Neutrophils recognize different substrates of microbial and response by sequestering the cargo via phagocytosis or by releasing bioactive factors outside the cell, thus changing and alerting the environment and bystander leukocytes 42 . Interestingly, the CC of GO analysis showed "Mitochondrial envelop", "Extrinsic component of plasma membrane", "Tans-Golgi network" and "B cell activation", suggesting that the neutrophil components were different between blood and synovium in RA patients. Thus, the microenvironmental condition is crucial for the construction and function of neutrophils.
In summary, we identi ed different genes in neutrophils from blood and synovium in severe RA patients. Immune dysfunction and microenvironment were two key differences of neutrophils in different locations. This study thus provides further insights into the features of neutrophils from different tissues in RA, which may facilitate the diagnosis and drug development.

Declarations
Declarations of interest: none