Dataset Information

Identification of mitochondrial dysfunction in rheumatoid arthritis patients

ABSTRACT: Immune cells are majorly dysregulated in rheumatoid arthritis patients and are a major cause of pathogenesis and progression of this autoimmune condition. As mitochondria are master regulators of metabolism of all cells present in human body, it is of prime importance to study mitochondrial homeostasis in immune cells for identifying any dysfunction contributing to pathogenesis or progression of RA. The objective of our study is to understand the mitochondrial and mitochondrially driven alterations in immune cells of RA patients which may cause them to perform abnormal functions and may have an important contribution in progression of the disease.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

DISEASE(S): Rheumatoid Arthritis

SUBMITTER: Bhawna Gupta

LAB HEAD: Dr. Bhawna Gupta

PROVIDER: PXD011934 | Pride | 2020-06-22

REPOSITORIES: Pride

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Publications

Altered mitochondrial proteome and functional dynamics in patients with rheumatoid arthritis.

Khanna Shweta S Padhan Prasanta P Jaiswal Kumar S KS Jain Ankit P AP Ghosh Arup A Tripathy Archana A Gowda Harsha H Raghav Sunil K SK Gupta Bhawna B

Mitochondrion 20200613

The autoimmune inflammatory disease, Rheumatoid arthritis (RA), has known imbalances in energy metabolism and superoxide levels thus may have an etiology associated with mitochondrial dysfunction. We thus evaluated the presence of a differential mitochondrial proteome as well as other characteristics including mitochondrial mass, membrane potential (Ψm), total cellular ATP and superoxide levels. Eighteen mitochondrial proteins were down-regulated while four were up-regulated in RA patients in co ...[more]

PMID: 32544466

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Project description:Background: Rheumatoid arthritis (RA) is a complex heterogenous autoimmune disease and achieving long term disease remission is an elusive goal for patients, thus implying improvement in drug targeting is necessary. Kinases are intracellular signalling mediators and key to sustaining the inflammatory process in RA. Therefore, oral inhibitors of specific kinases, such as Cyclin Dependent Kinases (CDKs) and Janus Kinase (JAKs), are under development or have been approved. However, the cell signalling mechanisms in treatment naïve RA patients is yet to be explored, which may facilitate targeted therapy stratification. Methods: We undertook phosphoproteome and total proteome analysis of 8 pre-treatment synovial biopsies of RA patients using label-free mass spectrometry. Results: The analysis revealed a clear separation of the phosphoproteome and proteome profile between the lymphoid and myeloid RA pathotypes. Differential expression analysis and function enrichment showed that the degree of inflammatory state and specific signalling activities are associated with different RA pathotypes. The lymphoid pathotype was enriched with immunological pathways and associated with Mammalian Target Of Rapamycin (MTOR) signalling, whereas the myeloid pathotype was associated with Mitogen-Activated Protein Kinase (MAPK) and CDK mediated signalling. This analysis also highlighted kinases not previously linked to RA, such as Serine/Threonine Protein Kinase N1 (PKN1) in the lymphoid pathotype and Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) in the myeloid pathotypes. Certain phosphosites were also highly correlated with clinical features, such as Disabled Homolog 2 (DAB2)-Ser723 with Disease Activity Score (DAS)-28, and so these may be potential biomarkers of disease progression and response. Conclusions: These data provide evidence that specific phosphoproteome and proteome signatures are associated with different RA pathotypes and may have clinical utility for stratifying patients as part of a personalised medicine approach.

Project description:Rheumatoid synoviocytes, which consist of fibroblast-like synoviocytes (FLS) and synovial macrophages (SM), are crucial for the progression of rheumatoid arthritis (RA). Particularly, FLS of RA patients (RA-FLS) exhibit invasive characteristics reminiscent of cancer cells, destroying cartilage and bone, although it remains unresolved how RA-FLS exhibit invasive phenotype. RA-FLS and SM originate differently from mesenchymal and myeloid cells, respectively, but share many pathologic functions. However, the molecular signatures and biological networks representing the distinct and shared features of the two cell types are unknown. Presently, we performed global transcriptome profiling of FLS and SM obtained from RA and osteoarthritis patients. By comparing the transcriptomes, we identified distinct molecular signatures and cellular processes defining invasiveness of RA-FLS and pro-inflammatory properties of RA synovial macrophages (RA-SM), respectively. Interestingly, under interleukin1β-stimulated condition, RA-FLS newly acquired pro-inflammatory signature mimicking RA-SM without losing invasive properties. We next reconstructed a network model that delineates the shared, RA-FLS-dominant (invasive), and RA-SM-dominant (inflammatory) processes. From the network model, we selected 13 genes, including POSTN and TWIST1, as novel regulator candidates responsible for FLS invasiveness. Of note, POSTN and TWIST1 expressions were elevated in independent RA-FLS and were further instigated by interleukin1β. In vitro functional assays demonstrated the requirement of POSTN and TWIST1 for migration and invasion of RA-FLS stimulated with interleukin1β. Taken together, our systems approach to rheumatoid synovitis provides a basis for identifying novel regulators responsible for pathological features of RA-FLS and RA-SM, demonstrating how a certain type of cells acquires functional redundancy under chronic inflammatory conditions. To identify molecular signatures of FLS and MLS in RA joints, we isolated FLS from synovial tissues of RA and osteoarthritis (OA) patients, obtained synovial macrophages from synovial fluid of RA patients, and differentiated control macrophages from peripheral blood of healthy subjects. Also, we stimulated FLS with IL1β, and then analyzed gene expression profiles of both IL1β-stimulated RA-FLS and OA-FLS

Project description:Cell-and context-specific activities of nuclear receptors may in part be due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We have shown previously that silencing of RIP140 enhances RA-induced differentiation and enhances the induction of model RA target genes in human embryonal carcinoma cells (EC). Through use of microarray technology we sought to elucidate in a de novo fashion the global role of RIP140 in RA-dependent signaling. RIP140-dependent gene expression was largely consistent with RIP140 functioning to limit RAR signaling. Few if any genes were regulated in a manner to support a role for RIP140 in â??active repressionâ??. Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140, suggesting that RIP140 may discriminate between different classes of RA target genes. RIP140 silencing also accelerated RA target gene activation and sensitized EC cells to low doses of RA. Together the data suggests that the RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation. RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. Experiment Overall Design: The current investigation sought to determine the global effects of RIP140 on RA-dependent and RAâ??independent gene expression. A dose of siRNA of 90 nM was chosen. Twelve independent hybridizations were performed from mRNA isolated from 12 independent samples. One scrambled and two independent RISC-free siRNA transfections and transfections with three distinct RIP140 siRNAs were performed in NT2/D1 cells. Each siRNA treatment was further treated with either 10 Î¼M RA or DMSO control for 24 hours. Hence, the design consisted of four groups, the three siRNA controls treated with vehicle, Group 1; the siRNA controls treated with RA, Group 2; siRNA to RIP140 treated with vehicle, Group 3; and siRNA to RIP140 treated with RA, Group 4 . Experiment Overall Design: The treatments were done in the presence of normal sera, since charcoal absorption of sera removes many small hydrophobic ligands to the nuclear receptor family in addition to retinoids. This measure was taken with the aim to perhaps identify target genes of other nuclear receptors including orphan receptors that could be regulated by RIP140 in the presence of low levels of their cognate ligands. RA treatment of 10 Î¼M for 24 hours was chosen since previous experience with NT2/D1 cells indicated that a reasonably sized set of altered genes would be obtained. In addition, NT2/D1 cells commit to RA-induced differentiation by 48 hours and RIP140 repression enhances and accelerates the effects of RA in these cells. Therefore, the 24-hour time point is well-within the range of the RA commitment â??windowâ?? and a larger percentage of direct RA-target genes could be expect to be uncovered compared to later time points. Experiment Overall Design: Based on the current understanding of the properties of RIP140, a number of potential outcomes were anticipated: 1) RIP140 may limit RA target gene activation by negative-feedback inhibition upon RA addition, 2) RIP140 may participate in â??active repressionâ?? of gene expression upon RA addition, 3) an unexpected outcome that RIP140 may participate in the activation of a subset of target genes, 4) RIP140 may regulate the transcription of target genes of other nuclear receptors or even non-nuclear receptor transcription factors. Experiment Overall Design: Total RNA was purified using RNeasy columns (Qiagen). Hybridizations were performed according to Affymetrix guidelines at the Dartmouth College Microarray Shared Resource using an Affymetrix GeneChip Workstation. Biotin-labeled cRNA was generated from 5 Î¼g of total RNA and hybridized to the Human Genome (HG) U133 Plus 2.0 chip A total of 12 hybridizations were performed comprising 12-independent biologic samples organized into the four groups of three.

			Action	DRS
	KIIT_iTRAQ_R.pdStudy	Other
	KIIT_iTRAQ_R1.raw	Raw
	KIIT_iTRAQ_R123.msf	Msf
	KIIT_iTRAQ_R123.pdResult	Other
	KIIT_iTRAQ_R2.raw	Raw