Beatrix Bartok

Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis

Summary:  Rheumatoid arthritis (RA) remains a significant unmet medical need despite significant therapeutic advances. The pathogenesis of RA is complex and includes many cell types, including T cells, B cells, and macrophages. Fibroblast‐like synoviocytes (FLS) in the synovial intimal lining also play a key role by producing cytokines that perpetuate inflammation and proteases that contribute to cartilage destruction. Rheumatoid FLS develop a unique aggressive phenotype that increases invasiveness into the extracellular matrix and further exacerbates joint damage. Recent advances in understanding the biology of FLS, including their regulation regulate innate immune responses and activation of intracellular signaling mechanisms that control their behavior, provide novel insights into disease mechanisms. New agents that target FLS could potentially complement the current therapies without major deleterious effect on adaptive immune responses.

PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

Phosphoinositide 3-Kinase δ Regulates Migration and Invasion of Synoviocytes in Rheumatoid Arthritis

Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of rheumatoid arthritis (RA). Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The class I PI3Ks control cell survival, proliferation, and migration, which might be involved in cartilage damage in RA. PI3Kδ isoform was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behavior. Therefore, we assessed the role of PI3Kδ in RA synoviocyte migration and invasion. We observed that PI3Kδ inhibition or small interfering RNA knockdown decreased platelet-derived growth factor (PDGF)–mediated migration and invasion of FLS. We then showed that PI3Kδ regulates the organization of actin cytoskeleton and lamellipodium formation during PDGF stimulation. To gain insight into molecular mechanisms, we examined the effect of PI3Kδ inhibition on Rac1/PAK, FAK, and JNK activation. Our studies suggest that Rac1/PAK is key target of PDGF-mediated PI3Kδ signaling, whereas FAK and JNK are not involved. Thus, PI3Kδ contributes to multiple aspects of the pathogenic FLS behavior in RA. These observations, together with previous findings that PI3Kδ regulates FLS growth and survival, suggest that PI3Kδ inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration, and invasion.

Protein Tyrosine Phosphatase Expression Profile of Rheumatoid Arthritis Fibroblast‐like Synoviocytes: A Novel Role of SH2 Domain–Containing Phosphatase 2 as a Modulator of Invasion and Survival

OBJECTIVE The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS. METHODS A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain-containing phosphatase 2 (SHP-2), a PTP that was up-regulated in RA, was then analyzed by knockdown using cell-permeable antisense oligonucleotides. RESULTS PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP-2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP-2 knockdown. RA FLS that were deficient in SHP-2 exhibited decreased activation of focal adhesion kinase and mitogen-activated protein kinases. CONCLUSION These findings indicate that SHP-2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA.

Integrative omics analysis of rheumatoid arthritis identifies non-obvious therapeutic targets.

Identifying novel therapeutic targets for the treatment of disease is challenging. To this end, we developed a genome-wide approach of candidate gene prioritization. We independently collocated sets of genes that were implicated in rheumatoid arthritis (RA) pathogenicity through three genome-wide assays: (i) genome-wide association studies (GWAS), (ii) differentially expression in RA fibroblast-like synoviocytes (FLS), and (iii) differentially methylation in RA FLS. Integrated analysis of these complementary data sets identified a significant enrichment of multi-evidence genes (MEGs) within pathways relating to RA pathogenicity. One MEG is Engulfment and Cell Motility Protein-1 (ELMO1), a gene not previously considered as a therapeutic target in RA FLS. We demonstrated in RA FLS that ELMO1 is: (i) expressed, (ii) promotes cell migration and invasion, and (iii) regulates Rac1 activity. Thus, we created links between ELMO1 and RA pathogenicity, which in turn validates ELMO1 as a potential RA therapeutic target. This study illustrated the power of MEG-based approaches for therapeutic target identification.

Novel Phosphoinositide 3-Kinase δ,γ Inhibitor: Potent Anti-Inflammatory Effects and Joint Protection in Models of Rheumatoid Arthritis

Phosphoinositide 3-kinases γ and δ (PI3Kγ and PI3Kδ) are expressed in rheumatoid arthritis (RA) synovium and regulate innate and adaptive immune responses. We determined the effect of a potent PI3Kδ,γ inhibitor, IPI-145, in two preclinical models of RA. IPI-145 was administered orally in rat adjuvant-induced arthritis (AA) and intraperitoneally in mouse collagen-induced arthritis (CIA). Efficacy was assessed by paw swelling, clinical scores, histopathology and radiography, and microcomputed tomography scanning. Gene expression and Akt phosphorylation in joint tissues were determined by quantitative real-time polymerase chain reaction and Western blot analysis. Serum concentrations of anti-type II collagen (CII) IgG and IgE were measured by immunoassay. T-cell responses to CII were assayed using thymidine incorporation and immunoassay. IPI-145 significantly reduced arthritis severity in both RA models using dosing regimens initiated before onset of clinical disease. Treatment of established arthritis with IPI-145 in AA, but not CIA, significantly decreased arthritis progression. In AA, histology scores, radiographic joint damage, and matrix metalloproteinase (MMP)-13 expression were reduced in IPI-145–treated rats. In CIA, joint histology scores and expression of MMP-3 and MMP-13 mRNA were lower in the IPI-145 early treatment group than in the vehicle group. The ratio of anti-CII IgG2a to total IgG in CIA was modestly reduced. Interleukin-17 production in response to CII was decreased in the IPI-145–treated group, suggesting an inhibitory effect on T-helper cell 17 differentiation. These data show that PI3Kδ,γ inhibition suppresses inflammatory arthritis, as well as bone and cartilage damage, through effects on innate and adaptive immunity and that IPI-145 is a potential therapy for RA.

Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy

Targeting joint-lining fibroblast-like synoviocytes reduces the severity of arthritis. Switching off arthritis In patients with rheumatoid arthritis (RA), joint-lining cells—fibroblast-like synoviocytes (FLS)—become activated and contribute to inflammation as well as cartilage and bone destruction. FLS express RPTPσ, which, in neurons, can be regulated by a proteoglycan switch. Doody et al. now find that FLS can also be regulated by this proteoglycan switch, and that an RPTPσ decoy protein can block this switch and decrease FLS invasiveness and severity of arthritis in human cells and a mouse model of RA. If these data hold true in humans, targeting this proteoglycan switch may add another option when treating patients with RA. Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

Differential distribution of PI3K isoforms in spinal cord and dorsal root ganglia: Potential roles in acute inflammatory pain

Summary Only spinal PI3K&bgr; antagonism reduced allodynia and spinal AMPA trafficking, while intraplantar PI3K&ggr; antagonism was the most effective anti‐allodynic. This reflected each isoforms unique distribution. ABSTRACT PI3‐kinases (PI3Ks) participate in nociception within spinal cord, dorsal root ganglion (DRG), and peripheral nerves. To extend our knowledge, we immunohistochemically stained for each of the 4 class I PI3K isoforms along with several cell‐specific markers within the lumbar spinal cord, DRG, and sciatic nerve of naive rats. Intrathecal and intraplantar isoform specific antagonists were given as pretreatments before intraplantar carrageenan; pain behavior was then assessed over time. The &agr;‐isoform was localized to central terminals of primary afferent fibers in spinal cord laminae IIi to IV as well as to neurons in ventral horn and DRG. The PI3K&bgr; isoform was the only class I isoform seen in dorsal horn neurons; it was also observed in DRG, Schwann cells, and axonal paranodes. The &dgr;‐isoform was found in spinal cord white matter oligodendrocytes and radial astrocytes, and the &ggr;‐isoform was seen in a subpopulation of IB4‐positive DRG neurons. No isoform co‐localized with microglial markers or satellite cells in naive tissue. Only the PI3K&bgr; antagonist, but none of the other antagonists, had anti‐allodynic effects when administered intrathecally; coincident with reduced pain behavior, this agent completely blocked paw carrageenan‐induced dorsal horn 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl) propanoic acid (AMPA) receptor trafficking to plasma membranes. Intraplantar administration of the &ggr;‐antagonist prominently reduced pain behavior. These data suggest that each isoform displays specificity with regard to neuronal type as well as to specific tissues. Furthermore, each PI3K isoform has a unique role in development of nociception and tissue inflammation.

Development of anti-CD20 therapy for multiple sclerosis.

The therapeutic utility of the targeting of B lymphocytes is currently being evaluated in a range of autoimmune diseases that include multiple sclerosis (MS). For MS, even though intrathecal immunoglobulin production is a hallmark of multiple sclerosis (MS), T cells have long been considered as the main effectors of pathogenesis. Recognition of the roles of autoreactive B cells has changed this conventional view of the disease and also provided a rationale for studies of anti-CD20 therapy in MS. Recent trials suggest that this approach may provide clinical benefits in some MS patients that equal or surpass currently approved approaches, yet not all patients may benefit. In this review we provide an overview on recent progress on these trials.

TGFβ responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness

Objective In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) β-target gene, PTPRK, promotes RA FLS invasiveness. Methods Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. Results PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. Conclusions By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFβ in RA FLS.