Muscle cell death in polymyositis is induced by CD8
cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8
...cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8
cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes.
Background
As glucocorticoids induce muscle atrophy during the treatment course of polymyositis (PM), novel therapeutic strategy is awaited that suppresses muscle inflammation but retains muscle ...strength. We recently found that injured muscle fibres in PM undergo FASLG‐mediated necroptosis, a form of regulated cell death accompanied by release of pro‐inflammatory mediators, contributes to accelerate muscle inflammation and muscle weakness. Glucagon‐like peptide‐1 receptor (GLP‐1R) agonists have pleiotropic actions including anti‐inflammatory effects, prevention of muscle atrophy, and inhibition of cell death, in addition to anti‐diabetic effect. We aimed in this study to examine the role of GLP‐1R in PM and the effect of a GLP‐1R agonist on in vivo and in vitro models of PM.
Methods
Muscle specimens of PM patients and a murine model of PM, C protein‐induced myositis (CIM), were examined for the expression of GLP‐1R. The effect of PF1801, a GLP‐1R agonist, on CIM was evaluated in monotherapy or in combination with prednisolone (PSL). As an in vitro model of PM, C2C12‐derived myotubes were treated with FASLG to induce necroptosis. The effect of PF1801 on this model was analysed.
Results
GLP‐1R was expressed on the inflamed muscle fibres of PM and CIM. The treatment of CIM with PF1801 in monotherapy (PF) or in combination with PSL (PF + PSL) suppressed CIM‐induced muscle weakness (grip strength, mean ± SD (g); PF 227 ± 6.0 (P < 0.01), PF + PSL 224 ± 8.5 (P < 0.01), Vehicle 162 ± 6.0) and decrease in cross‐sectional area of muscle fibres (mean ± SD (μm2); PF 1896 ± 144 (P < 0.05), PF + PSL 2018 ± 445 (P < 0.01), Vehicle 1349 ± 199) as well as the severity of histological inflammation scores (median, interquartile range; PF 0.0, 0.0–0.5 (P < 0.05), PF + PSL 0.0, 0.0–0.0 (P < 0.01), Vehicle 1.9, 1.3–3.3). PF1801 decreased the levels of inflammatory mediators such as TNFα, IL‐6, and HMGB1 in the serum of CIM. PF1801 inhibited necroptosis of the myotubes in an AMP‐activated protein kinase (AMPK)‐dependent manner. PF1801 activated AMPK and decreased the expression of PGAM5, a mitochondrial protein, which was crucial for necroptosis of the myotubes. PF1801 promoted the degradation of PGAM5 through ubiquitin‐proteasome activity. Furthermore, PF1801 suppressed FASLG‐induced reactive oxygen species (ROS) accumulation in myotubes, also crucial for the execution of necroptosis, thorough up‐regulating the antioxidant molecules including Nfe2l2, Hmox1, Gclm, and Nqo1.
Conclusions
GLP‐1R agonist could be a novel therapy for PM that recovers muscle weakness and suppresses muscle inflammation through inhi biting muscle fibre necroptosis.
Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must ...be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.
miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.
miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.
miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.
Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, ...the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1β. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes.
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•During inflammation, human fibroblasts upregulate LIF and STAT4•LIF acts in an autocrine manner via LIF receptor to promote STAT4 activation•Activated STAT4 together with NF-κB/p65-p52 and C/EBPβ enhances IL-6 transcription•LIFR/STAT4 circuit also regulates IL-8, G-CSF, IL-33, IL-11, IL-1α, and IL-1β
Growing evidence implicates fibroblasts as inflammatory cells in sites of peripheral inflammation. Nguyen and colleagues demonstrate that regulation of IL-6 along with a set of other inflammatory cytokines and chemokines is regulated by a positive feedback loop involving LIF, LIF receptor, and STAT4 that selectively operates in fibroblasts.
Fibroblasts regulate tissue homeostasis, coordinate inflammatory responses, and mediate tissue damage. In rheumatoid arthritis (RA), synovial fibroblasts maintain chronic inflammation which leads to ...joint destruction. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to pathology. Here, we show functional and transcriptional differences between fibroblast subsets from human synovial tissues using bulk transcriptomics of targeted subpopulations and single-cell transcriptomics. We identify seven fibroblast subsets with distinct surface protein phenotypes, and collapse them into three subsets by integrating transcriptomic data. One fibroblast subset, characterized by the expression of proteins podoplanin, THY1 membrane glycoprotein and cadherin-11, but lacking CD34, is threefold expanded in patients with RA relative to patients with osteoarthritis. These fibroblasts localize to the perivascular zone in inflamed synovium, secrete proinflammatory cytokines, are proliferative, and have an in vitro phenotype characteristic of invasive cells. Our strategy may be used as a template to identify pathogenic stromal cellular subsets in other complex diseases.
With advancement in the treatment options of rheumatoid arthritis (RA), optimising the outcomes of difficult-to-treat patients has become increasingly important in clinical practice. In particular, ...insensitivity to first-line biologic disease-modifying anti-rheumatic drugs (bDMARD) is becoming a significant problem because it may decrease the treatment adherence of patients. This study aimed to compare RA patients with an insensitivity and those with a poor response to initial treatment with tumour necrosis factor inhibitors (TNFis), which are the most frequently used bDMARDs.
This is a retrospective cohort study using clinical data from the FIRST registry. bDMARD-naïve RA patients treated with tumour necrosis factor inhibitors (TNFis) from August 2003 to May 2019 were included and categorised into three groups: TNFi insensitivity, poor response to TNFis and controls. TNFi insensitivity was defined as follows: (1) discontinuation of TNFi treatment within 22 weeks due to lack of any response, or (2) an increase in the disease activity score in 28 joints-C-reactive protein (DAS28-CRP) of > 0.6 at week 22 compared with week 0. Among the remaining patients, those with a DAS28-CRP > 2.6 at week 22 were categorised in the poor response group.
Of the included patients, 94 were classified in the insensitivity, 604 in the poor response and 915 in the control. A higher DAS28-CRP before treatment was a risk factor for a poor response but not for insensitivity. In contrast, dose escalation of infliximab decreased the risk of a poor response but not that of insensitivity.
In future research, poor and insensitivity to bDMARDs should be assessed separately to fully elucidate the aetiology of, and risk factors for, bDMARD refractoriness.
Objective
One of the histologic characteristics of systemic sclerosis (SSc) is an increased number of dermal myofibroblasts, and transforming growth factor β (TGFβ) plays a crucial role in the ...promotion of myofibroblast differentiation from fibroblasts, leading to dermal fibrosis. This study was undertaken to 1) examine whether inhibition of the cell cycle with a cyclin‐dependent kinase 4/6 (CDK4/6) inhibitor suppresses the proliferation of fibroblasts and their differentiation into myofibroblasts, and 2) assess the therapeutic effects of a CDK4/6 inhibitor, administered as monotherapy or in combination with a TGFβ receptor (TGFβR) inhibitor, on dermal fibrosis in murine models of SSc.
Methods
Fibroblasts obtained from the skin of patients with SSc were cultured in the presence or absence of TGFβ. The effects of palbociclib, a CDK4/6 inhibitor, on fibroblast proliferation and TGFβ‐induced differentiation into myofibroblasts were examined using bromodeoxyuridine uptake assays as well as immunofluorescence and immunoblotting analyses. Murine models of HOCl‐ and bleomycin‐induced dermal fibrosis were used to study the effect of a CDK4/6 inhibitor on dermal fibrosis, with the CDK4/6 inhibitor treatment administered as monotherapy or in combination with galunisertib, a TGFβR inhibitor.
Results
Addition of a CDK4/6 inhibitor to the cell cultures suppressed the proliferation of human dermal SSc fibroblasts and their TGFβ‐induced differentiation into myofibroblasts, without inhibiting canonical and noncanonical TGFβ signals. In murine models of dermal fibrosis, treatment of mice with a CDK4/6 inhibitor decreased dermal thickness and collagen content, as well as dermal fibroblast proliferation and the numbers of myofibroblasts. Combination therapy with the CDK4/6 inhibitor and TGFβR inhibitor resulted in additive antifibrotic effects. Mechanistically, the CDK4/6 inhibitor suppressed the expression of cellular communication network 2 and cadherin‐11, which are proteins that have important roles in the development and progression of fibrosis.
Conclusion
Results of this study demonstrate the therapeutic effect of a CDK4/6 inhibitor on dermal fibrosis when administered as monotherapy or in combination with a TGFβR inhibitor. CDK4/6 inhibitors, including palbociclib used in the present study, may represent novel agents for the treatment of SSc, which, if used in combination with a TGFβR inhibitor, might result in increased efficacy.
Abstract
Objective
Targeting synovial fibroblasts (SF) using a cyclin-dependent kinase (CDK) 4/6 inhibitor (CDKI) could be a potent therapy for RA via inhibition of proliferation and MMP-3 ...production. This study was designed to elucidate the mechanism of chondroprotective effects on SFs by CDK 4/6 inhibition.
Methods
CDK4/6 activity was inhibited using CDKI treatment or enhanced by adenoviral gene transduction. Chondroprotective effects were evaluated using a collagen-induced arthritis model (CIA). Gene and protein expression were evaluated with quantitative PCR, ELISA and Western blotting. The binding of nuclear extracts to DNA was assessed with an electrophoresis mobility shift assay. RNA-Seq was performed to identify gene sets affected by CDKI treatment.
Results
CDKI attenuated cartilage destruction and MMP-3 production in CIA. In RASFs, CDKI impaired the binding of AP-1 components to DNA and inhibited the production of MMP-1 and MMP-3, which contain the AP-1 binding sequence in their promoter. CDK4/6 protected JUN from proteasome-dependent degradation by inhibiting ubiquitination. The RNA-Seq analysis identified CDKI-sensitive inflammatory genes, which were associated with the pathway of RA-associated genes, cytokine-cytokine receptor interaction and IL-17 signalling. Notably, the AP-1 motif was enriched in these genes.
Conclusion
The mechanism of chondroprotective effects by CDK4/6 inhibition was achieved by the attenuation of AP-1 transcriptional activity via the impaired stability of JUN. Because the pharmacologic inhibition of CDK4/6 has been established as tolerable in cancer treatment, it could also be beneficial in patients with RA due to its chondroprotective and anti-inflammatory effects.
Synovial fibroblasts (SFs) in rheumatoid arthritis (RA) and osteoarthritis (OA) play biphasic roles in joint destruction and regeneration of bone/cartilage as mesenchymal stem cells (MSCs). Although ...MSCs contribute to joint homeostasis, such function is impaired in arthritic joints. We have identified functionally distinct three SF subsets characterized by the expression of CD34 and THY1 as follows: CD34
THY1
, CD34
THY1
, and CD34
THY1
. The objective of this study was to clarify the differentiation potentials as MSCs in each SF subset since both molecules would be associated with the MSC function.
SF subsets were isolated from synovial tissues of 70 patients (RA: 18, OA: 52). Expressions of surface markers associated with MSCs (THY1, CD34, CD73, CD271, CD54, CD44, and CD29) were evaluated in fleshly isolated SF subsets by flow cytometry. The differentiation potentials of osteogenesis, chondrogenesis, and adipogenesis were evaluated with histological staining and a quantitative polymerase chain reaction of differentiation marker genes. Small interfering RNA was examined to deplete THY1 in SFs.
The expression levels of THY1
, CD73
, and CD271
were highest and those of CD54
and CD29
were lowest in CD34
THY1
among three subsets. Comparing three subsets, the calcified area, alkaline phosphatase (ALP)-stained area, and cartilage matrix subset were the largest in the CD34
THY1
subset. Consistently, the expressions of differentiation markers of the osteoblasts (RUNX2, ALPL, and OCN) or chondrocytes (ACAN) were the highest in the CD34
THY1
subset, indicating that the CD34
THY1
subset possessed the highest osteogenic and chondrogenic potential among three subsets, while the differentiation potentials to adipocytes were comparable among the subsets regarding lipid droplet formations and the expression of LPL and PPARγ. The knockdown of THY1 in bulk SFs resulted in impaired osteoblast differentiation indicating some functional aspects in this stem-cell marker.
The CD34
THY1
SF subset has high osteogenic and chondrogenic potentials. The preferential enhancement of MSC functions in the CD34
THY1
subset may provide a new treatment strategy for regenerating damaged bone/cartilage in arthritic joints.
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