Mouse Model Of Arthritis Research Articles

Exosomes derived from mesenchymal stem cells primed with disease-condition-serum improved therapeutic efficacy in a mouse rheumatoid arthritis model via enhanced TGF-β1 production

BackgroundsRheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation-mediated progressive destruction of the cartilage and bone, resulting in reduced quality of life. We primed human telomerase reverse transcriptase-overexpressing immortalized human adipose tissue-derived mesenchymal stem cells (iMSCs) with serum derived from a non-human primate RA model and studied the immunomodulatory ability of exosomes obtained from primed iMSCs.MethodsAfter immunophenotyping, nanoparticle tracking analysis, and in vitro functional tests, Dulbecco’s phosphate-buffered saline (dPBS, Group C), exosomes derived from the supernatant of iMSCs (Exo-FBS, Group E), exosomes derived from the supernatant of iMSCs primed with RA serum (Exo-RA, Group F), and methotrexate (Group M) were administered in collagen-induced arthritis (CIA) model mice. dPBS was administered to the normal (N) group for comparison (n = 10/group).ResultsExo-RA had a significantly higher number of exosomes compared to Exo-FBS when measured with nanoparticle tracking analysis or exosome marker CD81, and Transforming growth factor-β1 amounts were significantly higher in Exo-RA than in Exo-FBS. When Exo-FBS or Exo-RA was administered to the collagen-induced arthritis model, serum interleukin (IL)-4 and the proportion of Th2 (CD4+CD25+GATA3+) and M2 (CD11c − CD206+ of CD45+CD64+) cells were significantly increased compared to the control group. Furthermore, Exo-RA could alleviate cartilage damage by significantly lowering the concentrations of proinflammatory cytokines such as tumor necrosis factor-α, keratinocyte chemoattractant, and IL-12p70.ConclusionExosomes derived from disease-condition-serum-primed iMSCs ameliorated cartilage damage in a RA model by enhancing TGF-β1 production, inducing Th2 and M2 polarization and lowering proinflammatory cytokines, TNF-α, KC, and IL-12p70 in the host. Patient-derived serum can be used as an iMSC priming strategy in iMSC-derived exosome treatment of RA.

Hepatorenal pathologies in TNF-transgenic mouse model of rheumatoid arthritis are alleviated by anti-TNF treatment

ObjectiveTo examine and quantify liver and kidney lesions and their response to anti-tumor necrosis factor (TNF) therapy in a TNF-Tg mouse model of rheumatoid arthritis (RA).MethodsFemale TNF-Tg (Tg3647) mice were used as the animal model for chronic RA. Ultrasound, immunofluorescence, histological staining, serology tests, and real-time RT-PCR were used to examine the pathological changes in the liver and kidney.ResultsTNF-Tg mice showed a significant decrease in the body weight and a dramatic increase in the volumes of the gallbladder, knee cavity, and popliteal lymph nodes. The liver and kidneys of TNF-Tg mice showed increased chronic inflammation and accumulation of immune cells and fibrosis, compared to wild-type (WT) mice. Moreover, upregulation of inflammatory factors and impaired normal function were observed in the liver and kidneys of TNF-Tg mice. Inflammatory infiltration and fibrosis of the liver and kidneys of female TNF-Tg mice were improved after anti-TNF treatment, and better treatment effects were achieved at 4.5-month-old mice when they were received 8 weeks of intervention.ConclusionsWe found that TNF drives the development of liver and kidney pathology in female TNF-Tg mice and that there are limitations to the loss of utility of anti-TNF for the prolonged treatment of RA-associated hepatic and renal injury. This study provides a reliable and clinically relevant animal model for further studies exploring the molecular mechanisms and drug discovery for hepatorenal pathologies in RA.

A Technique to Generate an Adjuvant-Induced Arthritis Mouse Model

A Technique to Generate an Adjuvant-Induced Arthritis Mouse Model

A Technique to Generate an Adjuvant-Induced Arthritis Mouse Model

A Technique to Generate an Adjuvant-Induced Arthritis Mouse Model

Epigenetic and transcriptional regulation of CCL17 production by glucocorticoids in arthritis

Glucocorticoids (GCs) are potent anti-inflammatory agents and are broadly used in treating rheumatoid arthritis (RA) patients, albeit with adverse side effects associated with long-term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action. We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CCL17 has a non-redundant role in inflammatory arthritis. Here, we provide molecular evidence that GCs can suppress GM-CSF-mediated upregulation of IRF4 and CCL17 expression via downregulating JMJD3 expression and activity. In mouse models of inflammatory arthritis, GC treatment inhibited CCL17 expression and ameliorated arthritic pain-like behavior and disease. Significantly, GC treatment of RA patient peripheral blood mononuclear cells exvivo resulted in decreased CCL17 production. This delineated pathway potentially provides new therapeutic options for the treatment of many inflammatory conditions, where GCs are used as an anti-inflammatory drug but without the associated adverse side effects.

Xanthohumol relieves arthritis pain in mice by suppressing mitochondrial-mediated inflammation.

Chronic pain is the most common symptom for people who suffer from rheumatoid arthritis and it affects approximately 1% of the global population. Neuroinflammation in the spinal cord induces chronic arthritis pain. In this study, a collagen-induced arthritis (CIA) mice model was established through intradermally injection of type II collagen in complete Freund's adjuvant solution. Following CIA inducement, the paws and ankles of mice were found to swell, mechanical pain and spontaneous pain were induced, and their motor coordination was impaired. The spinal inflammatory reaction was triggered, which presented as severe infiltration of inflammatory cells, and the expression levels of GFAP, IL-1β, NLRP3, and cleaved caspase-1 increased. Oxidative stress in the spinal cord of CIA mice was manifested as reduced Nrf2 and NDUFB11 expression and SOD activity, and increased levels of DHODH and Cyto-C. At the same time, spinal AMPK activity was decreased. In order to explore the potential therapeutic options for arthritic pain, Xanthohumol (Xn) was intraperitoneally injected into mice for three consecutive days. Xn treatment was found to reduce the number of spontaneous flinches, in addition to elevating mechanical pain thresholds and increasing latency time. At the same time, Xn treatment in the spinal cord reduced NLRP3 inflammasome-mediated inflammation, increased the Nrf2-mediated antioxidant response, and decreased mitochondrial ROS level. In addition, Xn was found to bind with AMPK via two electrovalent bonds and increased AMPK phosphorylation at Thr174. In summary, the findings indicate that Xn treatment activates AMPK, increases Nrf2-mediated antioxidant response, reduces Drp1-mediated mitochondrial dysfunction, suppresses neuroinflammation, and can serve to relieve arthritis pain.

Targeting the chemokine receptor CXCR4 with histamine analog to reduce inflammation in juvenile arthritis.

Among immune cells, activated monocytes play a detrimental role in chronic and viral-induced inflammatory pathologies, particularly in Juvenile Idiopathic Arthritis (JIA), a childhood rheumatoid arthritis (RA) disease. The uncontrolled activation of monocytes and excessive production of inflammatory factors contribute to the damage of bone-cartilage joints. Despite the moderate beneficial effect of current therapies and clinical trials, there is still a need for alternative strategies targeting monocytes to treat RA. To explore such an alternative strategy, we investigated the effects of targeting the CXCR4 receptor using the histamine analog clobenpropit (CB). Monocytes were isolated from the blood and synovial fluids of JIA patients to assess CB's impact on their production of key inflammatory cytokines. Additionally, we administered daily intraperitoneal CB treatment to arthritic mice to evaluate its effects on circulating inflammatory cytokine levels, immune cell infiltrates, joints erosion, and bone resorption, as indicators of disease progression. Our findings demonstrated that CXCR4 targeting with CB significantly inhibited the spontaneous and induced-production of key inflammatory cytokines by monocytes isolated from JIA patients. Furthermore, CB treatment in a mouse model of collagen-induce arthritis resulted in a significant decrease in circulating inflammatory cytokine levels, immune cell infiltrates, joints erosion, and bone resorption, leading to a reduction in disease progression. In conclusion, targeting CXCR4 with the small amino compound CB shows promise as a therapeutic option for chronic and viral-induced inflammatory diseases, including RA. CB effectively regulated inflammatory cytokine production of monocytes, presenting a potential targeted approach with potential advantages over current therapies. These results warrant further research and clinical trials to explore the full therapeutic potential of targeting CXCR4 with CB-like molecules in the management of various inflammatory diseases.

GRK2 inhibits Flt-1+ macrophage infiltration and its proangiogenic properties in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2f/fLyz2-Cre+/− mice. Synovial inflammation and M1 polarization were improved in GRK2f/fLyz2-Cre+/− mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1+ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.

Sesamol serves as a p53 stabilizer to relieve rheumatoid arthritis progression and inhibits the growth of synovial organoids

BackgroundRheumatoid arthritis (RA) is an autoimmune disease known as a leading cause of disability with considerable mortality. Developing alternative drugs and targets for RA treatment is an urgent issue. Sesamol is a phenolic compound isolated from natural food sesame (Sesamum indicum L.) with various biological activities. PurposeThe current research intended to illuminate the bioactivity and mechanisms of sesamol in RA fibroblast-like synoviocytes (FLS), and aimed to estimate the potential clinical application value of sesamol in RA treatment. MethodsCCK-8, EdU, and flow cytometry assays, as well as transwell tests were applied to observe the effects of sesamol on the abnormal functions of RA-FLS. Moreover, synovial organoids and a collagen-induced arthritis (CIA) mouse model were constructed to further explore the therapeutic capacity of sesamol on RA. Furthermore, RNA sequencing combined with quantitative real-time PCR assay, Western blot as well as co-immunoprecipitation were employed to clarify the mechanism of sesamol in regulating RA progression. ResultsSesamol suppressed the proliferation through inhibiting DNA replication, triggering cell cycle arrest and apoptosis of RA-FLS. Besides, sesamol impaired RA-FLS migration and invasion. Interestingly, sesamol inhibited the growth of constructed synovial organoids and alleviated RA symptoms in CIA mice. Moreover, RNA sequencing further implicated p53 signaling as a downstream pathway of sesamol. Furthermore, sesamol was shown to decrease p53 ubiquitination and degradation, thereby activating p53 signaling. Finally, bioinformatics analyses also highlighted the importance of sesamol-regulated networks in the progression of RA. ConclusionsOur investigation demonstrated that sesamol served as a novel p53 stabilizer to attenuate the abnormal functions of RA-FLS via facilitating the activation of p53 signaling. Moreover, our study highlighted that sesamol might be an effective lead compound or candidate drug and p53 could be a promising target for the therapy of RA.

Effects of alcohol on the symptoms of gouty arthritis and taxonomic structure of gut microbiota in C57BL/6 mice.

Gout is an acute arthritis caused by the elevated levels of serum uric acid (UA), and its prevalence has been rapidly increasing. Alcohol abuse could lead to a series of health problems. Multiple pieces of evidence suggest that alcohol intake affects the development and progression of gout, while the gut microbiota plays an important role in the development of gout and the long-term alcohol consumption could affect the stability of the gut microbiota. This study aimed to explore the effects of alcohol intake at different concentrations on gouty arthritis based on the gut microbiota. We investigated the effects of different concentrations of alcohol on gouty arthritis in mouse models of acute gouty arthritis established by injection of monosodium urate (MSU) crystals into C57BL/6 mice. The results indicated that the high-alcohol consumption not only exacerbated joint swelling and pain, increased the levels of UA, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), but also showed dramatic effects on the composition and structure of the gut microbiota in gouty mice. Two key microorganisms, Parasutterella and Alistipes, could aggravate gout symptoms through lipopolysaccharide biosynthesis, riboflavin metabolism, phenylalanine metabolism, and arginine and proline metabolisms. In conclusion, our study suggested that high-concentrations of alcohol altered the gut microbiota structure in gouty mice induced by MSU crystals, which could exacerbate gouty symptoms by enhancing pro-inflammatory pathways.

Sterile triggers drive joint inflammation in TNF- and IL-1β-dependent mouse arthritis models.

Arthritis is the most common extra-intestinal complication in inflammatory bowel disease (IBD). Conversely, arthritis patients are at risk for developing IBD and often display subclinical gut inflammation. These observations suggest a shared disease etiology, commonly termed "the gut-joint-axis." The clinical association between gut and joint inflammation is further supported by the success of common therapeutic strategies and microbiota dysbiosis in both conditions. Most data, however, support a correlative relationship between gut and joint inflammation, while causative evidence is lacking. Using two independent transgenic mouse arthritis models, either TNF- or IL-1β dependent, we demonstrate that arthritis develops independently of the microbiota and intestinal inflammation, since both lines develop full-blown articular inflammation under germ-free conditions. In contrast, TNF-driven gut inflammation is fully rescued in germ-free conditions, indicating that the microbiota is driving TNF-induced gut inflammation. Together, our study demonstrates that although common inflammatory pathways may drive both gut and joint inflammation, the molecular triggers initiating such pathways are distinct in these tissues.

Dysregulation of B lymphocyte development in the SKG mouse model of rheumatoid arthritis.

Rheumatoid arthritis is a chronic and systemic inflammatory disease that affects approximately 1% of the world's population and is characterised by joint inflammation, the destruction of articular cartilage and bone, and many potentially life-threatening extraarticular manifestations. B lymphocytes play a central role in the pathology of rheumatoid arthritis as the precursors of autoantibody secreting plasma cells, as highly potent antigen-presenting cells, and as a source of various inflammatory cytokines, however, the effects of rheumatoid arthritis on B lymphocyte development remain poorly understood. Here, we analyse B lymphocyte development in murine models of rheumatoid arthritis, quantifying all the subsets of B cell precursors in the bone marrow and splenic B cells using flow cytometry. We demonstrate a severe reduction in pre-B cells and immature B cells in the bone marrow of mice with active disease, despite no major effects on the mature naïve B cell numbers. The loss of B cell precursors in the bone marrow of the affected mice was associated with a highly significant reduction in the proportion of Ki67+ cells, indicating impaired cell proliferation, while the viability of the B cell precursors was not significantly affected. We also observed some mobilisation of the B cell precursor cells into the mouse spleen, demonstrated with flow cytometry and pre-B colony forming units assays. In summary, the current work demonstrates a severe dysregulation in B lymphocyte development in murine rheumatoid arthritis, with possible implications for B cell repertoire formation, tolerance induction, and disease mechanisms.

DKK-1 and Its Influences on Bone Destruction: A Comparative Study in Collagen-Induced Arthritis Mice and Rheumatoid Arthritis Patients.

Dickkopf-1 (DKK-1) has been considered a master regulator of bone remodeling. As precursors of osteoclasts (OCs), myeloid-derived suppressor cells (MDSCs) were previously shown to participate in the process of bone destruction in rheumatoid arthritis (RA). However, the role of DKK-1 and MDSCs in RA is not yet fully understood. We investigated the relevance between the level of DKK-1 and the expression of MDSCs in different tissues and joint destruction in RA patients and collagen-induced arthritis (CIA) mouse models. Furthermore, the CIA mice were administered recombinant DKK-1 protein. The arthritis scores, bone destruction, and the percentage of MDSCs in the peripheral blood and spleen were monitored. In vitro, the differentiation of MDSCs into OCs was intervened with recombinant protein and inhibitor of DKK-1. The number of OCs differentiated and the protein expression of the Wnt/β-catenin signaling pathway were explored. The level of DKK-1 positively correlates with the frequency of MDSCs and bone erosion in RA patients and CIA mice. Strikingly, recombinant DKK-1 intervention significantly exacerbated arthritis scores and bone destruction, increasing the percentage of MDSCs in the peripheral blood and spleen in CIA mice. In vitro experiments showed that recombinant DKK-1 promoted the differentiation of MDSCs into OCs, reducing the expression of β-catenin and TCF4 and increasing the expression of CyclinD1. In contrast, the DKK-1 inhibitor had the opposite effect. Our findings highlight that DKK-1 promoted MDSCs expansion in RA and enhanced the differentiation of MDSCs into OCs via targeting the Wnt/β-catenin pathway, aggravating the bone destruction in RA.

Eggerthella lenta augments preclinical autoantibody production and metabolic shift mimicking senescence in arthritis.

Although the etiology of rheumatoid arthritis (RA) is unknown, a strong genetic predisposition and the presence of preclinical antibodies before the onset of symptoms is documented. An expansion of Eggerthella lenta is associated with severe disease in RA. Here, using a humanized mouse model of collagen-induced arthritis, we determined the impact of E. lenta abundance on RA severity. Naïve mice gavaged with E. lenta produce preclinical rheumatoid factor and, when induced for arthritis, develop severe disease. The augmented antibody response was much higher in female mice, and among patients with RA, women had higher average load of E. lenta. Expansion of E. lenta increased CXCL5 and CD4 T cells, and both interleukin-17- and interferon-γ-producing B cells. Further, E. lenta gavage caused gut dysbiosis and decline in amino acids and nicotinamide adenine dinucleotide with an increase in microbe-dependent bile acids and succinyl carnitine causing systemic senescent-like inflammation.

A synthetic derivative of bioactive constituents from Isatis indigotica ameliorates hypersensitivity and arthritis by inhibiting JAK2-STAT3 pathway in mice

The JAK-STAT pathway plays a crucial role in the signaling cascade associated with various cytokines that have been implicated in the pathogenesis of inflammatory diseases and myeloproliferative neoplasms (MPN). Among the isoforms of JAKs, the JAK2 subtype is primarily responsible for the function of hematopoietic system cells, making it a significant target in the treatment of MPN. However, the precise regulatory role of JAK2 in inflammatory diseases requires further investigation and confirmation. The current study employed a selective JAK2 inhibitor, ZT55, derived from Isatis indigotica roots, to examine its regulatory effects on inflammatory and immune responses in delayed-type hypersensitivity (DTH) and arthritis in mice. To evaluate the efficacy of ZT55 treatment, DNFB-induced DTH and collagen-induced arthritis (CIA) mouse models were utilized. T cells were cultured and subsequently analyzed for proliferation and activation using flow cytometry and EdU assay. Additionally, the maturation and function of dendritic cells were assessed through flow cytometry and ELISA. Our findings indicate that ZT55 significantly reduced DNFB-induced DTH and attenuated inflammation, cartilage degradation, and bone destruction in CIA mice. Moreover, ZT55 was found to inhibit the proliferation and activation of T cells and the maturation of dendritic cells by regulating the JAK2-STAT3 signaling pathway. These results suggest that selectively targeting the JAK2 isoform could have anti-inflammatory and immunosuppressive effects by regulating the adaptive and innate immune responses via the JAK2-STAT3 signaling pathway. Therefore, ZT55 has the potential to be a promising pharmaceutical candidate for the treatment of inflammatory and autoimmune diseases.

Melittin acupoint injection in attenuating bone erosion in collagen-induced arthritis mice via inhibition of the RANKL/NF-κB signaling pathway.

Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. Bone erosion is the most serious pathological condition of RA and the main cause of joint deformities and disability. Melittin acupoint injection (MAI) is an effective traditional Chinese medicine (TCM) method for RA treatment. This study aimed to investigate the effect of MAI on RA bone erosion and to elucidate the underlying mechanism. A collagen-induced arthritis (CIA) mouse model was established as the experimental subject. MAI was administrated once every other day for 28 days to mice with CIA. The effects of MAI on joint diseases were assessed by body weight, arthritis index (AI) score, swollen joint count (SJC) score, and hind paw thickness. Ankle radiological changes were captured by micro-computed tomography (micro-CT) and histological changes were observed by pathological staining. Organ histological changes, spleen index, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Crea) levels of serum were tested to evaluate the toxicity of MAI. Cytokine expression levels were confirmed by enzyme-linked immunosorbent assay (ELISA) to evaluate the immunity of CIA mice. MAI administration markedly improved the clinical signs of CIA in mice, including hind paw thickness, AI, and the number of swollen paw joints (most of them P<0.05 or even <0.01). According to histopathological analysis, MAI ameliorated inflammatory cell infiltration, synovial hyperplasia, pannus formation, and bone erosion (all P<0.01). Micro-CT and tartrate-resistant acid phosphatase (TRAP) staining (P<0.01) also revealed that MAI could relieve bone erosion via reducing the formation of osteoclasts. Not only could MAI relieve the immunological boost [P<0.05 for the high-dose MAI (HM) group], but also it had no liver or kidney side effects (P>0.05). In addition, it decreased the serum levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) and increased the serum levels of IL-4 and IL-10 (the majority of P<0.05 or even <0.01). Transcriptome sequencing results indicated that MAI affected the expression of osteoclast differentiation pathway genes, which was connected with the receptor activator of the nuclear factor κB ligand/nuclear factor kappa B (RANKL/NF-κB) pathway. Based on our findings, MAI could suppress joint inflammation and inhibit RANKL/NF-κB-mediated osteoclast differentiation to rescue bone erosion in CIA mice, suggesting that MAI can be a potentially therapeutic substance for RA.

The interplay between Epstein-Barr virus DNA and gut microbiota in the development of arthritis in a mouse model.

Epstein-Barr virus (EBV) DNA may influence the development of autoimmune diseases by increasing the production of proinflammatory cytokines. Such cytokines have been associated with inducing the dysbiosis of colonic microbiota, which, in turn, is a risk factor for autoimmune diseases such as rheumatoid arthritis (RA). Therefore, we investigated the role that EBV DNA may play in modulating the intestinal microbiota and consequent exacerbation of arthritis in a mouse model. Mice were treated with collagen (arthritis-inducing agent), EBV DNA and collagen, EBV DNA, or water. Fecal samples were collected from arthritic and control mice, and 16S rRNA sequencing was performed to determine the effect of EBV DNA on the composition of colonic microbiota. EBV DNA causes a change in the alpha diversity of the microbiota resulting in an increased Chao1 microbial richness and decreased Shannon diversity index in the RA mouse model. In addition, the abundance of particular genera/genus clusters was significantly altered among the various groups, with the EBV DNA-exacerbated arthritic group having the highest number of altered genera/genus cluster abundances. This group also had the highest number of cells co-expressing IL-17A, FOXP3, and IFNγ in the colons. Antimicrobial-cleared mice transplanted with fecal samples from EBV DNA-exacerbated arthritic mice showed a higher incidence and enhanced severity of RA compared to those transplanted with fecal samples from water or collagen-treated mice. IMPORTANCE Epstein-Barr virus (EBV) DNA alters the composition and diversity of the gut microbiota in a rheumatoid arthritis (RA) mouse model. These induced changes are associated with enhanced severity of symptoms. This better understanding of the various factors involved in the development of RA will possibly help in creating individualized treatments for RA patients including target mediators triggered by viral DNA. Given that a large swathe of the population harbors EBV, a significant proportion of subjects with arthritis may benefit from possible approaches that target EBV or mediators triggered by this virus.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

Reactive Oxygen Species-Responsive Polymeric Prodrug Nanoparticles for Selective and Effective Treatment of Inflammatory Diseases.

It is challenging to manage inflammatory diseases using traditional anti-inflammatory drugs due to their limited efficacy and systemic side effects, which are a result of their lack of selectivity, poor stability, and low solubility. Herein, it reports the development of a novel nanoparticle system, called ROS-CA-NPs, which is formed using polymer-cinnamaldehyde (CA) conjugates and is responsive to reactive oxygen species (ROS). ROS-CA-NPs exhibit excellent drug stability, tissue selectivity, and controlled drug release upon oxidative stress activation. Using mouse models of chronic rheumatoid arthritis and acute ulcerative colitis, this study demonstrates that the systemic administration of ROS-CA-NPs results in their accumulation at inflamed lesions and leads to greater therapeutic efficacy compared to traditional drugs. Furthermore, ROS-CA-NPs present excellent biocompatibility. The findings suggest that ROS-CA-NPs have the potential to be developed as safe and effective nanotherapeutic agents for a broad range of inflammatory diseases.

Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.