Quantitative tracking of inflammatory activity at the peak and trough plasma levels of tofacitinib, a Janus kinase inhibitor, via in vivo 18 F-FDG PET.

PURPOSE: To assess the capability of in vivo positron emission tomography (PET) using 18 F-fluorodeoxyglucose (18 F-FDG) to quantify changes in inflammatory activity in response to tofacitinib, a Janus kinase (JAK) inhibitor, over a timeframe of a few hours to few days in a preclinical model of rheumatoid arthritis (RA). METHODS: Twenty-four mice with collagen-induced arthritis in the following groups were assessed: Group 1, where the changes in PET measures for the extremity joints were evaluated at the peak and trough plasma drug levels after administration of a single dose of tofacitinib (4 hours apart); Group 2, where joint PET measures were assessed before treatment and after 6 days of administration of a daily dose of tofacitinib; and group 3 (controls), where joint PET measures were derived from the same mice, 6 days apart. RESULTS: At about peak plasma levels of the drug after a single tofacitinib administration, there was a reduction in PET measures compared to pretreatment values, suggesting decreased inflammatory activity. These measures were equivalent to those obtained after 6 days of daily dosing by tofacitinib. However, PET measures at trough plasma levels of the drug from tofacitinib administration were significantly higher than those at peak plasma drug levels and equivalent to pretreatment measures. There were insignificant changes in PET measures for the control animals. CONCLUSION: 18 F-FDG PET can detect changes in inflammatory activity occurring in response to the JAK inhibitor tofacitinib: (a) during peak and trough plasma drug levels, that is within mere hours of treatment; and (b) over a span of days.

IL-9, a local growth factor for synovial T cells in inflammatory arthritis.

OBJECTIVE: The regulatory role of the Th9 cells along with its signature cytokine IL-9 in human immune system and its aberrant activation in autoimmune diseases is currently under investigation. We are reporting the functional significance of IL-9 in the pathogenesis of autoimmune inflammatory arthritis. METHODS: CD3(+) T cells were obtained from peripheral blood (PB) and synovial fluid (SF) of psoriatic arthritis (PsA), rheumatoid arthritis (RA), and osteoarthritis (OA) patients. MTT, FACS based CFSE dilution assay and apoptosis assay (Annexin-V) were performed to determine the pro-growth/survival effect of human recombinant IL-9 on activated CD3(+) T cells. Immunoblots were performed to determine the signaling proteins responsible for the progrowth/survival effect of IL-9. RESULTS: SF of PsA and RA was enriched with IL-9 producing CD3(+) T cells compared to the SF in OA. IL-9 level measured by ELISA was significantly elevated in PsA and RA patients compared to SF in OA (<.001). Activated T cells of PsA and RA had higher levels of IL-9 receptors. IL-9 promoted proliferation and survival of the CD3(+) T cells of PB and SF of PsA and RA and compared to untreated (media) controls (p<.005, t-test). IL-9 induced proliferation of T cells was dependent on PI3K/Akt/mTOR signaling pathway. CONCLUSION: IL-9 is functionally active, and is a pro-growth/survival factor for the localized pathologic T cells in the synovium of inflammatory arthritis. The pro-growth/survival effect is mediated by the activation of mTOR kinase cascade. To our knowledge, this is the first report of a functional role of IL-9 in human autoimmune arthritis.

In vivo quantification of mouse autoimmune arthritis by PET/CT.

AIM: To quantify the progression and severity of mouse collagen-induced arthritis (CIA) using an in vivo imaging tool, (18) F-fluorodeoxyglucose ((18) F-FDG) PET/CT and validate it against gold standard 'histopathological' evaluation. METHOD: The PET radiotracer (18) F-FDG, a marker for glucose metabolism, was injected in mice at different stages of CIA and the radiotracer distribution was imaged using a PET scanner. A sequential CT scan provided correlated anatomy. Radiotracer concentration was derived from PET/CT images for individual limb joints and on a per-limb basis at different stages of the disease. The imaging outcomes were subjected to correlation analysis with concurrently measured clinical and histological score. RESULTS: Clinical and histological score, and hence disease severity, showed a strong linear correlation (r(2)  = 0.71, P = 0.001 and r(2)  = 0.87, P < 0.001, respectively) with radiotracer concentration measured from PET/CT during the progression of CIA. CONCLUSIONS: The strong positive correlation of the (18) F-FDG PET/CT findings with the histopathological evaluation at different stages of the disease suggest the potential of this imaging tool for the non-invasive assessment of progression and severity in mouse autoimmune arthritis. Thus, in preclinical studies, (18) F-FDG PET/CT can be considered as a non-invasive tool to develop novel therapies of inflammatory arthritis.

Cross talk between neuroregulatory molecule and monocyte: nerve growth factor activates the inflammasome.

BACKGROUND: Increasing evidence points to a role for the extra-neuronal nerve growth factor (NGF) in acquired immune responses. However, very little information is available about its role and underlying mechanism in innate immunity. The role of innate immunity in autoimmune diseases is becoming increasingly important. In this study, we explored the contribution of pleiotropic NGF in the innate immune response along with its underlying molecular mechanism with respect to IL-1ß secretion. METHODS: Human monocytes, null and NLRP3 deficient THP-1 cell lines were used for this purpose. We determined the effect of NGF on secretion of IL-1ß at the protein and mRNA levels. To determine the underlying molecular mechanism, the effect of NGF on NLRP1/NLRP3 inflammasomes and its downstream key protein, activated caspase-1, were evaluated by ELISA, immunoflorescence, flow cytometry, and real-time PCR. RESULTS: In human monocytes and null THP-1 cell line, NGF significantly upregulates IL-1ß at protein and mRNA levels in a caspase-1 dependent manner through its receptor, TrkA. Furthermore, we observed that NGF induces caspase-1 activation through NLRP1/NLRP3 inflammasomes, and it is dependent on the master transcription factor, NF-κB. CONCLUSIONS: To best of our knowledge, this is the first report shedding light on the mechanistic aspect of a neuroregulatory molecule, NGF, in innate immune response, and thus enriches our understanding regarding its pathogenic role in inflammation. These observations add further evidence in favor of anti-NGF therapy in autoimmune diseases and also unlock a new area of research about the role of NGF in IL-1ß mediated diseases.

Kv1.3 in psoriatic disease: PAP-1, a small molecule inhibitor of Kv1.3 is effective in the SCID mouse psoriasis--xenograft model.

Kv1.3 channels regulate the activation/proliferation of effector memory T cells and thus play a critical role in the pathogenesis of autoimmune diseases. Using a combination of immunohistochemistry, confocal microscopy, flow cytometry and electrophysiology methods we observed a significant enrichment of activated Kv1.3(+) memory T cells in psoriasis plaques and synovial fluid from patients with psoriasis/psoriatic arthritis (PsA) compared to non-lesional psoriatic skin, normal skin or peripheral blood lympho-mononuclear cells. In in vitro studies performed with lesional mononuclear cells or T cells derived from skin and joints of psoriatic disease, the small molecule Kv1.3 blocker PAP-1 dose-dependently inhibited proliferation and suppressed IL-2 and IFN-γ production. To further substantiate the pathologic role of Kv1.3 high TEM cells in psoriatic disease we tested whether PAP-1 is able to improve psoriatic disease pathology in the SCID mouse-psoriasis skin xenograft model. Following four weeks of daily treatment with 2% PAP-1 ointment we noticed about 50% reduction in the epidermal thickness (rete peg length) and the number of CD3(+) lymphocytes/mm(2) of dermis decreased by 85%. Vehicle treated and untreated plaques in contrast remained unchanged and showed no reduction in epidermis thickness and infiltrating CD3(+) T cells and HLA-DR(+) T cells. Based on these results we propose the development of Kv1.3 targeted topical immunotherapy for psoriasis and possibly for other inflammatory skin conditions, where effector memory T cells are involved in the pathogenesis.

Severe combined immunodeficiency mouse-psoriatic human skin xenograft model: a modern tool connecting bench to bedside.

Psoriasis is a multifactorial chronic inflammatory disease. Research into the pathogenesis of this disease is hindered by the lack of a proper animal model. Over the past two decades, many scientists were involved in the development of animal models that nearly mirror the immunopathogenesis of psoriasis. One such model, which has opened doors to the study of molecular complexities of psoriasis as well as its treatment, is the severe combined immunodeficiency (SCID) mouse-human skin chimera model. This model not only mirrors the clinical and histopathological features of psoriasis but also help in the study of cell proliferation, angiogenesis, function of T cells, neurogenic inflammation and cytokines involved in inflammatory reactions. In this article, we have reviewed the prospects and the limitations of the SCID mouse model of psoriasis.

1,25-Dihydroxyvitamin D3-3-bromoacetate, a novel vitamin D analog induces immunosuppression through PI3K/Akt/mTOR signaling cascade.

PURPOSE: The molecular mechanism responsible for the immunomodulatory effect of 1,25-dihydroxyvitamin D3 (Vit-D) is still not well elucidated. Unavoidable systemic toxicity of Vit-D has encouraged to develop more potent and less toxic Vit-D analogs, such as 1,25-dihydroxyvitamin D3-3-bromoacetate (BE). Our aim was to explore the immunosuppressive effect of BE and its molecular mechanism in autoimmune diseases. METHOD: Magnetically sorted CD3(+) T cells (T cells) from PBMCs of psoriasis and autoimmune arthritis patients were cultured with/without BE and Vit-D followed by proliferation (MTT, CFSE dilution assays) and apoptosis assays (annexin V). Immunoblot was performed to determine the signaling cascade responsible for the antiproliferative effect. RESULTS: In MTT assay, BE (OD: 0.64±0.08) markedly inhibited the anti-CD3/CD28 stimulated proliferation of T cells (OD: 1.8±0.30, p<0.001) and at equivalent doses, the inhibitory effect was more than that of Vit-D (OD: 0.91±0.11, p<0.05). The antiproliferative effect of BE was extended to activated CD4(+) and CD8(+) memory T cells (CD45RA(-)CD11a(+)) without much effect on the naïve T cells. BE induced more apoptosis of T cells (45.01±4.27%, p<0.01) compared to untreated cells (3.45±1.8%), and the proapoptotic effect was markedly more than that of Vit-D (26.1±2.05%, p<0.05). BE effectively inhibited the anti-CD3/CD28-induced phosphorylation of Akt and mTOR and in both, BE showed more potency than Vit-D (p<0.05). CONCLUSION: Topical Vit-D is being used successfully in psoriasis for years. However, its potency is less compared to topical corticosteroids. The de novo BE showed significantly more immunosuppression than conventional Vit-D and the immunosuppressive effect is PI3K/Akt/mTOR dependent. Our results indicate that BE could be an effective therapeutic agent for psoriasis and other T-cell-mediated autoimmune diseases.

1α,25-Dihydroxyvitamin-D3-3-bromoacetate regulates AKT/mTOR signaling cascades: a therapeutic agent for psoriasis.

The efficacy of 1α,25-dihydroxyvitamin D3 (Vit-D) limits its topical use despite its profound effects on cellular differentiation, proliferation, and immunomodulation. Therefore, in search for a more effective analog of Vit-D, in this study we have evaluated the antiproliferative and proapoptotic effects of 1α,25-dihydroxyvitamin D3-3-bromoacetate (BE). Proliferation and apoptosis studies in normal human epidermal keratinocytes (NHEKs) were conducted by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), CFSE (carboxy fluorescein succinimidyl ester) dilution, and Annexin V assays. Western blot analysis and real-time PCR were performed to determine its effect on signal transduction. A reconstructed human epidermis (RHE) model was used to further validate the therapeutic role of BE in psoriasis. BE was significantly more potent than an equivalent concentration of Vit-D in inhibiting growth and survival of human keratinocytes. The antimitotic effect was found to be due to the inhibition of phosphorylation of serine/threonine protein kinase (AKT) and its downstream target, mammalian target of rapamycin (mTOR). In the RHE model, BE reversed IL-22-induced psoriasiform changes more effectively than Vit-D. Interestingly, BE inhibited the IL-22-induced gene expression of AKT1, MTOR, chemokines [IL-8 and RANTES (regulated upon activation, normal T-cell expressed and secreted)], and psoriasin (S100A7) more significantly than Vit-D. These results suggest the potential of BE as a prospective therapeutic agent for psoriasis.

FR255734, a humanized, Fc-Silent, Anti-CD28 antibody, improves psoriasis in the SCID mouse-psoriasis xenograft model.

In psoriasis, CD28/B7 costimulatory molecules are well characterized. Here, using the severe combined immunodeficient (SCID) mouse-psoriasis xenograft model, we report therapeutic efficacy of a humanized anti-CD28 monoclonal antibody (FR255734; Astellas Pharmaceuticals Inc., Tokyo, Japan). Transplanted psoriasis plaques on the SCID mouse were treated weekly for 4 weeks with intraperitoneal injections of FR255734 at 10, 3, and 1-mg kg(-1) doses. Groups treated with doses of 10 and 3 mg kg(-1) had significant thinning of the epidermis and reduced HLA-DR-positive lymphocytic infiltrates. The length of the rete pegs changed from 415.2+/-59.6 to 231.4+/-40.4 microm (P<0.005) in the 10-mg kg(-1) group, and from 323.4+/-69.6 to 237.5+/-73.6 microm in the 3-mg kg(-1) group (P=0.002). Positive controls treated with CTLA4-Ig and cyclosporine had significant histological improvement, whereas plaques treated with saline and isotype controls (human and mouse IgG2) remained unchanged. In vitro studies have shown that FR255734 effectively blocked T-cell proliferation and proinflammatory cytokine production. These observations warrant studies to evaluate the efficacy of FR255734 in human autoimmune diseases.

K252a, a high-affinity nerve growth factor receptor blocker, improves psoriasis: an in vivo study using the severe combined immunodeficient mouse-human skin model.

The peripheral nervous system, in addition to its sensory and motor functions, can induce a local inflammatory response known as neurogenic inflammation. This phenomenon plays a critical role in several inflammatory diseases, e.g., asthma, atopy, rheumatoid arthritis, psoriasis, and ulcerative colitis. Neurogenic inflammation and the role of nerve growth factor (NGF) have been extensively studied in psoriasis. There are increased levels of NGF in the keratinocytes and upregulation of NGF receptor (NGF-R) in the cutaneous nerves of psoriatic plaques. NGF can influence all the salient pathologic events noticed in psoriasis such as proliferation of keratinocytes, angiogenesis, T cell activation, expression of adhesion molecules, proliferation of cutaneous nerves, and upregulation of neuropeptides. In this double-blinded, placebo-controlled study, we addressed the role of NGF/NGF-R in psoriasis in an in vivo system using the severe combined immunodeficient (SCID) mouse-human skin model of psoriasis. The transplanted psoriatic plaques on the SCID mice (n=12) were treated with K252a, a high-affinity NGF receptor blocker. Psoriasis significantly improved following 2 wk of therapy. The length of the rete pegs changed from 308.57+/-98.72 to 164.64+/-46.78 microm (p<0.01, Student's t test). A similar improvement of psoriasis was observed by directly inhibiting NGF with NGF-neutralizing antibody. NGF-neutralizing antibody in normal saline at 10 ng (n=4) and 20 ng (n=4) per kilogram of body weight doses were used. Both doses of NGF-neutralizing antibody reduced rete peg lengths significantly, e.g., from 298.5+/-42.69 to 150.52+/-32.93 microm (p<0.05, Student's t test). This study provides evidence for the role of NGF and its high-affinity receptor in the pathogenesis of psoriasis and insights to develop novel therapeutic modalities.