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

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

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-1β 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-1β 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-1β 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-1β mediated diseases

In vivo quantification of mouse autoimmune arthritis by PET/CT

AimTo 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.MethodThe 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.ResultsClinical 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.ConclusionsThe 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

NGF induces activation of caspase-1 through NLRP1/NLRP3 inflammasomes in monocytic cell line.

<p><b>(A)</b> Magnetically sorted human monocytes (n = 8) were cultured 24 hrs with NGF (100 ng/ml) and activated caspase-1 was determined using FLICA kit. Representative immunofluorescent images (20x, Leica Microscope) showing more FLICA⁺ positive monocytes in NGF treated culture compared to untreated. <b>(B)</b> Monocytes cultured in similar settings with FLICA were subjected to flow cytometry to determine activated caspase-1 using FlowJo. First representative histogram is the stain-1 for FLICA using PI^- cells. NGF treatment showed more FLICA⁺ monocytes compared to untreated control. <b>(C)</b> Real-time PCR was done with extracted total RNA from null THP-1 cells cultured 24 hrs with or without NGF (100 ng/ml), K252a (100 ng/ml), PLX7486 (100 ng/ml) and PDTC (50 μM). 18S rRNA was used as an endogenous control. NGF significantly upregulated mRNA of NLRP1, which is effectively blocked by K252a, PLX7486 and PDTC (n = 6). <b>(D)</b> In similar setting, NGF/TrkA interaction significantly upregulated mRNA of NLRP3 in a NF-κB dependent manner (n = 6). Data expressed as Mean ± SEM. Mann Whitney U test was done to determine statistical significance.</p

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

PurposeTo 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).MethodsTwenty-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&nbsp;hours apart); Group 2, where joint PET measures were assessed before treatment and after 6&nbsp;days of administration of a daily dose of tofacitinib; and group 3 (controls), where joint PET measures were derived from the same mice, 6&nbsp;days apart.ResultsAt 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&nbsp;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.Conclusion18 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

Schematic diagram representing the NGF-induced activation of inflammasome complex and subsequent release of mature IL-1β.

<p>Briefly, NGF/TrkA interaction modulates the IL-1β release by acting at both transcriptional and posttranscriptional level. At transcriptional level, NGF upregulates the mRNA expression of NLRP1, NLRP3, procaspase-1 and proIL-1β. By upregulating the NLRP1/NLRP3 mRNA, NGF induces formation of active inflammasome complex, which converts pro IL-1β to mature IL-1β.</p

NGF/TrkA interaction induces IL-1β at protein and mRNA level in monocytic cell line through NF-κB.

<p><b>(A)</b> In null THP-1 cells, NGF (100 ng/ml) significantly induced mature IL-1β secretion compared to NLRP3 deficient THP-1 cells (DNLRP3). Prior treatment with TrkA inhibitors (K252a, 100 ng/ml and PLX7486, 100 ng/ml) or NF-κB inhibitor, PDTC (50 μM) effectively inhibited NGF induced IL-1β release (n = 6). <b>(B)</b> Western blot assay was done with null THP-1 cells cultured for 24 hrs with NGF (100 ng/ml) and LPS (1 μg/ml). Representative immunoblot and bar diagram (n = 6) showing significant phosphorylation of NF-κB p65 with NGF. LPS was used as a positive control. <b>(C)</b> Real-time PCR was performed with total RNA extracted from human monocytes cultured for 24 hrs with NGF (n = 8). NGF significantly upregulated IL-1β mRNA. <b>(D)</b> Magnetically sorted monocytes (n = 12) were cultured with or without NGF (100 ng/ml) and caspase-1 inhibitor (YVAD, 50 μM). Bar diagram showing significant induction of IL-1β with NGF and YVAD effectively blocked it. Data expressed as Mean±SEM. Mann Whitney U test was done to determine statistical significance.</p