Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome mediates caspase-1 activation and ...interleukin-1β release in immune cells but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3 inflammasome in AF.
NLRP3 inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal AF or long-standing persistent (chronic) AF. To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knockin mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electric activation pattern, Ca
spark frequency, atrial effective refractory period, and morphology of atria were evaluated in CM-KI mice and wild-type littermates.
NLRP3 inflammasome activity was increased in the atrial CMs of patients with paroxysmal AF and chronic AF. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3 inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic reticulum Ca
release, atrial effective refractory period shortening, and atrial hypertrophy. Adeno-associated virus subtype-9-mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF.
Our study establishes a novel pathophysiological role for CM NLRP3 inflammasome signaling, with a mechanistic link to the pathogenesis of AF, and establishes the inhibition of NLRP3 as a potential novel AF therapy approach.
Ion channels and anti-cancer immunity Panyi, Gyorgy; Beeton, Christine; Felipe, Antonio
Philosophical transactions - Royal Society. Biological sciences,
03/2014, Letnik:
369, Številka:
1638
Journal Article
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The outcome of a malignant disease depends on the efficacy of the immune system to destroy cancer cells. Key steps in this process, for example the generation of a proper Ca2+ signal induced by ...recognition of a specific antigen, are regulated by various ion channel including voltage-gated Kv1.3 and Ca2+-activated KCa3.1 K+ channels, and the interplay between Orai and STIM to produce the Ca2+-release-activated Ca2+ (CRAC) current required for T-cell proliferation and function. Understanding the immune cell subset-specific expression of ion channels along with their particular function in a given cell type, and the role of cancer tissue-dependent factors in the regulation of operation of these ion channels are emerging questions to be addressed in the fight against cancer disease. Answering these questions might lead to a better understanding of the immunosuppression phenomenon in cancer tissue and the development of drugs aimed at skewing the distribution of immune cell types towards killing of the tumour cells.
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups ...based on their structure and substrate specificity (collagenases, gelatinases, membrane type MT-MMP, stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel. Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.
Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone ...Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.
Electrophysiological and pharmacological studies coupled with molecular identification have revealed a unique network of ion channels—Kv1.3, KCa3.1, CRAC (Orai1 + Stim1), TRPM7, Clswell—in ...lymphocytes that initiates and maintains the calcium signaling cascade required for activation. The expression pattern of these channels changes during lymphocyte activation and differentiation, allowing the functional network to adapt during an immune response. The Kv1.3 channel is of interest because it plays a critical role in subsets of T and B lymphocytes implicated in autoimmune disorders. The ShK toxin from the sea anemone Stichodactyla helianthus is a potent blocker of Kv1.3. ShK-186, a synthetic analog of ShK, is being developed as a therapeutic for autoimmune diseases, and is scheduled to begin first-in-man phase-1 trials in 2011. This review describes the journey that has led to the development of ShK-186.
► ShK-186 is a 37-residue synthetic derivative of ShK, a peptide toxin from the sea anemone Stichodactyla helianthus. ► ShK-186 blocks Kv1.3 potassium channels with picomolar affinity and with a high degree of specificity. ► Kv1.3 potassium channels play a critical role in regulating the function of effector-memory T cells and class-switched memory B cells that are implicated in autoimmune diseases. ► ShK-186 has a good safety profile in rats and it ameliorates disease in rat models of multiple sclerosis and rheumatoid arthritis. ► ShK-186 is being developed as a therapeutic for autoimmune diseases and phase 1 human trials are planned for the near future.
Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes ...mellitus or rheumatoid arthritis (RA) are mainly CD4⁺CCR7⁻CD45RA⁻ effector memory T cells ($T_{EM}$cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naíve or central-memory ($T_{CM}$) cells. In$T_{EM}$cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvβ2, SAP97, ZIP,$p56^{Ick}$, and CD4. Although Kv1.3 inhibitors ShK(L5)-amide (SL5) and PAP1 do not prevent immunological synapse formation, they suppress Ca²⁺-signaling, cytokine production, and proliferation of autoantigen-specific$T_{EM}$cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.
How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice, N-glycan branching by the ...Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis, spontaneous inflammatory demyelination and neurodegeneration, the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3), including genetic variants in interleukin-7 receptor-α (IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3), optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation.
Fibroblast-like synoviocytes (FLS) and CCR7
effector memory T (T
) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly ...invasive, whereas T
cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and T
cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated T
cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and T
cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA.
We used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic T
cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies.
We show that the interactions of FLS from rats with CIA and of rat T
cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate T
cell proliferation and migration, and inhibiting Kv1.3 on T
cells reduces T
cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA.
Our results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and T
cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.
Allergic asthma is a chronic inflammatory disease of the airways. Of the different lower airway-infiltrating immune cells that participate in asthma, T lymphocytes that produce Th2 cytokines play ...important roles in pathogenesis. These T cells are mainly fully differentiated CCR7− effector memory T (TEM) cells. Targeting TEM cells without affecting CCR7+ naïve and central memory (TCM) cells has the potential of treating TEM-mediated diseases, such as asthma, without inducing generalized immunosuppression. The voltage-gated KV1.3 potassium channel is a target for preferential inhibition of TEM cells. Here, we investigated the effects of ShK-186, a selective KV1.3 channel blocker, for the treatment of asthma. A significant proportion of T lymphocytes in the lower airways of subjects with asthma expressed high levels of KV1.3 channels. ShK-186 inhibited the allergen-induced activation of peripheral blood T cells from those subjects. Immunization of F344 rats against ovalbumin followed by intranasal challenges with ovalbumin induced airway hyper-reactivity, which was reduced by the administration of ShK-186. ShK-186 also reduced total immune infiltrates in the bronchoalveolar lavage and number of infiltrating lymphocytes, eosinophils, and neutrophils assessed by differential counts. Rats with the ovalbumin-induced model of asthma had elevated levels of the Th2 cytokines IL-4, IL-5, and IL-13 measured by ELISA in their bronchoalveolar lavage fluids. ShK-186 administration reduced levels of IL-4 and IL-5 and induced an increase in the production of IL-10. Finally, ShK-186 inhibited the proliferation of lung-infiltrating ovalbumin-specific T cells. Our results suggest that KV1.3 channels represent effective targets for the treatment of allergic asthma.
CCR7− effector memory T lymphocytes are major players in lung inflammation that characterizes allergic asthma.
Blocking KV1.3 channels reduced the severity of an ovalbumin-induced model of asthma in rats.
KV1.3 channels are attractive targets for immunomodulation and the treatment of allergic asthma.
Selective KV1.3 channel blockers may prove beneficial in the treatment of asthma.
The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its ...application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShKK18A and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.
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