The NLRP3 Inflammasome Renders Cell Death Pro-inflammatory.

NLRP3 is the most studied inflammasome sensor due to its crucial involvement in sterile and infection-triggered inflammation. Although its molecular mode of activation remains to be defined, it is well established that low intracellular potassium concentrations result in its activation. This functionality allows the classical NLRP3 pathway to serve as a highly sensitive, but non-specific surveillance mechanism responding to any type of perturbation that breaches plasma membrane integrity and the associated potassium gradient across the membrane. Here, we review our current knowledge on potassium efflux-dependent NLRP3 activation, with a special focus on how major cell death programs are rendered pro-inflammatory by secondary NLRP3 activation. Apart from the "alternative inflammasome" as the major exception to the rule, this connection explains the fundamental importance of NLRP3 in cell death-associated inflammation and firmly establishes NLRP3 as a principal surveillance mechanism of cellular integrity.

Novel "Elements" of Immune Suppression within the Tumor Microenvironment.

Adaptive evolution has prompted immune cells to use a wide variety of inhibitory signals, many of which are usurped by tumor cells to evade immune surveillance. Although tumor immunologists often focus on genes and proteins as mediators of immune function, here we highlight two elements from the periodic table-oxygen and potassium-that suppress the immune system in previously unappreciated ways. While both are key to the maintenance of T-cell function and tissue homeostasis, they are exploited by tumors to suppress immuno-surveillance and promote metastatic spread. We discuss the temporal and spatial roles of these elements within the tumor microenvironment and explore possible therapeutic interventions for effective and promising anticancer therapies. Cancer Immunol Res; 5(6); 426-33. ©2017 AACR.

Potassium is a key signal in host-microbiome dysbiosis in periodontitis.

Dysbiosis, or the imbalance in the structural and/or functional properties of the microbiome, is at the origin of important infectious inflammatory diseases such as inflammatory bowel disease (IBD) and periodontal disease. Periodontitis is a polymicrobial inflammatory disease that affects a large proportion of the world's population and has been associated with a wide variety of systemic health conditions, such as diabetes, cardiovascular and respiratory diseases. Dysbiosis has been identified as a key element in the development of the disease. However, the precise mechanisms and environmental signals that lead to the initiation of dysbiosis in the human microbiome are largely unknown. In a series of previous in vivo studies using metatranscriptomic analysis of periodontitis and its progression we identified several functional signatures that were highly associated with the disease. Among them, potassium ion transport appeared to be key in the process of pathogenesis. To confirm its importance we performed a series of in vitro experiments, in which we demonstrated that potassium levels a increased the virulence of the oral community as a whole and at the same time altering the immune response of gingival epithelium, increasing the production of TNF-α and reducing the expression of IL-6 and the antimicrobial peptide human ß-defensin 3 (hBD-3). These results indicate that levels of potassium in the periodontal pocket could be an important element in of dysbiosis in the oral microbiome. They are a starting point for the identification of key environmental signals that modify the behavior of the oral microbiome from a symbiotic community to a dysbiotic one.

Mechanism and Regulation of NLRP3 Inflammasome Activation.

Members of the nucleotide-binding domain and leucine-rich repeat (LRR)-containing (NLR) family and the pyrin and HIN domain (PYHIN) family can form multiprotein complexes termed 'inflammasomes'. The biochemical function of inflammasomes is to activate caspase-1, which leads to the maturation of interleukin 1 beta (IL-1ß) and IL-18 and the induction of pyroptosis, a form of cell death. Unlike other inflammasomes, the NLRP3 inflammasome can be activated by diverse stimuli. The importance of the NLRP3 inflammasome in immunity and human diseases has been well documented, but the mechanism and regulation of its activation remain unclear. In this review we summarize current understanding of the mechanism and regulation of NLRP3 inflammasome activation as well as recent advances in the noncanonical and alternative inflammasome pathways.

Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux.

Recognition of microbe-associated molecular patterns or endogenous danger signals by a subset of cytosolic PRRs results in the assembly of multiprotein signaling complexes, the so-called inflammasomes. Canonical inflammasomes are assembled by NOD-like receptor (NLR) or PYHIN family members and activate caspase-1, which promotes the induction of pyroptosis and the release of mature interleukin-1ß/-18. Recently, a noncanonical inflammasome pathway was discovered that results in caspase-11 activation in response to bacterial lipopolysaccharide (LPS) in the cytosol. Interestingly, caspase-11 induces pyroptosis by itself, but requires NLRP3, the inflammasome adapter ASC, and caspase-1 to promote cytokine secretion. Here, we have studied the mechanism by which caspase-11 controls IL-1ß secretion. Investigating NLRP3/ASC complex formation, we find that caspase-11 functions upstream of a canonical NLRP3 inflammasome. The activation of NLRP3 by caspase-11 during LPS transfection is a cell-intrinsic process and is independent of the release of danger signals. Furthermore, we show that active caspase-11 leads to a drop of intracellular potassium levels, which is necessary to activate NLRP3. Our study, therefore, sheds new light on the mechanism of noncanonical inflammasome signaling.

K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling.

Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1-mediated production of IL-1ß and pyroptosis. However, the relative contributions of decreased cytosolic K(+) concentration versus increased cytosolic Ca(2+) concentration ([Ca(2+)]) remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca(2+)] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K(+) efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester. The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca(2+)] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent Leu-Leu-O-methyl ester. Second, agonists for three Ca(2+)-mobilizing G protein-coupled receptors (formyl peptide receptor, P2Y2 purinergic receptor, and calcium-sensing receptor) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared with the K(+) efflux agonists. Third, the intracellular Ca(2+) buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate, widely used reagents for disruption of Ca(2+)-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca(2+) homeostasis. The results indicate that the ability of K(+) efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca(2+)] as a necessary or sufficient signal.

Complement C5a potentiates uric acid crystal-induced IL-1ß production.

Anaphylatoxin C5a released upon complement activation is associated with both acute and chronic inflammations such as gout. The pathogenesis of gout was identified as uric acid crystal deposition in the joints that activates inflammasome, leading to IL-1ß release. However, little is known about the interaction between complement activation and monosodium urate/uric acid (MSU) crystal-induced inflammasome activation or IL-1ß production. Here, we report that MSU crystal-induced proinflammatory cytokines/chemokines in human whole blood is predominantly regulated by C5a through its interaction with C5a receptor. C5a induces pro-IL-1ß and IL-1ß production in human primary monocytes, and potentiates MSU or cholesterol crystals in IL-1ß production. This potentiation is caspase-1 dependent and requires intracellular Ca(2+) mobilization, K(+) efflux, and cathepsin B activity. Our results provide insight into the role of C5a as an endogenous priming signal that is required for the initiation of uric acid crystal-induced IL-1ß production. C5a could potentially be a therapeutic target together with IL-1ß antagonists for the treatment of complement-dependent and inflammasome-associated diseases.

Functional consequences of various leucine mutations in the M3/M4 loop of the Na+,K +-ATPase alpha-Subunit.

Leucines were mutated within the sequence L311 ILGYTWLE319 of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na+,K+-ATPase alpha-subunit. Replacement of Leu311 with Glu resulted in a considerable loss of Na+,K+-ATPase activity. Replacement of Leu313 with Glu shifted the equilibrium of E1P and E2P toward E1P and reduced the rate of the E1P to E2P transition. The reduction of the transition rate and stronger inhibition of Na+,K+-ATPase activity by Na+ at higher concentrations together suggest that there is interference of Na+ release on the extracellular side in the Leu313 mutant. Thus, Leu313 could be in the pathway of Na+ exit. Replacement of Leu318 with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K+, with an equilibrium shifted toward E2P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K+ (2)]E2 caused by inhibition of dephosphorylation through reduction of the K+ affinity of E2P. Thus, Leu318 may be a critical position in guiding external K+ to its binding site.

Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells that initiate the immune response by activating T lymphocytes. DCs express plasma membrane receptors for extracellular nucleotides named P2 receptors (P2Rs). Stimulation of P2Rs in these cells is known to cause chemotaxis, cytokine release, and cell death and to modulate LPS-dependent differentiation. Here we show that stimulation of the P2X(7) receptor subtype (P2X(7)R) causes fast microvesicle shedding from DC plasma membrane. Vesicle release occurs from both immature and mature DCs; however, only vesicles from mature DCs, due to their previous exposure to LPS, contain IL-1beta. Microvesicles, whether from immature or mature DCs, also contain caspase-1 and -3 and cathepsin D. They also express the P2X(7)R in addition to other P2Rs and known markers of immune cells such as major histocompatibility complex II (MHC II) and CD39. Activation of the P2X(7)R by extracellular ATP causes IL-1beta release from the vesicle lumen. Previous studies demonstrated that high extracellular K(+) inhibits IL-1beta processing and release; here we show that high ionic strength reduces microvesicle shedding when compared with a low ionic strength medium but strongly increases microvesicle IL-1beta loading.

Effect of malnutrition on K+ current in T lymphocytes.

Severe malnutrition in children is frequently associated with infectious diseases. Animal models have been useful for studying the effects of malnutrition. One of the immunosuppressive mechanisms of malnutrition is inhibition of the activation of T lymphocytes. The voltage-dependent K(V) potassium channels are vital for the activation of T lymphocytes. The blockade of K(V) channels inhibits the activation of T lymphocytes. Malnutrition could affect the suitable synthesis of K(V) channels in T lymphocytes, producing changes in the magnitude and/or dependency of the voltage of the K+ current. We reported a significant decrease in the K+ current and activation to a 20 mV more positive membrane potential in T lymphocytes of rats with severe malnutrition. These results indicate that the diminution in the K+ conductance by alteration of K(V) channels in severe malnutrition is one of the mechanisms that inhibit the activation of T lymphocytes.

Nervous immunity: neurotransmitters, extracellular K+ and T-cell function.

T cells are required to respond quickly to tissue injuries and dysregulations of varied complexity, and do so not under the direction of specific 'classical' immunological signals. This suggests that additional signals might be supplied by the nervous system and by physiological modulation of the ionic environment to activate T cells in a T-cell receptor independent manner.

Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins.

Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels.

Shifts of delayed immune response to persulphates and other allergens.

Certain chemicals such as persulphates and formaldehyde are able to induce both immediate and delayed type reactions in the skin. The levels of delayed type response to persulphates (at 48 and 74 h), obtained by epicutaneous tests were subjected to comparative studies of shifts (48 versus 72 h) and statistical analysis. The 95% confidence limit of the mean value of shifts to persulphates was significantly higher than that of a standard routine battery as a whole and, with the exception of formaldehyde, also to that of other individual constituents of this battery. In the case of persulphates, this difference in shifts was due to an extremely low mean value of delayed-type response read at 48 h. The possible underlying mechanisms and the possible limiting rôle of vaso-active amines in the early evolution of delayed-type response is discussed.

Interaction of HK and LK goat red blood cells with ouabain.

The characteristics of the interaction of Na-K pumps of high potassium (HK) and low potassium (LK) goat red blood cells with ouabain have been determined. The rate of inhibition by ouabain of the pump of HK cells is greater than the rate of inhibition of the pumps of LK cells. Treatment of LK cells with an antibody (anti-L) raised in HK sheep by injecting LK sheep red cells increases the rate of inhibition of the LK pumps by ouabain to that characteristic of HK pumps; reduction of intracellular K (K(c)) in LK cells increases the rate at which ouabain inhibits their pumps and exposure of these low K(c) cells to anti-L does not affect the rate of inhibition. There is considerable heterogeneity in the pumps of both HK and LK cells in the rate at which they interact with ouabain or the rate at which they pump or both. LK pumps which are sensitive to stimulation by anti-L bind ouabain less rapidly than the remainder of the LK pumps and exposure to antibody increases the rate at which ouabain binds to the sensitive pumps; the difference between the two types of pumps disappears if intracellular K is very low. The calculated number of ouabain molecules bound at 100% inhibition of the pump is about the same for HK and LK cells. Although exposure to anti-L increases the apparent number of ouabain binding sites in LK cells at normal K(c), it does not alter the apparent number of sites in LK cells when K(c) has been reduced.