Store-Operated Ca(2+) Entry in Follicular T Cells Controls Humoral Immune Responses and Autoimmunity.

T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription-factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.

The NLRP12 Sensor Negatively Regulates Autoinflammatory Disease by Modulating Interleukin-4 Production in T Cells.

Missense mutations in the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing family of gene 12 (Nlrp12) are associated with periodic fever syndromes and atopic dermatitis in humans. Here, we have demonstrated a crucial role for NLRP12 in negatively regulating pathogenic T cell responses. Nlrp12(-/-) mice responded to antigen immunization with hyperinflammatory T cell responses. Furthermore, transfer of CD4(+)CD45RB(hi)Nlrp12(-/-) T cells into immunodeficient mice led to more severe colitis and atopic dermatitis. NLRP12 deficiency did not, however, cause exacerbated ascending paralysis during experimental autoimmune encephalomyelitis (EAE); instead, Nlrp12(-/-) mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of balance. Enhanced T-cell-mediated interleukin-4 (IL-4) production promotes the development of atypical EAE disease in Nlrp12(-/-) mice. These results define an unexpected role for NLRP12 as an intrinsic negative regulator of T-cell-mediated immunity and identify altered NF-κB regulation and IL-4 production as key mediators of NLRP12-associated disease.

The inflammasome adaptor ASC regulates the function of adaptive immune cells by controlling Dock2-mediated Rac activation and actin polymerization.

The adaptor ASC contributes to innate immunity through the assembly of inflammasome complexes that activate the cysteine protease caspase-1. Here we demonstrate that ASC has an inflammasome-independent, cell-intrinsic role in cells of the adaptive immune response. ASC-deficient mice showed defective antigen presentation by dendritic cells (DCs) and lymphocyte migration due to impaired actin polymerization mediated by the small GTPase Rac. Genome-wide analysis showed that ASC, but not the cytoplasmic receptor NLRP3 or caspase-1, controlled the mRNA stability and expression of Dock2, a guanine nucleotide-exchange factor that mediates Rac-dependent signaling in cells of the immune response. Dock2-deficient DCs showed defective antigen uptake similar to that of ASC-deficient cells. Ectopic expression of Dock2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating Dock2-dependent Rac activation and actin polymerization in DCs and lymphocytes.

Signaling via the RIP2 adaptor protein in central nervous system-infiltrating dendritic cells promotes inflammation and autoimmunity.

Peripheral peptidolgycan (PGN) is present within antigen-presenting cells in the central nervous system (CNS) of multiple sclerosis (MS) patients, possibly playing a role in neuroinflammation. Accordingly, PGN is linked with disease progression in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), but the role of specific PGN-sensing proteins is unknown. Here we report that the progression of EAE was dependent on the intracellular PGN sensors NOD1 and NOD2 and their common downstream adaptor molecule, receptor interacting protein 2 (RIP2; also known as RIPK2 and RICK). We found that RIP2, but not toll-like receptor 2 (TLR2), played a critical role in the activation of CNS-infiltrating dendritic cells. Our results suggest that PGN in the CNS is involved in the pathogenesis of EAE through the activation of infiltrating dendritic cells via NOD1-, NOD2-, and RIP2-mediated pathways.

Selective ORAI1 Inhibition Ameliorates Autoimmune Central Nervous System Inflammation by Suppressing Effector but Not Regulatory T Cell Function.

The function of CD4(+) T cells is dependent on Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca(2+) influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4(+) T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.

Interference with Ca(2+) release activated Ca(2+) (CRAC) channel function delays T-cell arrest in vivo.

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.

Molecular regulation of CRAC channels and their role in lymphocyte function.

Calcium (Ca(2+)) influx is required for the activation and function of all cells in the immune system. It is mediated mainly by store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels located in the plasma membrane. CRAC channels are composed of ORAI proteins that form the channel pore and are activated by stromal interaction molecules (STIM) 1 and 2. Located in the membrane of the endoplasmic reticulum, STIM1 and STIM2 have the dual function of sensing the intraluminal Ca(2+) concentration in the ER and to activate CRAC channels. A decrease in the ER's Ca(2+) concentration induces STIM multimerization and translocation into puncta close to the plasma membrane where they bind to and activate ORAI channels. Since the identification of ORAI and STIM genes as the principal mediators of CRAC channel function, substantial advances have been achieved in understanding the molecular regulation and physiological role of CRAC channels in cells of the immune system and other organs. In this review, we discuss the mechanisms that regulate CRAC channel function and SOCE, the role of recently identified proteins and mechanisms that modulate the activation of ORAI/STIM proteins and the consequences of CRAC channel dysregulation for lymphocyte function and immunity.

Regulation of lymphocyte function by ORAI and STIM proteins in infection and autoimmunity.

Store-operated Ca(2+) entry (SOCE) in cells of the immune system is mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels that are formed by ORAI1 and its homologues ORAI2 and ORAI3. They are activated by stromal interaction molecules (STIM) 1 and 2 in response to depletion of endoplasmic reticulum Ca(2+) stores. Loss-of-function mutations in the human ORAI1 and STIM1 genes abolish CRAC channel function and SOCE in a variety of non-excitable cells including lymphocytes and other immune cells, resulting in a unique clinical syndrome termed CRAC channelopathy. It is dominated by severe immunodeficiency and autoimmunity due to impaired SOCE and defects in the function of several lymphocyte subsets. These include CD8(+) T cells, CD4(+) effector and regulatory T cells, natural killer (NK) cells and B cells. This review provides a concise discussion of the role of CRAC channels in these lymphocyte populations and the regulation of adaptive immune responses to infection, in autoimmunity and inflammation.

The inflammasome drives protective Th1 and Th17 cellular responses in disseminated candidiasis.

The Nlrp3 inflammasome has been proposed to play an important role in antifungal host defense. However, studies exploring the role of the inflammasome in antifungal host defense have been limited to the direct effects on IL-1ß processing. Although IL-1ß has important direct effects on the innate immune response, important effects of the caspase-1-dependent cytokines IL-1ß and IL-18 are exerted on the initiation of the adaptive Th1 and Th17 cellular responses. No studies have been employed to assess the impact of the inflammasome on the Th1/Th17 defense mechanisms in vivo during candidiasis. In the present study, we demonstrate an essential role for caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) in disseminated candidiasis through regulating antifungal Th1 and Th17 responses. Caspase-1(-/-) and ASC(-/-) mice display diminished Th1/Th17 responses, followed by increased fungal outgrowth and lower survival. These observations identify a critical role for the inflammasome in controlling protective adaptive immune responses during invasive fungal infection.

Cutting edge: critical role for PYCARD/ASC in the development of experimental autoimmune encephalomyelitis.

Multiple sclerosis is an autoimmune disease in which self-reactive T cells attack oligodendrocytes that myelinate axons in the CNS. Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is dependent on caspase-1; however, the role of Nod-like receptors upstream of caspase-1 is unknown. Danger- and pathogen-associated molecular patterns activate Nod-like receptor 3, which activates caspase-1 through the adaptor protein, apoptosis-associated speck-like protein containing CARD (ASC). We report that the progression of EAE is dependent on ASC and caspase-1 but not Nod-like receptor 3. ASC(-/-) mice were even more protected from the progression of EAE than were caspase-1(-/-) mice, suggesting that an inflammasome-independent function of ASC contributes to the progression of EAE. We found that CD4(+) T cells deficient in ASC exhibited impaired survival; accordingly, ASC(-/-) mice had fewer myelin oligodendrocyte glycoprotein-specific T cells in the draining lymph nodes and CNS.

NOD-like receptor (NLR) signaling beyond the inflammasome.

Recent years have witnessed a marked progress in our knowledge of NOD-like receptors (NLR), intracellular sensors with central roles in innate and adaptive immunity. A majority of the research has focused on caspase-1 inflammasomes. However, several members of the mammalian NLR family exert important roles in immunity beyond inflammasome signaling. Here we highlight the emerging roles of several of these NLR.

Role of the Nalp3 inflammasome in acetaminophen-induced sterile inflammation and liver injury.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure in the US and UK. Recent studies implied that APAP-induced injury is partially mediated by interleukin-1ß (IL-1ß), which can activate and recruit neutrophils, exacerbating injury. Mature IL-1ß is formed by caspase-1, dependent on inflammasome activation. The objective of this invetstigation was to evaluate the role of the Nalp3 inflammasome on release of damage associated molecular patterns (DAMPs), hepatic neutrophil accumulation and liver injury (ALT, necrosis) after APAP overdose. Mice deficient for each component of the Nalp3 inflammasome (caspase-1, ASC and Nalp3) were treated with 300mg/kg APAP for 24h; these mice had similar neutrophil recruitment and liver injury as APAP-treated C57Bl/6 wildtype animals. In addition, plasma levels of DAMPs (DNA fragments, keratin-18, hypo- and hyper-acetylated forms of high mobility group box-1 protein) were similarly elevated with no significant difference between wildtype and gene knockout mice. In addition, aspirin treatment, which has been postulated to attenuate cytokine formation and the activation of the Nalp3 inflammasome after APAP, had no effect on release of DAMPs, hepatic neutrophil accumulation or liver injury. Together, these data confirm the release of DAMPs and a sterile inflammatory response after APAP overdose. However, as previously reported minor endogenous formation of IL-1ß and the activation of the Nalp3 inflammasome have little impact on APAP hepatotoxicity. It appears that the Nalp3 inflammasome is not a promising therapeutic target to treat APAP overdose.

Identification of RIPK3 Type II Inhibitors Using High-Throughput Mechanistic Studies in Hit Triage.

Necroptosis has been implicated in a variety of disease states, and RIPK3 is one of the kinases identified to play a critical role in this signaling pathway. In an effort to identify RIPK3 kinase inhibitors with a novel profile, mechanistic studies were incorporated at the hit triage stage. Utilization of these assays enabled identification of a Type II DFG-out inhibitor for RIPK3, which was confirmed by protein crystallography. Structure-based drug design on the inhibitors targeting this previously unreported conformation enabled an enhancement in selectivity against key off-target kinases.

Biologic-like In Vivo Efficacy with Small Molecule Inhibitors of TNFα Identified Using Scaffold Hopping and Structure-Based Drug Design Approaches.

Scaffold hopping and structure-based drug design were employed to identify substituted 4-aminoquinolines and 4-aminonaphthyridines as potent, small molecule inhibitors of tumor necrosis factor alpha (TNFα). Structure-activity relationships in both the quinoline and naphthyridine series leading to the identification of compound 42 with excellent potency and pharmacokinetic profile are discussed. X-ray co-crystal structure analysis and ultracentrifugation experiments clearly demonstrate that these inhibitors distort the TNFα trimer upon binding, leading to aberrant signaling when the trimer binds to TNF receptor 1 (TNFR1). Pharmacokinetic-pharmacodynamic activity of compound 42 in a TNF-induced IL-6 mouse model and in vivo activity in a collagen antibody-induced arthritis model, where it showed biologic-like in vivo efficacy, will be discussed.

Tumor necrosis factor alpha is a proximal mediator of synergistic hepatotoxicity from trovafloxacin/lipopolysaccharide coexposure.

The use of trovafloxacin (TVX), a fluoroquinolone antibiotic, was severely restricted because of an association of TVX therapy with idiosyncratic hepatotoxicity in patients. The mechanisms underlying idiosyncratic toxicity are unknown; however, one hypothesis is that an inflammatory stress can render an individual sensitive to the drug. Previously, we reported that treatment of mice with TVX and lipopolysaccharide (LPS) induced tumor necrosis factor (TNF) alpha-dependent liver injury, whereas TVX or LPS treatment alone was nontoxic. The goal of this study was to elucidate the role of TNFalpha in TVX/LPS-induced liver injury. TNF receptor (TNFR) 1 p55(-/-) and TNFR2 (p75(-/-)) mice were protected from hepatotoxicity caused by TVX/LPS coexposure, suggesting that TVX/LPS-induced liver injury requires both TNF receptors. TNFalpha inhibition using etanercept significantly reduced the TVX/LPS-induced increases in the plasma concentrations of several cytokines around the time of onset of liver injury. However, despite the reduction in chemokines, etanercept treatment did not affect the TVX/LPS-induced hepatic accumulation of neutrophils. In addition, etanercept treatment attenuated TVX/LPS induction of plasminogen activator inhibitor-1, and this was associated with a reduction in hepatic fibrin deposition. Mice treated with TVX and a nontoxic dose of TNFalpha also developed liver injury. In summary, TNFalpha acts through p55 and p75 receptors to precipitate an innocuous inflammatory cascade. TVX enhances this cascade, converting it into one that results in hepatocellular injury.

Trovafloxacin enhances the inflammatory response to a Gram-negative or a Gram-positive bacterial stimulus, resulting in neutrophil-dependent liver injury in mice.

Trovafloxacin (TVX), a fluoroquinolone antibiotic, has been strongly linked with several cases of idiosyncratic hepatotoxicity in humans. Previous studies showed that a modest inflammatory stress induced by a Gram-negative bacterial stimulus [i.e., lipopolysaccharide (LPS)] rendered nontoxic doses of TVX hepatotoxic in mice. This study compared the interaction of TVX with Gram-negative and Gram-positive stimuli. Mice were given TVX 3 h before LPS (Gram-negative stimulus) or a peptidoglycan-lipoteichoic acid (PGN-LTA) mixture isolated from Staphylococcus aureus (Gram-positive stimulus). Administration of TVX, LPS, or PGN-LTA alone was nonhepatotoxic. However, TVX administration before PGN-LTA or LPS resulted in significant liver injury that occurred with similar time courses. TVX/PGN-LTA-induced hepatocellular necrosis was primarily localized to centrilobular regions, whereas that caused by TVX/LPS was predominantly midzonal. Administration of either LPS or PGN-LTA alone led to increased plasma concentrations of several cytokines and chemokines at a time near the onset of liver injury. TVX administration before LPS enhanced the concentrations of all of these cytokines, whereas TVX treatment before PGN-LTA increased all of the cytokines except tumor necrosis factor (TNF)-alpha and interferon-gamma. Liver injury was reduced in TVX/LPS- and TVX/PGN-LTA-treated mice given an antibody to CD18 and also in mice deficient in neutrophil [polymorphonuclear neutrophil (PMN)] elastase. Hepatic PMN accumulation and TNF-alpha production after TVX/PGN-LTA-, but not after TVX/LPS-coexposure, was CD18-dependent. In summary, TVX significantly enhanced the murine inflammatory response to either a Gram-negative or a Gram-positive stimulus and caused hepatotoxicity that developed similarly and was dependent on PMN activation in mice but that differed in lesion location and cytokine profile.

The role of the hemostatic system in murine liver injury induced by coexposure to lipopolysaccharide and trovafloxacin, a drug with idiosyncratic liability.

The use of the fluoroquinolone antibiotic trovafloxacin (TVX) was severely restricted in 1999 due to its association with idiosyncratic hepatotoxicity. Previously, we reported that a nontoxic dose of TVX interacts with a nontoxic dose of lipopolysaccharide (LPS) to cause robust hepatocellular injury in mice. This interaction with LPS was not seen in mice treated with levofloxacin (LVX), a fluoroquinolone not associated with hepatotoxicity in people. TVX/LPS-coexposure caused an increase in plasma alanine aminotransferase (ALT) activity as early as 4.5 h after LPS administration which progressed through 15 h.We examined the role of the hemostatic system in TVX/LPS-induced liver injury. At the onset of liver injury, coexposure to TVX/LPS, but not exposure to TVX, LVX, LPS or LVX/LPS, caused increased plasma concentration of thrombin-antithrombin dimers and decreased plasma circulating fibrinogen. LPS treatment induced a small increase in plasma plasminogen activator inhibitor-1 (PAI-1) concentration, and TVX pretreatment enhanced this effect. TVX/LPS coexposure also resulted in hepatic fibrin deposition. Anticoagulant heparin administration reduced TVX/LPS-induced hepatic fibrin deposition and liver injury. PAI-1-/- mice treated with TVX/LPS exhibited similar fibrin deposition to wild-type mice but had significantly reduced hepatocellular injury. PAI-1-/- mice, but not heparin-treated mice, had reduced plasma concentrations of several cytokines compared to TVX/LPS-treated controls. In summary, TVX/LPS-coexposure caused an imbalance in the hemostatic system, resulting in thrombin activation increased, plasma concentration of PAI-1 and hepatic fibrin deposition. Both thrombin activation and PAI-1 play critical roles in the progression of TVX/LPS-induced liver injury, but through different modes of action.

Lipopolysaccharide and trovafloxacin coexposure in mice causes idiosyncrasy-like liver injury dependent on tumor necrosis factor-alpha.

Idiosyncratic adverse drug reactions (IADRs) occur in a small subset of patients, are unrelated to the pharmacological action of the drug, and occur without an obvious relationship to dose or duration of drug exposure. The liver is often the target of these reactions. Why they occur is unknown. One possibility is that episodic inflammatory stress interacts with the drug to precipitate a toxic response. We set out to determine if lipopolysaccharide (LPS) renders mice sensitive to trovafloxacin (TVX), a fluoroquinolone antibiotic linked to idiosyncratic hepatotoxicity in humans and if the cytokine tumor necrosis factor-alpha (TNFalpha) is involved in the development of liver injury. Male mice were treated with a nontoxic dose of TVX followed 3 h later by a nonhepatotoxic dose of LPS. Coexposure to TVX and LPS led to a significant increase in liver injury as determined by plasma alanine aminotransferase activity and histopathological examination. In contrast, coexposure of mice to LPS and levofloxacin (LVX), a fluoroquinolone without liability for causing IADRs in humans, was not hepatotoxic. Measurements of TNFalpha concentration in the plasma revealed a significant, selective increase in TVX/LPS-treated mice at times prior to and at the onset of liver injury. Treatment with either pentoxifylline to inhibit TNFalpha transcription or etanercept to inhibit TNFalpha activity significantly reduced TVX/LPS-induced liver injury. The results suggest that the model in mice is able to distinguish between drugs with and without the propensity to cause idiosyncratic liver injury and that the hepatotoxicity is dependent on TNFalpha.

In vivo folding of recombinant metallo-beta-lactamase L1 requires the presence of Zn(II).

Metallo-beta-lactamase L1, secreted by pathogenic Stenotrophomonas maltophilia, is a dinuclear Zn(II)-containing enzyme that hydrolyzes almost all known penicillins, cephalosporins, and carbapenems. The presence of Zn(II) ions in both metal binding sites is essential for full enzymatic activity; however, the mechanism of physiological metal incorporation is unknown. To probe metal incorporation, L1 was over-expressed in minimal media with (mmL1+Zn) and without (mmL1-Zn) Zn(II) added to the media, and the resulting proteins were purified and characterized. The mmL1+Zn sample was bound by a Q-Sepharose column, exhibited steady-state kinetic properties, bound Zn(II), existed as a tetramer, and yielded fluorescence emission and CD spectra similar to L1 overexpressed in rich media. On the other hand, the mmL1-Zn sample did not bind to a Q-Sepharose column, and gel filtration studies demonstrated that this protein was monomeric. The mmL1-Zn sample exhibited a lower kcat value, bound less Zn(II), and yielded fluorescence emission and CD spectra consistent with this enzyme being folded improperly. Taken together, these data demonstrate that the proper folding of L1 requires the presence of Zn(II) and suggest that in vitro, thermodynamic metal binding studies do not accurately reflect physiological metal incorporation into L1.