Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO₃⁻); it thereby serves as a cellular sensor for HCO₃⁻, carbon dioxide (CO₂), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO₃⁻ binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO₃⁻ activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO₃⁻ sensing and a basis for targeting this system with drugs.The ubiquitous second messenger cAMP regulates diverse physiological processes, from fungal virulence to mammalian brain function (1, 2). In mammals, cAMP can be generated by any of 10 differently expressed and regulated adenylyl cyclases (ACs): nine transmembrane enzymes (tmACs) and one soluble AC (sAC) (3). TmACs reside in the cell membrane, where they mediate cellular responses to hormones acting through G protein-coupled receptors (4). In contrast, sAC functions in various intracellular locations, providing cell-specific spatial and temporal patterns of cAMP (5–7) in response to intracellular signals, including calcium, ATP, and bicarbonate (HCO₃⁻) (3, 8–10). HCO₃⁻ regulation of sAC enzymes is a direct effect on their catalytic domains and is conserved across bacterial, fungal, and animal kingdoms (1, 11–13). Via modulation of sAC, and sAC-like cyclase activities, HCO₃⁻ serves as an evolutionarily conserved signaling molecule mediating cellular responses to HCO₃⁻, CO₂, and pH (3, 14). In mammals, sAC acts as a CO₂/HCO₃⁻/pH sensor in processes such as sperm activation (15), acid-base homeostasis (16), and various metabolic responses (10, 17, 18). sAC has also been implicated in skin and prostate cancer and as a target for male contraceptives (19–21).All mammalian ACs are class III nucleotidyl cyclases sharing homologous catalytic domains. Their catalytic cores are formed through symmetrical or pseudosymmetrical association of two identical or highly similar catalytic domains, C₁ and C₂ (22–24); in mammalian ACs, both domains reside on a single polypeptide chain. Such C₁C₂ pseudoheterodimers form two pseudosymmetrical sites at the dimer interface: the active site and a degenerated, inactive pocket (3, 23). A conserved Lys and an Asp/Thr in the active site recognize the base of the substrate ATP, and two conserved Asp residues bind two divalent cations, normally Mg²⁺ (23). The ions, called ion A and ion B, coordinate the substrate phosphates and support the intramolecular 3′-hydroxyl (3′-OH) attack at the α-phosphorous to form cAMP and pyrophosphate (PP_i) (3). In tmACs, the degenerate site binds forskolin (24), a plant diterpene that activates tmACs but has no effect on sAC (25). The forskolin activation mechanism and the existence of physiological ligands for this site in tmACs or in sAC remain unclear.There are two sAC isoforms known to be generated by alternative splicing (26). Full-length sAC comprises N-terminal catalytic domains along with ∼1,100 residues with a little understood function except for an autoinhibitory motif and a heme-binding domain (3, 27, 28). Exclusion of exon 12 (26) generates a truncated isoform, sAC_t (residues 1–490), which comprises just the two sAC catalytic domains (sAC-cat) (25). sAC_t is widely expressed, and it is the isoform most extensively biochemically characterized (3, 8, 11). It is directly activated by Ca²⁺ and HCO₃⁻; Ca²⁺ supports substrate binding, and HCO₃⁻ increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3, 13, 29). In a homodimeric, HCO₃⁻-regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO₃⁻ appeared to facilitate an active site closure required for catalysis (13), but the HCO₃⁻ binding site and its mechanism of activation remained unknown.Here, we present crystal structures of the human sAC-cat in apo form and in complex with substrate, products, bicarbonate, and a pharmacological inhibitor. The structures reveal insights into binding sites and mechanisms for sAC catalysis and for its regulation by physiological and pharmacological small molecules.     

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1^−/−) develop spontaneous autoimmune diseases. PD-1^−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1^−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1^−/− recombination activating gene (RAG)2^−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1^+/+ RAG2^−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1^−/− RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells developed more severe EAE compared with the ones reconstituted with PD-1^+/+ RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells. We found that upon recognition of HKMTB, CD11b⁺ macrophages from PD-1^−/− mice produced very high levels of IL-6, which helped promote naive CD4⁺ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells.Autoimmune disease development is impacted by both genetic and environmental factors. Programmed cell death 1 (PD-1) is a type I membrane protein that delivers inhibitory signals to immune cells upon the binding of its ligand, PD-L1 or PD-L2 (1). PD-1 has been shown to be important for self-tolerance because spontaneous autoimmune diseases develop in PD-1^−/− mice (2–4). A single-nucleotide polymorphism that affects PD-1 expression is associated with autoimmune diseases in humans, such as systemic lupus erythematosus (5), type I diabetes (6), rheumatoid arthritis (7), and multiple sclerosis (MS) (8), suggesting that PD-1 deficiency may be a genetic factor involved in the development of autoimmunity.Experimental autoimmune encephalomyelitis (EAE) is a rodent model of T-cell–mediated inflammatory disease in the central nervous system (CNS), causing demyelination, axonal damage, and paralysis, and is a commonly used model for human MS. Previous reports suggested that PD-1 functions to attenuate EAE. PD-1 and its ligands were found to be strongly expressed on immune infiltrates in the CNS during the peak phase of EAE (9–11). In EAE studies, PD-1–deficient mice or the use of blocking antibodies that inhibit PD-1 engagement by ligands resulted in earlier disease onset, increased inflammatory infiltrates, and increased severity of clinical symptoms compared with normal disease progression (10–16). It has been demonstrated that ligand engagement of PD-1 inhibits T-cell activation, expansion, and cytokine production (17–19). Similarly, in EAE, PD-1 signaling in CNS-specific helper T cells may inhibit their expansion and secretion of inflammatory cytokines (10–12). Recently, T-helper 17 (Th17) cells were shown to be involved in EAE by producing IL-17 and GM-CSF (20, 21). Two reports showed that PD-1^−/− mice mount an augmented Th17 response to EAE induction (14, 16). However, the fundamental mechanisms by which PD-1 regulates antigen-specific Th17 cell differentiation, expansion, and effector function in EAE remain to be understood.To induce EAE, mice are immunized with myelin autoantigens in an emulsion of Mycobacterium tuberculosis (MTB)-derived adjuvants, causing a strong innate inflammatory response, leading to Th skewing (22). Curiously, recent studies showed that PD-1^−/− mice exhibited an altered response to infection with mycobacteria, characterized by uncontrolled bacterial burden; massive production of cytokines, termed “cytokine storm”; and early death (23–25). We wondered if this unique response of PD-1^−/− mice to mycobacteria contributed to their Th response in EAE.In this study, we took a combination of genetic and immunological approaches in which the innate response to MTB-derived adjuvant and antigen-specific T-cell polarization were separately analyzed. The present data suggest that an enhanced innate response of PD-1^−/− mice to MTB contributes to the susceptibility of these mice to severe EAE. We propose a previously undescribed function of PD-1 in controlling the basal state of the innate immune response, the failure of which can cause the activation of adaptive immune responses, provoking autoimmunity.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na⁺- and Ca²⁺-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.Although homeostatic mechanisms generally maintain the brain’s extracellular pH within narrow limits, neural activity can induce transient and localized pH fluctuations. For example, acidification may occur when synaptic vesicles, which have a pH of ∼5.2–5.7 (1–3), release their contents into the synapse. Studies of mammalian cone photoreceptors showed that synaptic vesicle exocytosis rapidly reduced synaptic cleft pH by an estimated 0.2–0.6 units (4–6). Transient synaptic cleft acidification also occurred with GABAergic transmission (7). Some, but not all, studies also reported that high-frequency stimulation (HFS) transiently acidified hippocampal brain slices, likely as a result of the release of synaptic vesicle contents (8, 9). Neurotransmission also induces a slower, more prolonged alkalinization (10, 11). In addition to release of synaptic vesicle protons, neuronal and glial H⁺ and HCO₃⁻ transporters, channels, H⁺-ATPases, and metabolism might influence extracellular pH (10–12).ASICs are potential targets of reduced extracellular pH. ASICs are Na⁺-permeable and, to a lesser extent, Ca²⁺-permeable channels that are activated by extracellular acidosis (13–19). In the brain, ASICs consist of homotrimeric and heterotrimeric complexes of ASIC1a, ASIC2a, and ASIC2b. The ASIC1a subunit is required for acid-activation in the physiological range (>pH 5.0) (20, 21). Several observations indicate that ASIC are located postsynaptically. ASICs are located on dendritic spines. Although similar to glutamate receptors, they are also present on dendrites and cell bodies (20, 22–24). ASIC subunits interact with postsynaptic scaffolding proteins, including postsynaptic density protein 95 and protein interacting with C-kinase-1 (20, 24–29). In addition, ASICs are enriched in synaptosome-containing brain fractions (20, 24, 30).Although these observations raised the possibility that protons might be a neurotransmitter, postsynaptic ASIC currents have not been detected in cultured hippocampal neurons (31, 32), and whether localized pH transients might play a signaling role in neuronal communication remains unclear. In previous studies of hippocampal brain slices, extracellular field potential recordings suggested impaired hippocampal long-term potentiation (LTP) in ASIC1a^−/− mice (20), although another study did not detect an effect of ASIC1a (33). Another study using microisland cultures of hippocampal neurons suggested that the probability of neurotransmitter release increased in ASIC1a^−/− mice (32).Here, we tested the hypothesis that protons are a neurotransmitter and that ASICs are the receptor. Criteria to identify substances as neurotransmitters have been proposed (34). Beg and colleagues (35) used these criteria to conclude that protons are a transmitter released from Caenorhabditis elegans intestine to cause muscle contraction. Key questions about whether protons meet criteria for a neurotransmitter are: Does presynaptic stimulation increase the extracellular proton concentration? Do protons activate currents in postsynaptic cells? Can exogenously applied protons reproduce effects of endogenous protons? What is the postsynaptic proton receptor? We studied lateral amygdala brain slices because amygdala-dependent fear-related behavior depends on a pH reduction (36). In addition, ASICs are abundantly expressed there, and ASIC1a^−/− mice have impaired fear-like behavior (36–38).     

Hyperglycemia in rodent models of type 2 diabetes requires insulin-resistant alpha cells

To determine the role of glucagon action in diet-induced and genetic type 2 diabetes (T2D), we studied high-fat-diet–induced obese (DIO) and leptin receptor-defective (LepR^−/−) rodents with and without glucagon receptors (GcgRs). DIO and LepR^−/−,GcgR^+/+ mice both developed hyperinsulinemia, increased liver sterol response element binding protein 1c, and obesity. DIO GcgR^+/+ mice developed mild T2D, whereas LepR^−/−,GcgR^+/+ mice developed severe T2D. High-fat–fed (HFF) glucagon receptor-null mice did not develop hyperinsulinemia, increased liver sterol response element binding protein 1c mRNA, or obesity. Insulin treatment of HFF GcgR^−/− to simulate HFF-induced hyperinsulinemia caused obesity and mild T2D. LepR^−/−,GcgR^−/− did not develop hyperinsulinemia or hyperglycemia. Adenoviral delivery of GcgR to GcgR^−/−,LepR^−/− mice caused the severe hyperinsulinemia and hyperglycemia of LepR^−/− mice to appear. Spontaneous disappearance of the GcgR transgene abolished the hyperinsulinemia and hyperglycemia. In conclusion, T2D hyperglycemia requires unsuppressible hyperglucagonemia from insulin-resistant α cells and is prevented by glucagon suppression or blockade.The prevalence of type 2 diabetes (T2D) in the United States was 29.1 million in 2012, and 37% of adults were identified as prediabetic (1). T2D is now present on every continent (2). Despite the magnitude of this threat to world physical and fiscal health, our understanding of the pathogenic pathway is vague and is based largely on epidemiologic correlations. For example, the correlation between T2D and obesity is so high that most obese Americans can be considered prediabetic, but the precise mechanism of this relationship is unknown. Although the “lipotoxic” effects of ectopic lipids were first suggested in 1994 (3) to link diet-induced obesity to T2D and other components of the metabolic syndrome (3–11), the relationship between IR and T2D is still poorly understood. Proposed hypothetical links range from beta cell “glucotoxicity” (12) to the action of modifier genes (13) to failure of redox control (14).It has recently been shown that glucagon receptor-null mice remain normoglycemic and nonketotic despite total insulin deficiency but that transduction of a glucagon receptor cDNA into their liver makes them severely diabetic (15, 16). This proves that, whether or not insulin action is present, suppression of glucagon action prevents hyperglycemia. It has long been known that insulin suppression of glucagon regulates alpha cell secretion (17, 18). Although the presence of hyperglucagonemia was established unequivocally in type 1 diabetes (T1D) (15, 16), direct evidence that it is essential for the hyperglycemia of T2D is lacking. However, it has long been known that glucagon is elevated in T2D (17, 19, 20) and is resistant to suppression by insulin.     

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O₂^•−) dismutase-like properties yet were inert to nitric oxide (NO^•) as well as peroxynitrite (ONOO⁻). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO₂ and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O₂^•− to O₂ by PEG-HCCs at >20,000 s⁻¹. The major products of this catalytic turnover are O₂ and H₂O₂, making the PEG-HCCs a biomimetic superoxide dismutase.Reactive oxygen species (ROS), such as superoxide (O₂^•−), hydrogen peroxide (H₂O₂), organic peroxides, and hydroxyl radical (^•OH), are a consequence of aerobic metabolism (1, 2). These ROS are necessary for the signaling pathways in biological processes (3, 4) such as cell migration, circadian rhythm, stem cell proliferation, and neurogenesis (5). In healthy systems, ROS are efficiently regulated by the defensive enzymes superoxide dismutase (SOD) and catalase, and by antioxidants such as glutathione, vitamin A, ascorbic acid, uric acid, hydroquinones, and vitamin E (6). When the production of ROS overwhelms the scavenging ability of the defense system, oxidative stress occurs, causing dysfunctions in cell metabolism (7–16).In addition to ROS, reactive nitrogen species (RNS) such as nitric oxide (NO^•), nitrogen dioxide, and dinitrogen trioxide can be found in all organisms. NO^• can act as an oxidizing or reducing agent depending on the environment (17), is more stable than other radicals (half-life 4–15 s) (18), and is synthesized in small amounts in vivo (17–22). NO^• is a potent vasodilator and has an important role in neurotransmission and cytoprotection (17, 18, 22, 23). Owing to its biological importance and the low concentration found normally in vivo, it is often important to avoid alteration of NO^• levels in biological systems to prevent aggravation of acute pathologies including ischemia and reperfusion.One way to treat these detrimental pathologies is to supply antioxidant molecules or particles that renormalize the disturbed oxidative condition. We recently developed a biocompatible carbon nanoparticle, the poly(ethylene glycolated) hydrophilic carbon cluster (PEG-HCC), which has shown ability to scavenge oxyradicals and protect against oxyradical damage in rodent models and thus far has demonstrated no in vivo toxicity in laboratory rodents (24–27). The carbon cores of PEG-HCCs are ∼3 nm wide and range from 30 to 40 nm long. Based on these data, we estimate that there are 2,000–5,000 sp² carbon atoms on each PEG-HCC core. We have demonstrated the efficacy of PEG-HCCs for normalizing in vivo O₂^•− in models of traumatic brain injury with concomitant hypotension. Simultaneously, we observed normalization in NO^• levels in these experiments (26, 27). A better understanding of these materials is necessary to potentially translate these therapeutic findings to the clinic.In the present work, we evaluated antioxidant properties of PEG-HCCs. Using spin-trap EPR spectroscopy, we demonstrate that PEG-HCCs scavenge O₂^•− with high efficiency. X-ray photoelectron spectroscopy (XPS) indicates that covalent addition of ROS to the PEG-HCCs is not responsible for the observed activity. Direct measurement of O₂^•− concentration using freeze-trap EPR demonstrates that PEG-HCCs behave as catalysts, and measurements made with a Clark oxygen electrode during the reaction reveal that the rate of production of O₂ is above that expected due to self-dismutation of O₂^•− in water. An equivalent amount of H₂O₂ is also simultaneously produced. Finally, selectivity for ROS is confirmed using a hemoglobin and a pyrogallol red assay; PEG-HCCs are unreactive to both NO^• and ONOO⁻. These results clarify the fundamental processes involved in the previously observed in vivo protection against oxygen damage (26, 27).     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg²⁺. Here we investigated the active forms of ATP and found that the action of MgATP²⁻ and ATP⁴⁻ differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP²⁻ promotes opening with very low efficacy. In contrast, both free ATP and MgATP²⁻ robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg²⁺ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP²⁻ and weak regulation by Mg²⁺. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP²⁻ and regulation by Mg²⁺, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.Seven subtypes of P2X receptors have been identified in mammals that can form either homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) or heteromeric (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/5, P2X2/6, P2X4/6, and possibly, P2X4/7) channels (1–8). These subtypes of P2X receptors have distinct gating properties, pharmacology, and cellular distributions. P2X1 and P2X3 receptors desensitize within a few hundred milliseconds when opened by ATP, and their distributions are restricted to either smooth muscle cells and platelets (P2X1) or a subset of sensory neurons (P2X3) (1, 9–14). P2X2 and P2X4 receptors exhibit slow desensitization during prolonged ATP application, and these receptors are the most abundant subtypes in the central nervous system (15). P2X2 subunits also express in a subset of sensory neurons; however, in these cells they only form heteromeric channels with P2X3 subunits (12, 16, 17). In sensory neurons, P2X3 homomeric channels together with P2X2/3 heteromeric channels play important roles in mediating the primary sensory effects of ATP, and knock-out animals with either P2X3 deletion or P2X2 and P2X3 double-deletions have revealed critical roles in taste, pain, oxygen sensing, and bladder filling (17–20).A long-standing conundrum in P2X receptor-mediated signaling concerns the forms of ATP that activate these channels. In neutral solutions, ATP is ionized and exists mostly as free ATP (ATP⁴⁻), an efficient chelator of divalent cations such as Mg²⁺, and to a lesser extent Ca²⁺ (21). In extracellular biological compartments, such as the synaptic cleft, Ca²⁺ and Mg²⁺ are present in the millimolar range, and therefore only a relatively small fraction of ATP released from vesicles is present in the free form. Although a range of important studies have explored the regulatory effects of Ca²⁺ and Mg²⁺ on P2X receptor channels (22–30), the essential question of which forms of ATP serve as agonists remains unresolved. Several previous studies have reported that P2X2, P2X7, and the native P2X receptors in cilia are activated by ATP in solutions containing low concentrations of divalent cations, and that the addition of divalent cations shifts the concentration dependence for activation of the channels to higher ATP concentrations, suggesting that either ATP⁴⁻ is the most active form of ATP or that divalent cations regulate those subtypes through allosteric mechanisms (27, 30–36). In the present study, we investigated the form(s) of ATP that serve as agonists for a range of subtypes of P2X receptor channels. Our primary focus was to determine whether ATP⁴⁻ or MgATP²⁻ are the principal agonists and to explore whether Mg²⁺ might serve specific regulatory roles. Our results demonstrate that the action of MgATP²⁻ and ATP⁴⁻ differ between subtypes of P2X receptors, and reveal that heteromeric channels can have unique hybrid phenotypes, findings that will be crucial for understanding the physiological functions of these channels in both the peripheral and central nervous systems.     

Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1^−/− mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER^T2Ripk1^fl/fl mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1^fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1^fl/fl mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1^fl/fl Ripk3^−/− progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.The proinflammatory cytokine TNF stimulates receptor-interacting serine/threonine-protein kinase 1 (RIPK1) ubiquitination, NFκB and MAPK activation, and induction of apoptosis or necroptosis (1, 2). TNF signaling via TNF receptor 1 (TNFR1) is highly regulated and results in the recruitment of several adapter proteins including TNFR1-associated death domain (TRADD) protein, the E3 ubiquitin ligases cellular inhibitor of apoptosis protein-1 and -2 (cIAP1/2), and TNFR-associated factor 2 (TRAF2) or 5, and the serine threonine death domain-containing kinase RIPK1 (complex I) (1). We have demonstrated that the kinase activity of RIPK1 is not required for NFκB activation (3); rather, RIPK1 is modified by the addition of Lys63-linked and linear polyubiquitin chains (3–6). Polyubiquitinated RIPK1 then recruits NEMO/IκB kinase-γ (IKKγ) to mediate IKK activation and TAK1/TAB2/3 to mediate MAPK activation, resulting in antiapoptotic and proinflammatory gene expression (7, 8). Deubiquitination of RIPK1 by cylindromatosis (CYLD) results in the formation of a cytosolic complex containing TRADD, Fas-associated death domain protein (FADD), caspase-8, and RIPK1 (complex IIa) (2). Caspase-8 cleaves and inactivates RIPK1 and CYLD and stimulates apoptosis (9–11). In the absence of caspase-8 or the presence of caspase inhibitors, TNF family members and potentially other ligands stimulate the kinase activity of RIPK1 to induce necroptosis (9, 11–16). RIPK1 also is recruited to the Toll-like receptor adapter TRIF via the Rip homotypic interaction motif (RHIM) to mediate NFκB activation (17) and, under conditions of caspase-8 inhibition, initiates necroptosis (14, 16). Necrostatin-1 (Nec-1), an allosteric RIPK1 inhibitor, inhibits necroptosis induced by TNF or the TLR3 ligand poly I:C and abolishes the formation and activation of an RIPK1/3 complex (13–16, 18). Although the molecular details whereby RIPK1 initiates necroptosis are unclear, RIPK3 and the pseudo kinase MLKL appear to be required (2).Genetic studies in mice have revealed cross-regulation between the apoptotic and necroptotic pathways. For example, the FADD/caspase-8/FLICE-like inhibitory protein long form (FLIP_L) complex regulates RIPK1 and RIPK3 activity during development, because the embryonic lethality associated with a caspase-8 deficiency is completely rescued by the absence of RIPK3 (19, 20). Similarly, RIPK1 deficiency rescues FADD-associated embryonic lethality (21). Thus, in the absence of FADD or caspase-8, embryos succumb to RIPK1- and RIPK3-dependent necroptosis. However, Fadd^−/−/Ripk1^−/− mice, die perinatally (21, 22), as do Ripk1^−/− mice, revealing that RIPK1 has prosurvival roles beyond the regulation of the FADD/caspase-8/FLIP_L complex.We have demonstrated that complete RIPK1 deficiency results in increased TNF-induced cell death that can be rescued, in part, by the absence of the TNFR1 (22, 23). However, Ripk1^−/−Tnfr1^−/− animals still succumb (23), indicating that other death ligands/pathways contribute to the RIPK1-associated lethality. Consistent with this hypothesis, RIPK3 deficiency recently has been shown to rescue the perinatal lethality observed in Ripk1^−/−Tnfr1^−/− mice (24, 25). Similarly, combined caspase-8 and RIPK3 deficiency also rescues the RIPK1-associated lethality (24–26). Collectively, these genetic studies in mice reveal that the perinatal death of Ripk1^−/− mice reflects TNF-induced apoptosis and RIPK3-mediated necroptosis. The nature of the ligand(s) or the trigger(s) of RIPK3-mediated necroptosis in vivo remain unclear. However, Ripk1^−/− MEFs are prone to necroptosis induced by poly I:C or by treatment with type I or type II IFN (24, 25), suggesting that these pathways contribute. Although these studies reveal a regulatory role for RIPK1, the multiorgan cell death and inflammation observed in the complete and compound RIPK1-knockout strains have made it difficult to discern the specific tissues that require RIPK1 for survival.     

Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle

Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb^−/− mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb^−/− oocytes to produce essential oocyte-secreted factors or of Fshb^−/− cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb^+/− females, these increases fail to occur in Fshb^−/− females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb^−/− females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility.Fertility in mammals depends on the coordinated execution of multiple events within the fully grown ovarian follicle at the time of ovulation (1, 2). The oocyte undergoes meiotic maturation, during which it progresses to metaphase II of meiosis and acquires the ability to begin embryonic development (3). Concomitantly, the layer of granulosa cells (GCs) immediately surrounding the oocyte, termed the “cumulus,” undergoes a process termed “expansion,” which is required for sperm to penetrate this layer and reach the oocyte (4–7). At the perimeter of the follicle, an inflammatory response associated with rupture of the follicular wall permits the cumulus–oocyte complex (COC) to escape from the follicle and enter the oviduct where fertilization will occur. These events are triggered by the preovulatory release of luteinizing hormone (LH), which acts on LH receptors (LHCGR) on the mural GCs that line the interior wall of the fully grown follicle (8).Recent studies have identified a key downstream effector of LH activity at ovulation. Binding of LH to LHCGR triggers the release of the EGF-related peptides amphiregulin (AREG, betacellulin (BTC), and epiregulin (EREG) (9–11). These bind to EGF receptors (EGFRs) located on both the mural and cumulus GCs (12–19) and activate MAPK3/1 as well as other signaling networks (20–28). Considerable evidence supports the view that the EGFR signaling mediates many or most ovulatory events. First, the release of the EGFR ligands follows the LH surge but precedes the LH-dependent responses (9–11). Second, EGF and the EGFR ligands can induce cumulus expansion and oocyte maturation in vitro, independently of LH (9, 10, 20, 29). Third, these events are impaired in mice bearing a hypomorphic Egfr allele that reduces EGFR activity by about one-half and in mice in which Egfr has been selectively inactivated in GCs through a targeted mutation (22, 23). Thus, the activation of EGFR signaling in GCs of mature follicles appears to be a major effector of the ovulatory response to LH.FSH binds to receptors located on GCs and induces the expression of numerous genes, including Lhcgr (8, 30). Lhcgr expression is impaired substantially in mice that lack either FSH, because of targeted mutation of the Fshb gene that encodes its β-subunit, or the FSH receptor and in humans bearing spontaneous mutations; these individuals fail to ovulate (31–34). Thus, the ovulatory response to LH depends strictly on the prior FSH-dependent expression of Lhcgr, and in this manner FSH indirectly controls the LHCGR-regulated release of the EGFR ligands. We report here that FSH also drives an increase in EGFR expression during late folliculogenesis and provide evidence that this increase is essential to enable the ovulatory response to EGF. By coordinating the expression of EGFR and the release of its ligands, FSH endows full-grown follicles with the capacity to activate EGFR signaling at ovulation.     

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2^−/− mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins (1, 2). Ulcerative colitis (UC) and Crohn disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries (3). Furthermore, IBD is a risk factor for the development of colon cancer (4). Although the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer development (5).Neutrophil recruitment and activation are key steps in the intestinal innate immune response observed in IBD (6–8), and studies with animal models of colitis highlight the relationship between neutrophil infiltration and disease severity (9–11). We recently reported results of a comprehensive analysis of histopathology, changes in gene expression, and nucleic acid damage occurring during progression of lower bowel disease in Rag2^−/− mice infected with Helicobacter hepaticus (Hh) (10). This mouse model emulates many aspects of human IBD, and infected mice develop severe colitis that progress into colon carcinoma, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages (12, 13).Phagocytes produce strong oxidants and radicals that damage cellular macromolecules and promote tissue damage at sites of inflammation (14–16). Myeloperoxidase (MPO) is an abundant enzyme in neutrophils that produces hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride ion (17, 18). HOCl can oxidize and chlorinate DNA, proteins, and lipids (19, 20). A prominent target of HOCl is tyrosine, which leads to the formation of the stable aromatic residue, 3-chlorotyrosine (Cl-Tyr) (21, 22). MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2^−/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10, 24, 25).Macrophages also contribute to the array of oxidants and radicals at sites of inflammation through release of nitric oxide (NO) generated by the inducible NO synthase (iNOS) enzyme. NO reacts with superoxide anion (O₂^−•) at diffusion-controlled rates to yield highly reactive peroxynitrite (ONOO⁻) (26, 27). MPO also reacts H₂O₂ with nitrite (NO₂⁻, the endpoint of cellular NO oxidation) to produce the strong nitrating agent, nitrogen dioxide radical (NO₂^•) (28). Both NO₂^• and ONOO⁻ can react with tyrosine residues to generate the stable tyrosine nitration product, 3-nitrotyrosine (Nitro-Tyr) (29, 30).Multiple MS methods have been applied for determination of Cl-Tyr and Nitro-Tyr levels in biological systems (10, 31–38), and both have been detected in inflamed tissues from animals and humans (11, 39). The presence of Nitro-Tyr has been demonstrated in colon tissue of IBD patients by immunohistochemistry, and levels were reported to correlate with disease activity (40, 41). We undertook the present study to test the null hypothesis that the H. hepaticus-infected mouse model of colitis and colitis-associated carcinoma represents a useful surrogate of human IBD. To examine this hypothesis, we first quantified levels of Nitro-Tyr and Cl-Tyr in proteins and 5-Cl-dC in DNA of colon tissues of IBD patients. Comparison of these data with our previous findings (10) further assessed the validity of this animal model. We then tested the hypothesis that inflammation-induced damage in the colon would be reflected in changes in serum constituents, and would therefore serve as a noninvasive measure of IBD activity. For this purpose, we determined levels of protein chlorination and nitration products, acute-phase proteins, cytokines, and chemokines in human and mouse sera. In addition, gene expression of several inflammatory signaling molecules was monitored in mice colons to determine whether colonic inflammation was directly associated with serum cytokine levels. We then used multivariate analysis to determine which systemic inflammatory markers in serum were most closely associated with disease activity and were also common to human IBD and H. hepaticus-associated colitis in Rag2^−/− mice.     

iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17^−/− mice die perinatally with open eyes, like Egfr^−/− mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2^−/− mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2^−/− double knockout mice resemble Adam17^−/− and Egfr^−/− mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2^−/− tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.ADAM17 (a disintegrin and metalloprotease 17) is a membrane-anchored metalloproteinase that controls two major signaling pathways with important roles in development and disease, the EGF receptor (EGFR) pathway and the proinflammatory tumor necrosis factor α (TNF-α) pathway (1–5). Mice lacking ADAM17 resemble mice with defects in EGFR signaling in that they have open eyes at birth, enlarged semilunar heart valves, and enlarged hypertrophic zones in long bone growth plates, most likely caused by a lack of ADAM17-dependent release of the EGFR ligands transforming growth factor α (TGF-α) and heparin-binding epidermal growth factor (HB-EGF) (3, 6–14). In humans, defects in skin and intestinal barrier protection have been reported in a patient lacking ADAM17 (15) and in patients treated with EGFR inhibitors (16, 17), and similar skin defects were recently identified in a patient with defective EGFR signaling (18). Mouse models of ADAM17/EGFR signaling appear to recapitulate these mechanisms, because defects in skin barrier protection can be observed by inactivating either ADAM17 or the EGFR in keratinocytes (19), as well as in mice expressing very low levels of ADAM17, which also have increased susceptibility to intestinal inflammation (20). A hallmark feature of ADAM17 is its rapid response to various activators of cellular signaling pathways (21–23), which is presumably important to allow a rapid response to injury and to maintain the skin and intestinal barrier. The rapid activation of ADAM17 is controlled by its transmembrane domain whereas the cytoplasmic domain is dispensable in this context (22), suggesting that ADAM17 is regulated by one or more other membrane proteins, yet the underlying mechanism has remained enigmatic.Recent studies have shown that the maturation and function of ADAM17 in myeloid cells depend on inactive Rhomboid 2 (iRhom2), a catalytically inactive member of the Rhomboid family of seven membrane-spanning intramembrane serine proteinases (24–28). Myeloid cells lacking iRhom2 release very little TNF-α in response to activation of Toll-like receptor 4 by lipopolysaccharide (LPS) (24, 26, 28). Therefore, mice lacking iRhom2 are protected from the detrimental effects of TNF-α in mouse models for septic shock and inflammatory arthritis, similar to conditional knockout mice lacking ADAM17 in myeloid cells (11, 26, 29). However, iRhom2^−/− (iR2^−/−) mice are viable with no evident spontaneous pathological phenotypes (26, 29), whereas Adam17^−/− (A17^−/−) mice die shortly after birth (3). A major unresolved question has therefore been whether iRhom2 and the related iRhom1 are the long-sought-after regulators of the function of ADAM17-dependent EGFR signaling in vivo. Here we generate iRhom1^−/− (iR1^−/−) mice, which are viable and healthy, and report that iR1/2^−/− double knockout mice closely resemble mice lacking ADAM17 or the EGFR, providing the first genetic evidence, to our knowledge, that the principal function of iRhoms1/2 during mouse development is to control ADAM17/EGFR signaling.     

Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ^−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ^−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ^−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ^−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ^−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.Protein tyrosine phosphatase (PTP)σ, encoded by PTPRS (1), consists of a cell adhesion molecule-like ectodomain containing three immunoglobulin (Ig)-like and three to eight fibronectin type III repeats, a transmembrane domain, and a cytosolic region with two PTPase domains, of which the first (D1) is catalytically active (2). PTPσ expression is developmentally regulated and found primarily in the nervous system and specific epithelia (3, 4). It was previously shown to play a role in axon growth and path finding (5–7), neuroregeneration (5, 8, 9), autophagy (10), and neuroendocrine development (11–13).To investigate the function of PTPσ in vivo, our group (11) and Tremblay and coworkers (12) generated PTPσ^−/− mice. These mice exhibited high neonatal mortality, various neurological and neuroendocrine defects, colitis, and cachexia (5, 11, 13, 14). Analysis of the intestinal tissue in surviving mice by our group revealed the presence of mucosal inflammation, intestinal crypt branching, and villus blunting: all features of colitis similar to the enteropathy associated with human inflammatory bowel disease (IBD) (15). Notably, PTPσ^−/− mice also showed increased susceptibility to chemical and infectious models of murine colitis, specifically treatment with dextran sodium sulfate (DSS) or infection with Citrobacter rodentium (15). The intestinal phenotype in the mice strongly inferred a connection between PTPσ and IBD.IBD is a chronic, idiopathic, relapsing disorder affecting the gastrointestinal tract, where Crohn disease and ulcerative colitis (UC) are the two major forms (16). In IBD pathogenesis, the presence of environmental factors together with polymorphisms in IBD-susceptibility genes cause an abnormal innate and adaptive host immune response to commensal gut bacteria, leading to sustained and deleterious inflammation (17). Chronic infection (18, 19), dysbiosis (19), defective mucosal barrier defense (20), and insufficient microbial clearance (19) have all been implicated as factors contributing to IBD pathogenesis. The disease is known to have a strong genetic component, as evidenced by specific populations exhibiting a disproportionately high incidence (21) and the high disease concordance between monozygotic twins (22). Genome-wide association studies and associated metaanalyses have implicated several genes and pathways in IBD, notably genes associated with intestinal barrier defense [MYO9B (23), PARD3 (24), MAGI2 (24), CDH1 (25, 26)].Through SNP analysis of IBD patients, we showed that PTPRS is genetically associated with UC (15). The identified SNP polymorphism leads to alternative splicing in the extracellular region of the epithelial isoform of PTPσ, causing loss of the third Ig domain (15). This splicing might potentially lead to altered ligand recognition or may affect receptor dimerization (27). In addition, through an interaction-trap assay, we identified the apical junction complex (AJC) proteins E-cadherin (CDH1) and β-catenin (CTNNB1) as colonic substrates of PTPσ (15). Interestingly, recent large-scale genetic studies have identified over 160 loci that affect risk of developing IBD, many of which involved in barrier regulation (24, 28, 29).The AJC confers polarity to epithelial cells and maintains intestinal barrier integrity (30). Defective regulation of AJC proteins creates disrupted epithelial barriers, permeability defects, and aberrant intestinal morphology (30, 31), similar to defects seen in IBD. The connection between the AJC and IBD is further demonstrated by our earlier SNP analysis, which revealed a haplotype polymorphism in CDH1 that is associated with Crohn disease, leading to a truncated E-cadherin protein that fails to localize to the plasma membrane (PM), as also observed in IBD patient biopsy samples (25). Thus, we postulate that PTPσ regulates epithelial barrier integrity through regulation of AJC proteins and that defective PTPσ function may contribute to IBD.In this report, we demonstrate an intestinal epithelial barrier defect in the PTPσ^−/− mice and identify the AJC protein ezrin as an in vivo colonic substrate for PTPσ. We further demonstrate that dephosphorylation of ezrin-Y353 or -Y145 by PTPσ leads to its redistribution from the PM to the cytosol, similar to its localization following induction of IBD in mice.     

PirB regulates a structural substrate for cortical plasticity

Experience-driven circuit changes underlie learning and memory. Monocular deprivation (MD) engages synaptic mechanisms of ocular dominance (OD) plasticity and generates robust increases in dendritic spine density on L5 pyramidal neurons. Here we show that the paired immunoglobulin-like receptor B (PirB) negatively regulates spine density, as well as the threshold for adult OD plasticity. In PirB^−/− mice, spine density and stability are significantly greater than WT, associated with higher-frequency miniature synaptic currents, larger long-term potentiation, and deficient long-term depression. Although MD generates the expected increase in spine density in WT, in PirB^−/− this increase is occluded. In adult PirB^−/−, OD plasticity is larger and more rapid than in WT, consistent with the maintenance of elevated spine density. Thus, PirB normally regulates spine and excitatory synapse density and consequently the threshold for new learning throughout life.Experience generates both functional and structural changes in neural circuits. The learning process is robust at younger ages during developmental critical periods and continues, albeit at a lower level, into adulthood and old age (1–3). For example, young barn owls exposed to horizontally shifting prismatic spectacles can adapt readily to altered visual input, but adult owls cannot. The experience in the young owls results in a rearranged audiovisual map in tectum that is accompanied by ectopic axonal projections (1). Experience-dependent structural changes have also been observed in the mammalian cerebral cortex. Enriched sensory experience or motor learning are both associated with an increase in dendritic spine density, and a morphological shift from immature thin spines to mushroom spines which harbor larger postsynaptic densities (PSDs) and stronger synapses (4–7). On the flip side, bilateral sensory deprivation induces spine loss (8, 9). Abnormal sensory experience also results in structural modification of inhibitory synapses and circuitry that is temporally and spatially coordinated with changes in excitatory synapses on dendritic spines (10–13).These experience-driven spine changes are thought to involve synaptic mechanisms of long-term potentiation (LTP) and long-term depression (LTD). In hippocampal slices, induction of LTP causes new spines to emerge, as well as spine head enlargement on existing spines (14–16); induction of LTD results in rapid spine regression (14, 17). Importantly, the emergence or regression of spines starts soon after the induction of LTP or LTD, suggesting that these structural changes underlie the persistent expression of long-term plasticity (14, 17).Little is known about molecular mechanisms that restrict experience-dependent plasticity at circuit and synaptic levels and connect it to spine stability. Paired Ig-like receptor B (PirB), a receptor expressed in cortical pyramidal neurons, is known to limit ocular dominance (OD) plasticity both during the critical period and in adulthood (18). PirB binds major histocompatibility class I (MHCI) ligands, whose expression is regulated by visual experience and neural activity (19–21) and thus could act as a key link connecting functional to structural plasticity. If so, mice lacking PirB might be expected to have altered synaptic plasticity rules on the one hand and changes in the density and stability of dendritic spines on the other.     

Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice

Cannabinoid CB₂ receptors (CB₂Rs) have been recently reported to modulate brain dopamine (DA)-related behaviors; however, the cellular mechanisms underlying these actions are unclear. Here we report that CB₂Rs are expressed in ventral tegmental area (VTA) DA neurons and functionally modulate DA neuronal excitability and DA-related behavior. In situ hybridization and immunohistochemical assays detected CB₂ mRNA and CB₂R immunostaining in VTA DA neurons. Electrophysiological studies demonstrated that activation of CB₂Rs by JWH133 or other CB₂R agonists inhibited VTA DA neuronal firing in vivo and ex vivo, whereas microinjections of JWH133 into the VTA inhibited cocaine self-administration. Importantly, all of the above findings observed in WT or CB₁^−/− mice are blocked by CB₂R antagonist and absent in CB₂^−/− mice. These data suggest that CB₂R-mediated reduction of VTA DA neuronal activity may underlie JWH133''s modulation of DA-regulated behaviors.The presence of functional cannabinoid CB₂ receptors (CB₂Rs) in the brain has been controversial. When CB₂Rs were first cloned, in situ hybridization (ISH) failed to detect CB₂ mRNA in brain (1). Similarly, Northern blot and polymerase chain reaction (PCR) assays failed to detect CB₂ mRNA in brain (2–5). Therefore, CB₂Rs were considered “peripheral cannabinoid receptors” (1, 6).In contrast, other studies using ISH and radioligand binding assays detected CB₂ mRNA and receptor binding in rat retina (7), mouse cerebral cortex (8), and hippocampus and striatum of nonhuman primates (9). More recent studies using RT-PCR also detected CB₂ mRNA in the cortex, striatum, hippocampus, amygdala, and brainstem (9–14). Immunoblot and immunohistochemistry (IHC) assays detected CB₂R immunoreactivity or immunostaining in various brain regions (13, 15–20). The specificities of the detected CB₂R protein and CB₂-mRNA remain questionable, however, owing to a lack of controls using CB₁^−/− and CB₂^−/− mice in most previous studies (21). A currently accepted view is that brain CB₂Rs are expressed predominantly in activated microglia during neuroinflammation, whereas brain neurons, except for a very small number in the brainstem, lack CB₂R expression (21).On the other hand, we recently reported that brain CB₂Rs modulate cocaine self-administration and cocaine-induced increases in locomotion and extracellular dopamine (DA) in the nucleus accumbens in mice (22). This finding is supported by recent studies demonstrating that systemic administration of the CB₂R agonist O-1966 inhibited cocaine-induced conditioned place preference in WT mice, but not in CB₂^−/− mice (23), and that increased CB₂R expression in mouse brain attenuates cocaine self-administration and cocaine-enhanced locomotion (19). In addition, brain CB₂Rs may be involved in several DA-related CNS disorders, such as Parkinson’s disease (24), schizophrenia (25), anxiety (26), and depression (27). The cellular mechanisms underlying CB₂R modulation of DA-related behaviors and diseases are unclear, however. Given that midbrain DA neurons of the ventral tegmental area (VTA) play an important role in mediating the reinforcing and addictive effects of drugs of abuse (28, 29), we hypothesized that brain CB₂Rs, similar to other G protein-coupled receptors, are expressed in VTA DA neurons, where they modulate DA neuronal function and DA-related behaviors.In the present study, we tested this hypothesis using multiple approaches. We first assayed for CB₂ mRNA and protein expression in brain and in VTA DA neurons using quantitative RT-PCR (qRT-PCR), ISH, and double-label IHC techniques. We then examined the effects of the selective CB₂R agonist JWH133 and several other CB₂R agonists on VTA DA neuronal firing in both ex vivo and in vivo preparations using electrophysiological methods. Finally, we observed the effects of microinjections of JWH133 into the VTA on intravenous cocaine self-administration to study whether activation of VTA CB₂Rs modulates DA-dependent behavior. This multidisciplinary approach has provided evidence of functional CB₂Rs in VTA DA neurons. Importantly, all findings observed in WT or CB₁^−/− mice were blocked by a CB₂R antagonist and/or absent in CB₂^−/− mice, suggesting that CB₂Rs expressed in VTA DA neurons play an important role in modulating DA neuronal activity and DA-related functions.