受体缺乏改变小鼠脑内组胺含量及昼夜节律

在乙谜麻醉下,分别于明时(8:00 a.m.)及暗时(8:00 p.m.)断头处死野生型及组胺H1R基因敲除型小鼠,迅速取出脑组织并分离出皮层、纹状体、海马、下丘脑、丘脑、中脑及脑干等脑区.这些脑组织被制成匀浆并用HPLC荧光检测法测量其组胺含量.结果显示暗时处死时,H1R基因敲除型小鼠海马、丘脑、中脑及脑干中的组胺含量明显低于野生型小鼠.明时处死时,野生型小鼠各脑区组胺含量均较暗时处死显著降低,但这一变化在H1R基因敲除型小鼠中并未观察到.这些表明作为组胺的功能靶,H1R不仅介导组胺的功能,而且调节大脑中组胺含量与释放的昼夜节律.     

组胺H1受体缺乏改变小鼠脑内组胺含量及昼夜节律

在乙谜麻醉下,分别于明时(8:00 a.m.)及暗时(8:00 p.m.)断头处死野生型及组胺H1R基因敲除型小鼠,迅速取出脑组织并分离出皮层、纹状体、海马、下丘脑、丘脑、中脑及脑干等脑区.这些脑组织被制成匀浆并用HPLC荧光检测法测量其组胺含量.结果显示暗时处死时,H1R基因敲除型小鼠海马、丘脑、中脑及脑干中的组胺含量明显低于野生型小鼠.明时处死时,野生型小鼠各脑区组胺含量均较暗时处死显著降低,但这一变化在H1R基因敲除型小鼠中并未观察到.这些表明作为组胺的功能靶,H1R不仅介导组胺的功能,而且调节大脑中组胺含量与释放的昼夜节律.     

Involvement of receptor-interacting protein 2 in innate and adaptive immune responses

Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-kappaB, whereas dominant negative Rip2 inhibited NF-kappaB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-gamma production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.     

Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway

High-dose intravenous immunoglobulin is a widely used therapeutic preparation of highly purified immunoglobulin G (IgG) antibodies. It is administered at high doses (1-2 grams per kilogram) for the suppression of autoantibody-triggered inflammation in a variety of clinical settings. This anti-inflammatory activity of intravenous immunoglobulin is triggered by a minor population of IgG crystallizable fragments (Fcs), with glycans terminating in α2,6 sialic acids (sFc) that target myeloid regulatory cells expressing the lectin dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN; also known as CD209). Here, to characterize this response in detail, we generated humanized DC-SIGN mice (hDC-SIGN), and demonstrate that the anti-inflammatory activity of intravenous immunoglobulin can be recapitulated by the transfer of bone-marrow-derived sFc-treated hDC-SIGN(+) macrophages or dendritic cells into naive recipients. Furthermore, sFc administration results in the production of IL-33, which, in turn, induces expansion of IL-4-producing basophils that promote increased expression of the inhibitory Fc receptor FcγRIIB on effector macrophages. Systemic administration of the T(H)2 cytokines IL-33 or IL-4 upregulates FcγRIIB on macrophages, and suppresses serum-induced arthritis. Consistent with these results, transfer of IL-33-treated basophils suppressed induced arthritic inflammation. This novel DC-SIGN-T(H)2 pathway initiated by an endogenous ligand, sFc, provides an intrinsic mechanism for maintaining immune homeostasis that could be manipulated to provide therapeutic benefit in autoimmune diseases.     

NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens

Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of nuclear factor-κB (NF-κB), type I interferon and inflammasome signalling. Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis, but the role of NLRP6 in microbial infections and the nature of the inflammatory signalling pathways regulated by NLRP6 remain unclear. Here we show that Nlrp6-deficient mice are highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signalling in both haematopoietic and radioresistant cells contributed to increased susceptibility. Nlrp6 deficiency enhanced activation of mitogen-activated protein kinase (MAPK) and the canonical NF-κB pathway after Toll-like receptor ligation, but not cytosolic NOD1/2 ligation, in vitro. Consequently, infected Nlrp6-deficient cells produced increased levels of NF-κB- and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signalling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and -negative bacterial pathogens.     

Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells

The regulation of the subclass of immunoglobulin secreted by B cells has been studied in vitro in polyclonal systems using mitogens, such as lipopolysaccharide (LPS), to bypass the requirement for cognate interaction between antigen-specific T and B cells. In these systems, interleukin-(IL)-4 induces the secretion of IgG1 (ref. 1) and IgE (ref. 2); IL-5 enhances the secretion of IgA, and interferon-gamma (IFN-gamma) enhances the secretion of IgG2a (ref. 5). Clones of murine TH cells can be divided into two subsets, TH1 and TH2 (ref. 6). Both subsets synthesize IL-3 and granulocyte-monocyte colony-stimulating factor (GM-CSF), but only TH1 clones produce IL-2, IFN-gamma, and lymphotoxin (LT) and TH2 clones produce IL-4 and IL-5 (ref. 7). We have examined the role of clones of antigen-specific TH1 and TH2 cells in the regulation of the subclasses of IgG antibody secreted by antigen-specific B cells. Our results show that both types of TH cells induce the secretion of IgM and IgG3, whereas clones of TH1 and TH2 cells specifically induce antigen-specific B cells to secrete IgG2a and IgG1, respectively. We also demonstrate that regulation of commitment to the secretion of a particular IgG isotype occurs in two distinct stages: cognate interaction between T and B cells and interaction between T-cell-derived lymphokines and B cells.     

Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1

Activated T lymphocytes differentiate into effector cells tailored to meet disparate challenges to host integrity. For example, type 1 and type 2 helper (T(H)1 and T(H)2) cells secrete cytokines that enhance cell-mediated and humoral immunity, respectively. The chemokine monocyte chemoattractant protein-1 (MCP-1) can stimulate interleukin-4 production and its overexpression is associated with defects in cell-mediated immunity, indicating that it might be involved in T(H)2 polarization. Here we show that MCP-1-deficient mice are unable to mount T(H)2 responses. Lymph node cells from immunized MCP-1(-/-) mice synthesize extremely low levels of interleukin-4, interleukin-5 and interleukin-10, but normal amounts of interferon-gamma and interleukin-2. Consequently, these mice do not accomplish the immunoglobulin subclass switch that is characteristic of T(H)2 responses and are resistant to Leishmania major. These effects are direct rather than due to abnormal cell migration, because the trafficking of naive T cells is undisturbed in MCP-1(-/-) mice despite the presence of MCP-1-expressing cells in secondary lymphoid organs of wild-type mice. Thus, MCP-1 influences both innate immunity, through effects on monocytes, and adaptive immunity, through control of T helper cell polarization.     

Pathogen-induced human TH17 cells produce IFN-γ or IL-10 and are regulated by IL-1β

IL-17-producing CD4+ T helper cells (TH17) have been extensively investigated in mouse models of autoimmunity. However, the requirements for differentiation and the properties of pathogen-induced human TH17 cells remain poorly defined. Using an approach that combines the in vitro priming of naive T cells with the ex vivo analysis of memory T cells, we describe here two types of human TH17 cells with distinct effector function and differentiation requirements. Candida albicans-specific TH17 cells produced IL-17 and IFN-γ, but no IL-10, whereas Staphylococcus aureus-specific TH17 cells produced IL-17 and could produce IL-10 upon restimulation. IL-6, IL-23 and IL-1β contributed to TH17 differentiation induced by both pathogens, but IL-1β was essential in C. albicans-induced TH17 differentiation to counteract the inhibitory activity of IL-12 and to prime IL-17/IFN-γ double-producing cells. In addition, IL-1β inhibited IL-10 production in differentiating and in memory TH17 cells, whereas blockade of IL-1β in vivo led to increased IL-10 production by memory TH17 cells. We also show that, after restimulation, TH17 cells transiently downregulated IL-17 production through a mechanism that involved IL-2-induced activation of STAT5 and decreased expression of ROR-γt. Taken together these findings demonstrate that by eliciting different cytokines C. albicans and S. aureus prime TH17 cells that produce either IFN-γ or IL-10, and identify IL-1β and IL-2 as pro- and anti-inflammatory regulators of TH17 cells both at priming and in the effector phase.     

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.     

Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.     

Excitatory effect of histamine on neuronal activity of rat cerebellar fastigial nucleus in vitro

The cerebellar fastigial nucleus (FN) holds an important role in motor control and body balance. Previous studies have revealed that the nucleus is innervated by direct hypothalamocerebellar histaminergic fibers. However, the functional role of histaminergic projection in cerebellar FN has never been established. In this study, we investigated the effect of histamine on neuronal firing of cerebellar FN by using slice preparations. Sixty-five FN cells were recorded from 47 cerebellar slices, and a vast majority of the cells responded to histamine stimulation with an excitatory response (58/65, 89.2%). Perfusing slices with low-Ca2 /high-Mg2 medium did not block the histamine-induced excitation (n=10), supporting a direct postsynaptic action of histamine on the cells. Furthermore, the excitatory effect of histamine on FN neurons was not blocked by selective histamine H1 receptor antagonist triprolidine (n=15) or chlorpheniramine (n=10), but was effectively suppressed by ranitidine (n=15), a highly selective histamine H2 receptor antagonist. On the other hand, highly selective histamine H2 receptor agonist dimaprit (n=20) instead of histamine H1 receptor agonist 2-pyridylethylamine (n=16) mimicked the ex- citatory effect of histamine on FN neurons. The dimaprit-induced FN neuronal excitation was effectively antagonized by selective histamine H2 receptor antagonist ranitidine (n=13) but not influenced by se- lective histamine H1 receptor antagonist triprolidine (n=15). These results demonstrate that histamine excites cerebellar FN cells via the histamine H2 receptor mechanism and suggest that the hypotha- lamocerebellar histaminergic fibers may modulate cerebellar FN-mediated sensorimotor integration through their excitatory innervations on FN neurons.     

Extrathymically generated regulatory T cells control mucosal TH2 inflammation

A balance between pro- and anti-inflammatory mechanisms at mucosal interfaces, which are sites of constitutive exposure to microbes and non-microbial foreign substances, allows for efficient protection against pathogens yet prevents adverse inflammatory responses associated with allergy, asthma and intestinal inflammation. Regulatory T (T(reg)) cells prevent systemic and tissue-specific autoimmunity and inflammatory lesions at mucosal interfaces. These cells are generated in the thymus (tT(reg) cells) and in the periphery (induced (i)T(reg) cells), and their dual origin implies a division of labour between tT(reg) and iT(reg) cells in immune homeostasis. Here we show that a highly selective blockage in differentiation of iT(reg) cells in mice did not lead to unprovoked multi-organ autoimmunity, exacerbation of induced tissue-specific autoimmune pathology, or increased pro-inflammatory responses of T helper 1 (T(H)1) and T(H)17 cells. However, mice deficient in iT(reg) cells spontaneously developed pronounced T(H)2-type pathologies at mucosal sites--in the gastrointestinal tract and lungs--with hallmarks of allergic inflammation and asthma. Furthermore, iT(reg)-cell deficiency altered gut microbial communities. These results suggest that whereas T(reg) cells generated in the thymus appear sufficient for control of systemic and tissue-specific autoimmunity, extrathymic differentiation of T(reg) cells affects commensal microbiota composition and serves a distinct, essential function in restraint of allergic-type inflammation at mucosal interfaces.     

A novel class (H3) of histamine receptors on perivascular nerve terminals

Two types of histamine receptor, the H1- and H2-receptors, are found not only on vascular smooth muscle cells but on the perivascular autonomic nerve terminals. Activation of the prejunctional histamine receptors modifies transmitter release from the nerve terminals. Recently, histamine was shown to inhibit its own release from depolarized slices of rat cerebral cortex. This phenomenon was found to be mediated by a novel class of histamine receptor, the H3-receptor, that was pharmacologically distinct from the H1- and H2-receptors. Up to now, there has been no indication whether this third class of histamine receptor is present in any tissue other than the brain. We report here that histamine depresses sympathetic neurotransmission in the guinea-pig mesenteric artery by interacting with histamine H3-receptors on the perivascular nerve terminals. The pharmacological properties of these receptors are similar to those reported for the H3-receptors in the brain. Our data provide evidence for the existence of H3-receptors in the autonomic nervous system.     

Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor

Although histaminergic neurones have not yet been histochemically visualized, there is little doubt that histamine (HA) has a neurotransmitter role in the invertebrate and mammalian central nervous system. For example, a combination of biochemical, electrophysiological and lesion studies in rats have shown that histamine is synthesized in and released from a discrete set of neurones ascending through the lateral hypothalamic area and widely projecting in the telencephalon. Histamine acts on target cells in mammalian brain via stimulation of two classes of receptor (H1 and H2) previously characterized in peripheral organs and probably uses Ca2+ and cyclic AMP, respectively, as second messengers. It is well established that several neurotransmitters affect neuronal activity in the central nervous system through stimulation not only of postsynaptic receptors, but also of receptors located presynaptically which often display distinct pharmacological specificity and by which they may control their own release. Such 'autoreceptors' have been demonstrated (or postulated) in the case of noradrenaline, dopamine, serotonin, acetylcholine and gamma-aminobutyric acid (GABA) neurones but have never been demonstrated for histamine. We show here that histamine inhibits its own release from depolarized slices of rat cerebral cortex, an action apparently mediated by a class of receptor (H3) pharmacologically distinct from those previously characterized, that is, the H1 and H2 receptors.     

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.     

RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems

The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.