Critical in vivo roles of histamine and histamine receptor signaling in animal models of metabolic syndrome

Histamine, a classic low‐molecular‐weight amine, is synthesized from L‐histidine by histidine decarboxylase (HDC), and histamine‐specific receptors (HRs) are essential for its actions. Our serial in vivo studies have uniquely reported that expression of histamine/HRs is variably identified in atherosclerotic lesions, and that HDC‐gene knockout mice without histamine/HRs signaling show a marked reduction of atherosclerotic progression. These data have convinced us that histamine plays a pivotal role in the pathogenesis of atherosclerosis. Among four subclasses of HRs, the expression profile of the main receptors (H1/2R) has been shown to be switched from H2R to H1R during monocyte to macrophage differentiation, and H1R is also predominant in smooth muscle and endothelial cells of atheromatous plaque. Using various animal models of H1/2R–gene knockout mice, H1R and H2R were found to reciprocally but critically regulate not only hypercholesterolemia‐induced atherosclerosis and injury‐induced arteriosclerosis, but also hyperlipidemia‐induced nonalcoholic fatty liver disease (NAFLD). Metabolic syndrome manifests obesity, dyslipidemia, insulin resistance, atherosclerosis, and/or NAFLD, i.e. the dysregulation of lipid/bile acid/glucose metabolism. Therefore, although its etiology is complicated and multifactorial, histamine/HRs signaling has a close relationship with the development of metabolic syndrome. We herein review diverse, key in vivo roles of histamine/HR signaling in the pathogenesis of metabolic syndrome.     

Histamine receptors expressed in circulating progenitor cells have reciprocal actions in ligation‐induced arteriosclerosis

Histamine is synthesized as a low‐molecular‐weight amine from L‐histidine by histidine decarboxylase (HDC). Recently, we demonstrated that carotid artery‐ligated HDC gene‐deficient mice (HDC^–/–) showed less neointimal formation than wild‐type (WT) mice, indicating that histamine participates in the process of arteriosclerosis. However, little is known about the roles of histamine‐specific receptors (HHRs) in arteriosclerosis. To define the roles of HHRs in arteriosclerosis, we investigated intimal remodeling in ligated carotid arteries of HHR‐deficient mice (H1R^–/– or H2R^–/–). Quantitative analysis showed that H1R^–/– mice had significantly less arteriosclerogenesis, whereas H2R^–/– mice had more, as compared with WT mice. Bone marrow transplantation from H1R^–/– or H2R^–/– to WT mice confirmed the above observation. Furthermore, the increased expression of monocyte chemoattractant protein (MCP‐1), platelet‐derived growth factor (PDGF), adhesion molecules and liver X receptor (LXR)‐related inflammatory signaling factors, including Toll‐like receptor (TLR3), interleukin‐1 receptor (IL‐1R) and tumor necrosis factor receptor (TNF‐R), was consistent with the arteriosclerotic phenotype of H2R^–/– mice. Peripheral progenitor cells in H2R^–/– mice accelerate ligation‐induced arteriosclerosis through their regulation of MCP‐1, PDGF, adhesion molecules and LXR‐related inflammatory signaling factors. In contrast, peripheral progenitor cells act to suppress arteriosclerosis in H1R^–/– mice, indicating that HHRs reciprocally regulate inflammation in the ligation‐induced arteriosclerosis.     

Novel function of histamine signaling via histamine receptors in cholesterol and bile acid metabolism: Histamine H2 receptor protects against nonalcoholic fatty liver disease

We have reported that the function of histamine and its receptors (HRs) has a close relationship with the development of nonalcoholic fatty liver disease (NAFLD). However, much less is known regarding its pathogenic and molecular mechanism(s), including the early stage of hepatic and intestinal function for lipid and bile acid (BA) metabolism. We used H1R and H2R knockout mice (H1/2R‐KO) to clarify those pivotal roles in cholesterol/BA metabolism, in which H1/2R‐KO mice were separately fed a short‐term 1% cholesterol or cholic acid (CA) diet. [³H]Cholesterol absorption study revealed that significantly enhanced accumulation occurred in the jejunum, blood and liver, but not in the feces, of H2R‐KO mice, compared to wild‐type and H1R‐KO mice. Furthermore, four weeks after the high‐cholesterol diet, the H2R‐KO jejunum but not liver exhibited increased expressions of cholesterol transporters, consistent with higher plasma lipoprotein levels. Five days after CA diet, the H2R‐KO mice showed significantly higher expressions of ileal BA‐reabsorption and hepatic BA‐efflux factors, corresponding to higher serum but lower fecal BA levels. The following long‐term CA diets resulted in severe injury to the H2R‐KO liver. Histamine/H2R signaling might have a protective role in the initial phase during NAFLD progression, correlated with cholesterol and BA metabolism in the liver/intestine.     

Histamine H4 receptors modulate dendritic cell migration through skin--immunomodulatory role of histamine

Background: Dendritic cells (DC) are the major antigen‐presenting cells and play a key role in adaptive immunity as they are able to activate naive T cells. It was recently described, that the histamine H₄ receptor (H4R) is present on human monocyte‐derived DC and that chemotaxis and T‐helper (Th)1–Th2 polarization is mediated by this receptor. However, the distribution of histamine receptors on murine DC has not been studied yet. Methods: The histamine receptor expression on murine bone marrow (BM)‐derived DC and effects of histamine and H4R agonism on DC migration through skin were studied. As it was demonstrated in scratching experiments that NMRI mice are more susceptible to H4R‐mediated itch than BALB/c mice, DC function of NMRI and BALB/c mice was compared. Results: The mRNA of the H1R, H2R and H4R could be detected in murine BM‐derived DC, while mRNA of the H3R was found to be low or undetectable. There were no distinct differences in mRNA expression and in H4R protein level (flow cytometry) between NMRI compared with BALB/c mice indicating, that a higher susceptibility is not associated with a generally higher H4R expression in all cell types. Histamine as well as the H4R agonist clobenpropit induced an enhanced chemotaxis in the skin DC migration assay. The enhanced chemotaxis was blocked by the H4R antagonist JNJ7777120. This finding was confirmed by in vitro migration experiments with BM‐derived DC. Conclusion: Referring to DC migration, blocking the H4R on inflammatory cells might be a promising anti‐inflammatory, immunomodulatory strategy.     

Histamine H4 receptor activation on human slan-dendritic cells down-regulates their pro-inflammatory capacity

6‐Sulpho LacNAc dendritic cells (slanDC) are a major population of human blood DC that are highly pro‐inflammatory, as characterized by their outstanding capacity to produce tumour necrosis factor‐α and interleukin‐12 (IL‐12) and to prime antigen‐specific T‐cell responses. SlanDC were found to be present in inflamed tissue such as atopic dermatitis, where high levels of histamine are also present. As histamine is an important regulator of allergic inflammation we investigated the role of histamine receptors, particularly the most recently identified histamine H₄ receptor (H₄R), in modulating the pro‐inflammatory function of slanDC. The expression of H₄R was evaluated by real‐time PCR and flow cytometry. Cytokine production in response to H₄R stimulation was assessed by intracellular flow cytometric staining and enzyme‐linked immunosorbent assay. We show that slanDC express the H₁R, H₂R and H₄R on mRNA and the H₄R on protein level. No differences were observed in basal H₄R expression in patients with atopic dermatitis and psoriasis, but in atopic dermatitis patients the H₄R was up‐regulated by interferon‐γ. When stimulated with lipopolysaccharide in the presence of histamine, slanDC produced substantially lower levels of the pro‐inflammatory cytokines tumour necrosis factor‐α and IL‐12, mediated solely via the H₄R and via the combined action of H₂R and H₄R, respectively. In contrast, the production of IL‐10 was not affected by histamine receptor activation on slanDC. The slanDC express the H₄R and its stimulation leads to reduced pro‐inflammatory capacity of slanDC. Hence, H₄R agonists might have therapeutic potential to down‐regulate immune reactions, e.g. in allergic inflammatory skin diseases.     

Histamine induces Th2 activation through the histamine receptor 1 in house dust mite rhinitic but not asthmatic patients

Background Effects of mast cell‐released histamine on smooth muscle and endothelial cells are considered as responsible of immediate symptoms of anaphylaxis. However, little is known about histamine effects on Th2 lymphocytes, which orchestrate the allergic reaction upstream of mast cells. Objective We addressed this question in house dust mite (HDM) allergics, according to the presence of rhinitis or asthma and allergen stimulation. Methods Peripheral blood mononuclear cell from 15 rhinitic and 14 asthmatic HDM‐allergic subjects and 16 controls were cultured with Der p 1 or histamine. The effect of Der p 1 on histamine receptor (H1R and H2R) expression was studied. T‐cell cytokine production was studied upon Der p 1 or histamine stimulation. The role of H1R in histamine effects was assessed with levocetirizine. Results H1R and H2R are overexpressed on T cells from asthmatic but not from rhinitic subjects. Der p 1 increases H1R expression on CD4⁺ cells from both allergic groups, and decreases it in controls, on CD4⁺ and CD8⁺ subsets. Der p 1 decreases T‐cell H2R expression in asthmatics. Allergen increases IL‐4 and IL‐13 in both allergic groups. Histamine increases Th2 cytokines in rhinitics only, and levocetirizine abolishes this effect. In asthmatics and controls, histamine decreases T‐cell cytokines through a non‐H1R dependent pathway. Conclusion In rhinitis but not in asthma, histamine is able to increase allergic inflammation by increasing Th2 cytokine production in a positive feedback dependent on H1R. This result could explain in part why H1R antagonists, are very efficient in rhinitis, but not in asthma. Cite this as: K. Botturi, Y. Lacoeuille, D. Vervloet and A. Magnan, Clinical & Experimental Allergy, 2010 (40) 755–762.     

Suppressive and pro‐inflammatory roles for IL‐4 in the pathogenesis of experimental drug‐induced liver injury

The pathogenesis of immune‐mediated drug‐induced liver injury (DILI) following halogenated anesthetics, carbamazepine or alcohol has not been fully elucidated. Detecting cytochrome P450 2E1 (CYP2E1) IgG4 auto‐antibodies in anesthetic DILI patients suggests a role for IL‐4 in this hapten‐mediated process. We investigated IL‐4‐mediated mechanisms using our model of experimental DILI induced by immunizing BALB/c (WT) and IL‐4^?/? (KO) mice with S100 liver proteins covalently modified by a trifluoroacetyl chloride (TFA) hapten formed following halogenated anesthetic metabolism by CYP2E1. WT mice developed more hepatitis, TFA and S100 antibodies (p<0.01), as well as T‐cell proliferation to CYP2E1 and TFA (p<0.01) than KO mice. Additionally, WT CD4⁺ T cells adoptively transferred hepatitis to naïve Rag^?/? mice (p<0.01). Pro‐inflammatory cytokines were expectedly decreased in TFA hapten‐stimulated KO splenocyte supernatants (p<0.001); however, IL‐2 and IFN‐γ (p<0.05), as well as IL‐6 and IL‐10 (p<0.001) levels were elevated in CYP2E1‐stimulated KO splenocyte supernatants, suggesting dual IL‐4‐mediated pro‐inflammatory and regulatory responses. Anti‐IL‐10 administered to KO mice increased hepatitis, TFA and CYP2E1 antibodies in KO mice confirming a critical role for IL‐4. This is the first demonstration of dual roles for IL‐4 in the pathogenesis of immune‐mediated DILI by suppressing auto‐antigen‐induced regulatory responses while promoting hapten‐induced pro‐inflammatory responses.     

Neurometabolic and structural alterations in rat brain due to acute hypobaric hypoxia: in vivo 1H MRS at 7 T

In response to hypobaric hypoxia (HH), which occurs at high altitude, the brain undergoes deleterious changes at the structural and metabolite level. In vivo T₂ weighted imaging (T2WI) and ¹H‐MRS was performed to understand the structural and metabolic changes in the hippocampus region of rat brain. Data were acquired pre‐exposure (baseline controls), immediately after exposure and subsequently at the first, fourth, seventh and 14th days post exposure at normoxia. T₂ weighted images of rat brain showed hyperintensity in the CA2/CA3 region of the hippocampus 7 d after acute HH, which persisted till 14 d, probably indicating structural changes in the hippocampus. ¹H‐MRS results showed no change in metabolite level immediately after acute HH exposure, but on the first day of normoxia the myo‐inositol level was significantly decreased, possibly due to altered astrocyte metabolism. Metabolic alterations showing an increase in choline and decrease in glutamate on the fourth day of normoxia may be seen as a process of demyelination and loss of glutamate pool respectively. On the seventh and 14th days of normoxia, decreases in N‐acetylaspartate, creatine and glutamine + glutamate were observed, which might be due to decreased viability of glutamatergic neurons. In vivo ¹H‐MRS demonstrated early neurometabolic changes prior to probable structural changes post acute HH exposure. The extension of these studies will help in early risk assessment, developing intervention and strategies for combating HH related changes. Copyright © 2014 John Wiley & Sons, Ltd.     

High‐density lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation

Interleukin‐1β (IL‐1β), a potent pro‐inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL‐1β is controlled by a multi‐protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High‐density lipoprotein (HDL) has consistently been shown to be anti‐atherogenic and to have anti‐inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL‐1β secretion in response to cholesterol crystals in THP‐1 cells and in human‐monocyte‐derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro‐IL‐1β and NLRP3, and reducing caspase‐1 activation. In summary, we have described a novel anti‐inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.     

Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid‐rich necrotic cores in hypercholesterolemic mice

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide‐binding oligomerization domain‐containing protein (NOD)‐like receptor pathways. However, the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10‐week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid‐rich necrotic cores in Ldlr^?/? mice. Myeloid‐specific ablation of NOD2, but not its downstream kinase, receptor‐interacting serine/threonine‐protein kinase 2, restrained the expansion of the lipid‐rich necrotic core in Ldlr^?/? chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low‐density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP‐binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF‐κB‐mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid‐rich necrotic core and promotes vascular inflammation in atherosclerosis.     

Elevated CD14 (Cluster of Differentiation 14) and Toll‐Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice

Apical periodontitis (periapical lesions) is an infection‐induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll‐like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild‐type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection‐induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF‐kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency‐dependent elevation of CD14 (cluster of differentiation 14), which is a co‐receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency‐dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281–1292, 2016. © 2016 Wiley Periodicals, Inc.     

Deletion of tenascin-C gene exacerbates atherosclerosis and induces intraplaque hemorrhage in Apo-E-deficient mice

AimsTenascin-C (TNC), a matricellular protein, is up-regulated in atherosclerotic plaques. We investigated whether the deletion of TNC gene affects the development of atherosclerosis in a murine model.MethodsTNC?/?/apo E?/? mice were generated and used for atherosclerosis studies. We compared these results to those observed in control groups of apo E?/? mice.ResultsThe en face analysis of aortic area showed that the mean aortic lesion area of the double knockout (KO) mice was significantly higher than that of control mice at different times after feeding of atherogenic diet; the accumulation of lesional macrophages and lipids was significantly higher. Analysis of cell adhesion molecules revealed that vascular cell adhesion molecule-1 (VCAM-1), but not intercellular adhesion molecule-1, was up-regulated 1 week after feeding of atherogenic diet in the double KO mouse as compared to apo E?/? mouse. Cell culture studies revealed that the expression of VCAM-1 in endothelial cells isolated from the double KO mouse is more sensitive to the tumor necrosis factor α stimulation than the cells isolated from apo E?/? mice. Cell adhesion studies showed that the adherence of RAW monocytic cells to the endothelial cells was significantly enhanced in the cultured endothelial cells from the TNC gene-deleted cells. Following the prolonged feeding of an atherogenic diet (28–30 weeks), the aortic and carotid atherosclerotic lesions frequently demonstrated large grossly visible areas of intraplaque hemorrhage in the double KO mice compared to control.ConclusionsThese data unveil a protective role for TNC in atherosclerosis and suggest that TNC signaling may have the potential to reduce atherosclerosis, in part by modulating VCAM-1 expression.     

Local effects of human PCSK9 on the atherosclerotic lesion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low‐density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE^?/? and LDLR^?/? mice, which were then placed on a high‐fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow‐derived PCSK9 progressively accumulated in lesions of apoE^?/? recipient mice, while increasing the infiltration of Ly6C^hi inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b‐ and Ly6C^hi‐positive cell numbers in spleens of apoE^?/? mice. In vitro, expression of hPCSK9 in LPS‐stimulated macrophages increased mRNA levels of the pro‐inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti‐inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR‐dependent, as PCSK9 protein was not detected in lesions of LDLR^?/? recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR‐dependent but cholesterol‐independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Histamine protects against the acute phase of experimentally-induced hepatic ischemia/re-perfusion

Histamine, involved in many inflammatory reactions and immune responses, is reported to suppress—via H4R stimulation—injury concomitant with the late phase of warm hepatic ischemia/re-perfusion (I/R). The current study investigated the possible effects of histamine on the acute phase of hepatic I/R injury, and the possible underlying mechanisms like oxidative stress and release of inflammatory cytokines (e.g., tumor necrosis factor (TNF)-α nd interleukin [IL]-12). Rats were divided into naïve, sham-operated, and I/R groups. The I/R group was divided into sub-groups and pre-treated with histaminergic ligands before induction of ischemia. Anesthetized rats were subjected to warm ischemia for 30 min by occlusion of the portal vein and hepatic artery, then re-perfused for 90 min. Rats in the control I/R group showed significant increases in hepatic malondialdehyde (MDA), TNFα, and IL-12 contents, and in plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, along with significant decreases in hepatic reduced glutathione (GSH) content and marked diffuse histopathologic damage. Pre-treatment with histamine resulted in significant mitigation of each of these end-points. The protective effect of histamine was not antagonized by pre-treatment with mepyramine (H1R antagonist) or ranitidine (H2R antagonist) and completely reversed by pre-treatment with thioperamide (H3R and H4R antagonist). In addition, the histamine protective effect was mimicked by pre-treatment of rats with clozapine (H4R agonist). These observations strongly suggested that histamine has a protective effect against hepatic I/R-mediated tissue injury during the acute phase, and this effect was mediated through an H4R stimulation that led to a decrease in IL-12 and TNFα production—outcomes that consequently decreased localized oxidative stress and afforded hepatic protection in general.     

Histamine potentiates acid-induced responses mediating transient receptor potential V1 in mouse primary sensory neurons

In inflamed tissues, extracellular pH decreases and acidosis is an important source of pain. Histamine is released from mast cells under inflammatory conditions and evokes the pain sensation in vivo, but the cellular mechanism of histamine-induced pain has not been well understood. In the present study, we examined the effects of histamine on [Ca²⁺]_i and membrane potential responses to acid in isolated mouse dorsal root ganglion (DRG) neurons. In capsaicin-sensitive DRG neurons from wild-type mice, acid (>pH 5.0) evoked [Ca²⁺]_i increases, but not in DRG neurons from transient receptor potential V1 (TRPV1) (−/−) mice. Regardless of isolectin GS-IB4 (IB4)-staining, histamine potentiated [Ca²⁺]_i responses to acid (≥pH 6.0) that were mediated by TRPV1 activation. Histamine increased membrane depolarization induced by acid and evoked spike discharges. RT-PCR indicated the expression of all four histamine receptors (H1R, H2R, H3R, H4R) in mouse DRG. The potentiating effect of histamine was mimicked by an H1R agonist, but not H2R-H4R agonists and was inhibited only by an H1R antagonist. Histamine failed to potentiate the [Ca²⁺]_i response to acid in the presence of inhibitors for phospholipase C (PLC) and protein kinase C (PKC). A lipoxygenase inhibitor and protein kinase A inhibitor did not affect the potentiating effects of histamine. Carrageenan and complete Freund's adjuvant produced inflammatory hyperalgesia, but these inflammatory conditions did not change the potentiating effects of histamine in DRG neurons. The present results suggest that histamine sensitizes acid-induced responses through TRPV1 activation via H1R coupled with PLC/PKC pathways, the action of which may be involved in the generation of inflammatory pain.     

The histamine H4‐receptor (H4R) regulates eosinophilic inflammation in ovalbumin‐induced experimental allergic asthma in mice

Via the histamine H₄‐receptor (H₄R), histamine promotes the pathogenesis of experimental allergic asthma in mice. Application of H₄R antagonists during sensitization as well as during provocation reduces the severity of the disease. However, the specific cell types functionally expressing H₄R in experimental allergic asthma have not been well characterized in vivo. In this study, we identified the cell type(s) responsible for H₄R activity in experimental asthma and related physiological mechanisms. Using H₄R‐deficient mice, we studied the role of H₄R in the sensitization and effector phase. DCs lacking H₄R expression during the in vitro sensitization reaction resulted in effector T cells unable to induce an entire eosinophilic inflammation in the lung upon adoptive transfer in vivo. Recipient mice lacking H₄R expression, which were adoptively transferred with H₄R^+/+ T cells polarized in the presence of H₄R^+/+ DCs, showed reduced signs of inflammation and ameliorated lung function. Here, we provide in vivo evidence that in experimental asthma in mice the H₄R specifically regulates activation of DCs during sensitization, while in the effector phase the H₄R is active in cells involved in the activation of eosinophils, and possibly other cells. A putative therapy targeting the H₄R may be an option for asthma patients developing IL‐5‐dependent eosinophilia.     

Type 1 innate lymphoid cell aggravation of atherosclerosis is mediated through TLR4

ILC populations elaborate a similar cytokine expression pattern with helper T cell subsets Th1, Th2 and Th17. Recent studies indicate that CD 25+ILC 2 could alleviate atherosclerosis by altering lipid metabolism, whereas the depletion of CD 90‐expressing ILC s had no influence on atherosclerosis. Thus, these findings raise the question of whether ILC 1 cells react on atherosclerosis. Hence, our group attempted to explore the role of ILC 1 cells in atherosclerosis. We found that ILC 1 cells have a high Th1‐like gene expression of T‐bet and IFN ‐γ, which is distinct from ILC 2, ILC 3 or conventional NK (cNK ) cells. Moreover, atherosclerotic lesions were greatly reduced in ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs for depleting ILC 1 cells (ILC 1+cNK cells), compared to ApoE−/−Rag1−/− mice treated with anti‐IL ‐15R mA bs for depleting cNK cells, and these effects could be fully rescued through the adoptive transfer of ILC 1 cells sorted from the spleen of ApoE−/−TLR 4+/+ mice into ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs. However, the adoptive transfer of ILC 1 cells sorted from the spleen of ApoE−/−TLR 4−/− mice into ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs blocked the progression of atherosclerosis, indicating that the pro‐atherosclerotic role of ILC 1 cells is dependent on TLR 4. Furthermore, oxLDL ‐induced increase in IFN ‐γ expression from ApoE−/− ILC 1 cells was correlated with the decrease in BACH 2 expression. Taken together, ILC 1 cells exist in atherosclerosis and aggravate atherosclerosis via increasing pro‐inflammatory cytokine expression in a TLR 4/BACH 2‐dependent manner.     

DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR‐activated murine macrophages

Delta‐like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR‐induced expression of key pro‐inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1‐deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN‐β and other pro‐inflammatory cytokines, including TNF‐α, IL‐12, and IL‐23. The expression of key proteins involved in IFN‐β signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL‐12 and IL‐23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS‐induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH‐mediated, pro‐inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.     

The functional role of the T1R family of receptors in sweet taste and feeding

The discovery of the T1R family of Class C G protein-coupled receptors in the peripheral gustatory system a decade ago has been a tremendous advance for taste research, and its conceptual reach has extended to other organ systems. There are three proteins in the family, T1R1, T1R2, and T1R3, encoded by their respective genes, Tas1r1, Tas1r2, and Tas1r3. T1R2 combines with T1R3 to form a heterodimer that binds with sugars and other sweeteners. T1R3 also combines with T1R1 to form a heterodimer that binds with l-amino acids. These proteins are expressed not only in taste bud cells, but one or more of these T1Rs have also been identified in the nasal epithelium, gut, pancreas, liver, kidney, testes and brain in various mammalian species. Here we review current perspectives regarding the functional role of these receptors, concentrating on sweet taste and feeding. We also discuss behavioral findings suggesting that a glucose polymer mixture, Polycose, which rodents avidly prefer, appears to activate a receptor that does not depend on the combined expression of T1R2 and T1R3. In addition, although the T1Rs have been implicated as playing a role in glucose sensing, T1R2 knock-out (KO) and T1R3 KO mice display normal chow and fluid intake as well as normal body weight compared with same-sex littermate wild type (WT) controls. Moreover, regardless of whether they are fasted or not, these KO mice do not differ from their WT counterparts in their Polycose intake across a broad range of concentrations in 30-minute intake tests. The functional implications of these results and those in the literature are considered.     

Functional characterization of histamine H4 receptor on human mast cells

Among the four different types of histamine receptors (H1-H4), H4R is predominantly expressed in immune cells and involved in immunomodulatory response. Here, in this study we determined the expression of H4R in human mast cells (HMC-1, LAD-2 and primary cord blood derived CD34+ human mast cells) and characterized its functional properties. Interestingly, we found that human mast cells responded to both histamine (natural ligand) and 4-methylhistamine (selective H4R agonist) for sustained intracellular calcium mobilization, degranulation and cytokine production. However, only histamine induced the release of cAMP, but 4-methylhistamine down regulates cAMP indicating that H4R mediates its effect through Gαi/o protein and H1R via Gαq protein. Furthermore, both histamine and 4-methylhistamine induced the production of cysteinyl leukotrienes and LTB4. Using human inflammation antibody array membrane, we found that H4R induced the expression of various inflammatory proteins, involving pro-inflammatory cytokines and chemokines and these are TGF-β1, TNF-α, TNF-β, PDGF-BB, TIMP-2, M-CSF, IP-10, IL-16, IL-6, IL-3, IL-10, MIP-1α, IL-1α, ICAM-1, Eotaxin-2, RANTES, IL-8, MCP-1, and IL-6sR. We also quantified the level of various inflammatory cytokines produced by human mast cells through H4R. It was observed that, the production level of Th2 cytokines IL-4(401.34 pg/ml), IL-5 (64.21 pg/ml) and IL-13 (1044 pg/ml) and classical proinflammatory cytokines IL-6 (221.27 pg/ml) and IL-1β (34.24 pg/ml) and chemokines MCP-1(106 pg/ml) and IL-8 (818.32 pg/ml). Furthermore, activation of H4R caused the phosphorylation of ERK and PI3 K in a time dependent manner. Taken together these data demonstrate that, the activation of H4R in human mast cells produced not only inflammatory mediators that are associated with allergic reactions but also other inflammatory conditions.