Sh Mice Research Articles

Repeated exposure to novelty promotes resilience against the amyloid-beta effect through dopaminergic stimulation.

The accumulation of beta-amyloid peptide (Aβ) in the forebrain leads to cognitive dysfunction and neurodegeneration in Alzheimer's disease. Studies have shown that individuals with a consistently cognitively active lifestyle are less vulnerable to Aβ toxicity. Recent research has demonstrated that intrahippocampal Aβ can impact catecholaminergic release and spatial memory. Interestingly, exposure to novelty stimuli has been found to stimulate the release of catecholamines in the hippocampus. However, it remains uncertain whether repeated enhancing catecholamine activity can effectively alleviate cognitive impairment in individuals with Alzheimer's disease. Our primary aim was to investigate whether repeated exposure to novelty could enable cognitive resilience against Aβ. This protection could be achieved by modulating catecholaminergic activity within the hippocampus. To investigate this hypothesis, we subjected mice to three different conditions-standard housing (SH), repeated novelty (Nov), or daily social interaction (Soc) for one month. We then infused saline solution (SS) or Aβ (Aβ1-42) oligomers intrahippocampally and measured spatial memory retrieval in a Morris Water Maze (MWM). Stereological analysis and extracellular baseline dopamine levels using in vivo microdialysis were assessed in independent groups of mice. The mice that received Aβ1-42 intrahippocampal infusions and remained in SH or Soc conditions showed impaired spatial memory retrieval. In contrast, animals subjected to the Nov protocol demonstrated remarkable resilience, showing strong spatial memory expression even after Aβ1-42 intrahippocampal infusion. The stereological analysis indicated that the Aβ1-42 infusion reduced the tyrosine hydroxylase axonal length in SH or Soc mice compared to the Nov group. Accordingly, the hippocampal extracellular dopamine levels increased significantly in the Nov groups. These compelling results demonstrate the potential for repeated novelty exposure to strengthen the dopaminergic system and mitigate the toxic effects of Aβ1-42. They also highlight new and promising therapeutic avenues for treating and preventing AD, especially in its early stages.

Connective tissue mast cells store and release noradrenaline

Mast cells are present in mucosal and connective tissues throughout the body. They synthesize and release a wide variety of bioactive molecules, such as histamine, proteases, and cytokines. In this study, we found that a population of connective tissue mast cells (CTMCs) stores and releases noradrenaline, originating from sympathetic nerves. Noradrenaline-storing cells, not neuronal fibers, were predominantly identified in the connective tissues of the skin, mammary gland, gastrointestinal tract, bronchus, thymus, and pancreas in wild-type mice but were absent in mast cell–deficient W-sash c-kit mutant KitW−sh/W−sh mice. In vitro studies using bone marrow–derived mast cells revealed that extracellular noradrenaline was taken up but not synthesized. Upon ionomycin stimulation, noradrenaline was released. Electron microscopy analyses further suggested that noradrenaline is stored in and released from the secretory granules of mast cells. Finally, we found that noradrenaline-storing CTMCs express organic cation transporter 3 (Oct3), which is also known as an extraneuronal monoamine transporter, SLC22A3. Our findings indicate that mast cells may play a role in regulating noradrenaline concentration by storing and releasing it in somatic tissues.

Regulation of Syk activity by antiviral adaptor MAVS in FcεRI signaling pathway.

Mast cells are the major effector cell type for IgE-mediated allergic reactions. Recent studies revealed a role for mast cells in orchestrating the host response to viral infections. We studied the relationship between FcεRI (high-affinity IgE receptor) and RIG-I-like receptor (RLR)-mediated antiviral signaling pathways. Mast cells (BMMCs) were cultured from bone marrow cells from mice deficient in MAVS or other RLR signaling molecules. MAVS expression was restored by retroviral transduction of MAVS-deficient BMMCs. These cells were stimulated with IgE and antigen and their activation (degranulation and cytokine production/secretion) was quantified. FcεRI-mediated signaling events such as protein phosphorylation and Ca2+ flux were analyzed by western blotting and enzyme assays. WT and mutant mice as well as mast cell-deficient KitW-sh/W-sh mice engrafted with BMMCs were subjected to passive cutaneous anaphylaxis. Unexpectedly, we found that mast cells devoid of the adaptor molecule MAVS exhibit dramatically increased cytokine production upon FcεRI stimulation, despite near-normal degranulation. Consistent with these observations, MAVS inhibited tyrosine phosphorylation, thus catalytic activity of Syk kinase, the key signaling molecule for FcεRI-mediated mast cell activation. By contrast, mast cells deficient in RIG-I, MDA5 or IRF3, which are antiviral receptor and signaling molecules upstream or downstream of MAVS, exhibited reduced or normal mast cell activation. MAVS-deficient mice showed enhanced late-phase responses in passive cutaneous anaphylaxis. This study demonstrates that the adaptor MAVS in the RLR innate immune pathway uniquely intersects with the adaptive immune FcεRI signaling pathway.

The ketone body β-hydroxybutyrate mitigates ILC2-driven airway inflammation by regulating mast cell function.

Ketone bodies are increasingly understood to have regulatory effects on immune cell function, with β-hydroxybutyrate (BHB) exerting a predominantly anti-inflammatory response. Dietary strategies to increase endogenous ketone body availability such as the ketogenic diet (KD) have recently been shown to alleviate inflammation of the respiratory tract. However, the role of BHB has not been addressed. Here, we observe that BHB suppresses group 2 innate lymphoid cell (ILC2)-mediated airway inflammation. Central to this are mast cells, which support ILC2 proliferation through interleukin-2 (IL-2). Suppression of the mast cell/IL-2 axis by BHB attenuates ILC2 proliferation and the ensuing type 2 cytokine response and immunopathology. Mechanistically, BHB directly inhibits mast cell function in part through GPR109A activation. Similar effects are achieved with either the KD or 1,3-butanediol. Our data reveal the protective role of BHB in ILC2-driven airway inflammation, which underscores the potential therapeutic value of ketone body supplementation for the management of asthma.

Mouse Model of Weak Depression Exhibiting Suppressed cAMP Signaling in the Amygdala, Lower Lipid Catabolism in Liver, and Correlated Gut Microbiota

To establish a mouse model of weak depression, we raised 6-week-old C57BL/6N mice in single (SH) or group housing (GH) conditions for 2 weeks. The SH group showed less social interaction with stranger mice, learning disability in behavioral tests, and lower plasma corticosterone levels. The cecal microbiota of the SH group showed significant segregation from the GH group in the principal coordinate analysis (PCoA). Transcriptome analysis of the amygdala and liver detected multiple differentially expressed genes (DEGs). In the amygdala of SH mice, suppression of the cyclic adenine monophosphate (cAMP) signal was predicted and confirmed by the reduced immunoreactivity of phosphorylated cAMP-responsive element-binding protein. In the liver of SH mice, downregulation of beta-oxidation was predicted. Interestingly, the expression levels of over 100 DEGs showed a significant correlation with the occupancy of two bacterial genera, Lactobacillus (Lactobacillaceae) and Anaerostipes (Lachnospiraceae). These bacteria-correlated DEGs included JunB, the downstream component of cAMP signaling in the amygdala, and carnitine palmitoyltransferase 1A (Cpt1a), a key enzyme of beta-oxidation in the liver. This trans-omical analysis also suggested that nicotinamide adenine dinucleotide (NAD) synthesis in the liver may be linked to the occupancy of Lactobacillus through the regulation of nicotinamide phosphoribosyltransferase (NAMPT) and kynureninase (KYNU) genes. Our results suggested that SH condition along with the presence of correlated bacteria species causes weak depression phenotype in young mice and provides a suitable model to study food ingredient that is able to cure weak depression.

Dextromethorphan - A widely-used cough suppressant - Induces local anaphylaxis via MRGPRX2 on mast cells

Dextromethorphan (DM) is a cough suppressant available in many prescribed and over-the-counter medications. Adverse reactions induced by DM have been regularly reported, including allergic skin reactions in some cases. However, the underlying mechanisms of local anaphylaxis induced by DM have not been elucidated. In this study, we found that DM could activate mast cells to increase calcium mobilization and release β-hexosaminidase, histamine, tumor necrosis factor-α, MCP-1, and IL-8 in a dose-dependent manner. The allergic reactions were confirmed by hind paw swelling and extravasation assay in vivo. Furthermore, DM was revealed to induce local anaphylaxis via MRGPRX2 by the mast cell-deficient kitW-sh/W−sh mice and MRGPRX2 knockdown mast cells. And the MRGPRX2-HEK293/CMC analysis and frontal analysis also showed that DM has a considerable affinity with MRGPRX2. Together, our findings suggest that close monitoring should be drawn on patients with DM for its potential anaphylaxis via MRGPRX2.

Mast cells as mediators of astrocyte morphology associated with neuroinflammatory disease states

Abstract Morphological deficits observed in astrocytes, the most abundant glial cell type in the central nervous system, are associated with various neuroinflammatory diseases. The morphological and molecular astrocyte changes associated with multiple sclerosis are well-characterized and concentrated in the white matter of the brain. While the exact mechanisms behind this autoimmune disease are not fully understood, mast cells play a key role in the progression of white matter demyelination. We hypothesize mast cells orchestrate morphological astrocyte changes that are associated with neuroinflammatory disease states. To test this hypothesis, we used GFAP as a marker to visualize astrocyte morphology in white matter tracts of wild type (WT) and mast cell deficient KitW–sh/W–sh (sash) mice. Using skeleton analysis, we quantified astrocyte characteristics such as number of endpoints, branches, and branch length. Compared with WT mice, sash mice exhibited fewer branches (77 vs. 54 branches/cell) and endpoints per cell (49.5 vs. 24.5 P<0.001). These findings suggest that mast cells may drive altered astrocyte morphology and function in white matter. In order to determine whether this effect is due to altered development, studies using sash mice at time points across the lifespan are in progress. In addition, we are using diphtheria toxin in combination with novel genetic lines to ablate mast cells acutely in adult mice. Along with new molecular techniques, this will allow us to better characterize the interaction between mast cells and astrocytes in the adult and developing brain. Unraveling this interplay will be critical to understanding the role of mast cells in neurodevelopment and neuroinflammatory diseases such as multiple sclerosis.

Mast cells as Immune modulators of behavior and brain cellular composition

Abstract Mast cells (MC) are involved in peripheral disease such as allergy/anaphylaxis, but they are also present in brain where they can modulate behavior. MC deficient KitW–sh/W–sh (sash) mice have been reported to show increased anxiety-like behaviors compared to wild type (WT) mice, but these studies did not consider potential interactions with sex and/or stress, which is relevant considering that MCs are sexually dimorphic and stress-sensitive. Here, wild type (WT) and sash male and female mice were exposed to control handling or mild environmental stress and then phenotyped for behaviors relevant to human mood disorders. Stress did not alter anxiety or motivated behaviors in WT. Compared to WT, sash showed increased anxiety, which was independent of sex or stress. A measure of depressive-like behavior in rodents is preference for sucrose water over regular water. In females, sash showed reduced sucrose preference compared to WT, which was exacerbated by stress. In males, control sash and WT showed similar sucrose preference, but stress induced sucrose anhedonia only in sash. To explore the underlying mechanisms, we studied the Nucleus Accumbens, a brain area involved in anxiety and motivation. We found that sash have a reduced number of astrocytes and a two-fold higher number of cells expressing FosB, a transcription factor induced by stress and involved in synaptic plasticity and behavior. Together, these data implicate MCs as key immune modulators of brain cellular composition and activity. Our ongoing investigations aim at deeply understanding the role that MCs play on mood and cognition, which could provide significant insights into the mechanisms underlying human psychiatric disease and inform new therapeutic targets.

Identification of a new strain of mouse kidney parvovirus associated with inclusion body nephropathy in immunocompromised laboratory mice

ABSTRACT Inclusion body nephropathy (IBN) and kidney fibrosis in aged immunodeficient mice and, to lesser extent, in immunocompetent mice have been recently linked to infection of mouse kidney parvovirus (MKPV), also known as murine chapparvovirus (MuCPV). Knowledge about its prevalence and the complete genome sequence of more MKPV strains is essential for understanding phylogenetic relationships and pathogenicity among MKPV strains. In the present study using PCR and genome walking, we determined the complete 4440-nucleotide genome of a new MKPV strain, namely MIT-WI1, which was identified in IBN-affected Il2rg−/–Rag2−/– c-Kit W–sh/W–sh mice housed in the vivarium at Whitehead Institute for Biomedical Research (WI). The overall nucleotide (>94%) and deduced amino acid sequences (>98%) of p10, p15, NS1 (replicase), NS2 and VP1 (capsid protein) within the MIT-WI1 genome, are closely related to MKPV/MuCPV strains described in laboratory and wild Mus musculus mice. In addition, PCR and qPCR assays using newly designed primers conserved among the known MKPV/MuCPV genomes were developed and utilized to assess MKPV status in selected laboratory mice. MKPV was also detected in immunodeficient (NSG) and immunocompetent (Crl:CD1(ICR), UTXflox) mouse strains/stocks. The abundance of the MKPV genome copies was significantly correlated with the severity of IBN. Our data indicate that MKPV is present in selected mouse strains/stocks, and provides new insights into the genome evolution of MKPV.

Genetic Deficiency of T‐bet Protects Against Chronic Renal Injury in Murine Renal Artery Stenosis

AimTh1 cells have potent pro‐inflammatory properties and have been shown to play an important role in the development and progression of acute renal injury to chronic renal disease. The aim of this study is to test the hypothesis that chronic renal damage in mice with renal artery stenosis (RAS) would be ameliorated in mice with homozygous deletion of the Th1 transcription factor T‐bet.MethodsC57BL6/J (WT) and C57BL6/J mice with homozygous deletion of T‐bet (KO) were obtained from Jackson Laboratory. Renal artery stenosis surgery (RAS) was performed by placement of a polytetrafluoroethylene cuff on the right renal artery of KO (KO RAS group, n=5) and WT mice (WT RAS group, n=6). Sham (SH) surgery was performed with manipulation of the right renal artery without cuff placement in KO (KO SH group, n=4) and WT mice (WT SH group, n=5). Renal blood perfusion and oxygenation (R2*) were measured using 16.4T MRI. Blood pressure measurements were done using tail‐cuff method. Kidneys were harvested at 28 days after RAS for assessment of histology.ResultsBaseline intra‐renal perfusion in both KO RAS and WT RAS mice was reduced to a similar extent following cuff placement. In KO RAS group, baseline intra‐renal perfusion (795 ± 57 ml/100g/min) was reduced to 234 ± 22 ml/100g/min (p<0.0001 vs baseline, mean reduction 71%) and in WT RAS group, baseline intra‐renal perfusion (836 ± 87 ml/100g/min) was reduced to 220 ± 21 ml/100g/min (p=0.002 vs baseline, mean reduction 74%). Both KO RAS and WT RAS mice developed hypertension, which persisted throughout the 4 week study. WT RAS group showed significant elevation in cortical R2*, a measure of hypoxia, following RAS compared to their baseline (p=0.002). KO RAS mice showed slightly higher R2* value, which was not significantly different from their baseline values. The weight of the stenotic kidney of the KO RAS group (105 ± 11.6 mg) was significantly lower than the KO SH mice (178 ± 14.1 mg, p=0.0021) but significantly higher than the WT RAS group (51.7 ± 7.9 mg, p=0.0010). On histologic and immune‐histochemical analysis, we observed that the stenotic kidney of the KO RAS group had significantly lower (%) cortical tubular atrophy (15% ± 6.7, p<0.0001) compared to WT RAS mice (81.7% ± 7). KO RAS group had significantly lower macrophage (MQ) (p=0.0016) and T cell influx (p=0.0052) compared to the WT RAS, as assessed by the % of cortical surface areas staining positive for F4/80 (MQ) and CD3 (T cell). When compared to their respective shams, KO RAS showed no significant differences in the % cortical tubular atrophy, MQ and CD3+ T cell influx.ConclusionWe conclude that deficiency of Th1 transcription factor T‐bet, protects against chronic renal injury as evidenced by the less cortical atrophy in KO RAS compared to WT RAS group, despite a similar elevation in blood pressure and reduction in perfusion. This model may provide a novel therapeutic target to prevent renal disease progression in patients with renovascular hypertension.Support or Funding InformationNational Institutes of Health Grant AI 100911This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of the interscutularis model as an outcome measure for facial nerve surgery

IntroductionAnimal models for the study of facial paralysis have been well developed, but concern has arisen regarding the accuracy of eye closure and whisker movement as outcome measures due to new data regarding interconnectivity between facial nerve branches and autonomic innervation. The posterior auricular nerve (PAN) is an isolated branch of the facial nerve which has been confirmed as the sole motor innervat or of the interscutularis muscle. This study was designed to develop a model for facial nerve palsy utilizing the PAN and interscutularis muscle. MethodsA custom-made automated video capture system was built into a poly methyl methacrylate cage using a high definition monochrome digital camera and image sensor to record the animal as it drank from a water feeder. A copper floor pad and copper collar around the water feeder were connected to an electrical circuit for automatic saving of the video recording 10 s prior to and 30 s following the drinking event. A pre-operative baseline recording of ear movement during drinking was captured. Female YFP-16 mice at 6 weeks were assigned to sham (Sh, n = 5), nerve excision (Ex, n = 10), or nerve crush (Cr, n = 10) groups with all interventions performed on the right PAN. Sh mice were irrigated with 10 ml normal saline as were the Ex and Cr mice following operative intervention. In Ex mice, a 3 mm section of the PAN was sharply excised and nerve gap was confirmed with fluorescent microscopy. In Cr mice, the PAN was crushed 3 mm from the origin of the facial nerve trunk with size 5 jeweler’s forceps for two periods of 20 s. Post-operative video recordings were collected on post-operative days (POD) 1, 10, 20, and 30. To determine the change in ear movement, the right ear was graphically compared to the left control side. ResultsSh animals exhibited a statistically significant reduction in ear movement at POD01 compared to other POD recordings (p < 0.05), but no significant change in right ear movement following POD05. Ex animals had a significant reduction in right ear movement at all PODs in comparison to the left ear (p < 0.05) with no significant change in right ear movement during the study period (p = 0.94). Cr animals showed a significant reduction in right ear movement compared to the left at POD01, POD10, and POD20 (p < 0.05). At POD30, there was no significant difference between ear movement on either side (p = 0.35). There was a significant change in right ear movement during the data collection period (p < 0.05). ConclusionThe results show that significant differences were demonstrated between the experimental groups and that significant changes within the crush group were identifiable making this an acceptable model to develop as an accurate outcome measure following rodent facial nerve surgery.

Disruption of a CD1d-mediated interaction between mast cells and NKT cells aggravates atherosclerosis

Background and aimsThe development of atherosclerosis is tightly regulated by the innate and adaptive immune system. Communication between these two compartments occurs, among others, upon presentation of lipid antigens to the NKT cell population by CD1d-expressing antigen-presenting cells. Recent evidence states that also mast cells express CD1d and can directly communicate with NKT cells. However, no such relationship has been reported in atherosclerosis. Here, we aimed to elucidate in vivo the CD1d-mediated interaction between mast cells and NKT cells upon atherosclerosis progression. MethodsWe adoptively transferred CD1d−/− or control mast cells to mast cell-deficient apoE−/−KitW-sh/W−sh mice and subsequently placed the animals on a Western-type diet for 10 weeks. ResultsAt the end of the Western-type diet period, the aortic root of CD1d−/− mast cell-reconstituted mice displayed increased plaque size, with less collagen deposition and higher intraplaque CD4+ T cells, as compared to control mice. In addition, T cells inside the aortic arch showed higher pro-inflammatory cytokine production in the form of IFNγ, TNFα and IL-17. Finally, T-bet expression was found elevated in both CD4+ and CD8+ circulating T cells. ConclusionsThis study is the first to illustrate that disruption of the CD1d communication pathway between mast cells and NKT cells aggravates atherosclerosis, through a shift towards pro-inflammatory T cell responses. This ability of mast cell action during plaque progression sheds new light on their role in atherosclerosis.

Kit W-sh Mutation Prevents Cancellous Bone Loss during Calcium Deprivation.

Calcium is essential for normal bone growth and development. Inadequate calcium intake increases the risk of osteoporosis and fractures. Kit ligand/c-Kit signaling plays an important role in regulating bone homeostasis. Mice with c-Kit mutations are osteopenic. The present study aimed to investigate whether impairment of or reduction in c-Kit signaling affects bone turnover during calcium deprivation. Three-week-old male WBB6F1/J-Kit W /Kit W-v /J (W/W v ) mice with c-Kit point mutation, Kit W-sh /HNihrJaeBsmJ (W sh /W sh ) mice with an inversion mutation in the regulatory elements upstream of the c-Kit promoter region, and their wild-type controls (WT) were fed either a normal (0.6% calcium) or a low calcium diet (0.02% calcium) for 3weeks. μCT analysis indicated that both mutants fed normal calcium diet had significantly decreased cortical thickness and cancellous bone volume compared to WT. The low calcium diet resulted in a comparable reduction in cortical bone volume and cortical thickness in the W/W v and W sh /W sh mice, and their corresponding controls. As expected, the low calcium diet induced cancellous bone loss in the W/W v mice. In contrast, W sh /W sh cancellous bone did not respond to this diet. This c-Kit mutation prevented cancellous bone loss by antagonizing the low calcium diet-induced increase in osteoblast and osteoclast numbers in the W sh /W sh mice. Gene expression profiling showed that calcium deficiency increased Osx, Ocn, Alp, type I collagen, c-Fms, M-CSF, and RANKL/OPG mRNA expression in controls; however, the W sh mutation suppressed these effects. Our findings indicate that although calcium restriction increased bone turnover, leading to osteopenia, the decreased c-Kit expression levels in the W sh /W sh mice prevented the low calcium diet-induced increase in cancellous bone turnover and bone loss but not the cortical bone loss.

Evidence for mast cell-mediated zinc homeostasis: Increased labile zinc in the hippocampus of mast-cell deficient mice

The dentate gyrus of the hippocampus is a site of adult neurogenesis, and is also known to contain one of the highest concentrations of labile brain zinc (Zn), thought to aid in learning and memory by supporting neurogenesis. At the same time, it is known that unbound Zn, when present at excessive levels, decreases the formation of new neurons. Since mast cells contain Zn transporters capable of moving this essential element across their plasma membrane, as well as Zn-rich granules that are dispelled upon secretion, we reasoned that mast cells contribute to Zn homeostasis in this area of the brain, as they are found in greatest numbers in and around the dentate gyrus. This line of evidence was tested by comparing Timm-stained hippocampal sections of mast cell-deficient C57BL/6-KitW−sh/W−sh (Sash−/−) mice to those of mast cell-containing wild type (Sash+/+) animals. Mast cell deficient mice were found to have significantly increased Timm-positive staining as compared to controls, reflecting an increase in labile or bioactive Zn in this region. As we observed no change in total brain Zn (protein-bound plus unbound Zn), these increases indicate that mast cells may serve to bind what would otherwise be excessive or deleterious levels of labile Zn, or that they are able to recruit metallothionein proteins. Because elevated levels of labile Zn are observed in the brains of patients with neurodegenerative diseases such as Alzheimer’s, the potential contribution of mast cells to these diseases remains a compelling one. Overall, these data support a role for mast cells in either establishing or maintaining Zn homeostasis in the brain in the service of health, while Zn dysregulation has the potential to reduce learning, memory, and ultimately organismal survival.

Disodium cromoglycate inhibits asthma-like features induced by sphingosine-1-phosphate

Compelling evidence suggests the involvement of sphingosine-1-phosphate (S1P) in the pathogenesis of asthma. The systemic administration of S1P causes asthma like features in the mouse involving mast cells. In this study we investigated whether disodium cromoglycate (DSCG), administered as a preventative treatment as in human therapy, could affect S1P effects on airways. BALB/c mice, treated with DSCG, received subcutaneous administration of S1P. Bronchi and pulmonary tissues were collected and functional, molecular and cellular studies were performed. DSCG inhibited S1P-induced airway hyper-reactivity as well as pulmonary inflammation. DSCG decreased the recruitment of solely mast cells and B cells in the lung. IgE serum levels, prostaglandin D2, mucus production and IL-13 were also reduced when mice were pretreated with DSCG. S1P induced pulmonary expression of CD23 on T and B cells, that was reversed by DSCG. Conversely, S1P failed to upregulate CD23 in mast cell-deficient Kit W−sh/W−sh mice. In conclusion we have shown that DSCG inhibits S1P-induced asthma like features in the mouse. This beneficial effect is due to a regulatory action on mast cell activity, and in turn to an inhibition of IgE-dependent T and B cells responses.

Mammary tumor growth and metastasis are reduced in c-Kit mutant Sash mice.

Besides its well‐known function in allergic response, mast cell, one of the key immune cells present in tumor microenvironment, plays important roles in cancer progression. However, the functional role of mast cells in breast cancer development and metastasis is not well understood. To test the involvement of mast cells in breast cancer, we examined the effects of loss of mast cells on mammary tumor development by crossing the well‐known mast cell deficient mouse strain sash (KitW‐sh/W‐sh) with the mammary tumor transgenic mouse strain MMTV‐Polyoma Middle T antigen (PyMT). Although mammary tumor onset was not affected in the absence of mast cells, mammary growth and metastasis were reduced in PyMT/KitW‐sh/W‐sh mice compared with PyMT/wild‐type mice (WT). Histological and immunofluorescent analyses showed that tumors from PyMT/KitW‐sh/W‐sh mice showed largely differentiated morphology with reduced angiogenesis compared with MMTV‐PyMT/WT mice. Our results suggest that mast cells may promote breast cancer growth and metastasis. Agents that can block mast cells growth are potential new therapies to treat metastatic breast cancer.

S1P-induced airway smooth muscle hyperresponsiveness and lung inflammation in vivo: molecular and cellular mechanisms.

Sphingosine-1-phosphate (S1P) has been shown to be involved in the asthmatic disease as well in preclinical mouse experimental models of this disease. The aim of this study was to understand the mechanism(s) underlying S1P effects on the lung. BALB/c, mast cell-deficient and Nude mice were injected with S1P (s.c.) on days 0 and 7. Functional, molecular and cellular studies were performed. S1P administration to BALB/c mice increased airway smooth muscle reactivity, mucus production, PGD2 , IgE, IL-4 and IL-13 release. These features were associated to a higher recruitment of mast cells to the lung. Mast cell-deficient Kit (W) (-sh/) (W) (-sh) mice injected with S1P did not display airway smooth muscle hyper-reactivity. However, lung inflammation and IgE production were still present. Treatment in vivo with the anti-CD23 antibody B3B4, which blocks IgE production, inhibited both S1P-induced airway smooth muscle reactivity in vitro and lung inflammation. S1P administration to Nude mice did not elicit airway smooth muscle hyper-reactivity and lung inflammation. Naïve (untreated) mice subjected to the adoptive transfer of CD4+ T-cells harvested from S1P-treated mice presented all the features elicited by S1P in the lung. S1P triggers a cascade of events that sequentially involves T-cells, IgE and mast cells reproducing several asthma-like features. This model may represent a useful tool for defining the role of S1P in the mechanism of action of currently-used drugs as well as in the development of new therapeutic approaches for asthma-like diseases.

C-kit plays a critical role in induction of intravenous tolerance in experimental autoimmune encephalomyelitis.

c-kit (CD117) is a tyrosine kinase receptor found in various types of immune cells. It has been shown that c-kit plays a role in the pathogenesis of multiple sclerosis, an inflammatory demyelinating disorder of the CNS. Recent data have suggested an immunoregulatory effect of c-kit. We therefore examined the role of c-kit in autoantigen-induced i.v. tolerance in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Our results show that induction of intravenous tolerance against EAE in B6 mice is characterized by increased numbers of CD117(+) cells and altered mast cell-associated molecules in the periphery and in the CNS. W(-sh) (c-kit-deficient) mice were resistant to i.v autoantigen-induced tolerance, with increased proinflammatory cytokine production in the periphery. I.v. autoantigen in WT mice suppressed the production of proinflammatory cytokines IFN-γ and IL-6 and up-regulated the expression of FoxP3, a transcription factor of Tregs; however, in W(-sh) mice, IFN-γ and IL-6 were increased with a failure of FoxP3 induction upon i.v. autoantigen injection and is thus a mechanism for resistance to i.v. tolerance induction in these mice. We conclude that c-kit signaling has a regulatory role in i.v. tolerance and could be a target for potential immunotherapy in autoimmune disorders.

Hepatoprotective effect of Schisandra chinensis (Turcz.) Baill. lignans and its formula with Rubus idaeus on chronic alcohol-induced liver injury in mice.

This study aimed to investigate the liver protection effect of Schisandra chinensis (Turcz.) Baill. (SC) lignans and its combination with Rubus idaeus (RI) on chronic alcohol-induced mice. A low level of SC lignans (SL) was prepared from the clear juice of sarcocarp. Lignans were further extracted from the SC seeds and added to the SL to form high-level SC lignans (SH). Moreover, RI clear juice lyophilized powder was mixed with SL (SR), and the liver protection effects of SL, SH and SR were investigated. Male ICR mice were administered with the corresponding samples and gastrically infused with 50% alcohol (1 h later) once per day for 60 d. In the in vitro study, the characteristic lignans in the SC clear juice and the seed extract were analyzed by high performance liquid chromatography (HPLC). The total phenolic content (TPC) and antioxidant capability of SL, SH, and SR were determined. The results of the in vivo study showed that SC lignans exhibited a dose-dependent effect on the regulation of hepatic antioxidant status, serum transaminases levels, hyperlipidemia and hepatic fat deposition in mice. However, hepatic lesions were observed in the SH mice, which indicated a potential side effect caused by long-term consumption of SH under chronic alcohol administration. By contrast, SR exhibited a similar hepatoprotective effect as SH without any abnormality found in the histological analysis. After analysis with HPLC, Schizandrol A and Schizandrol B were identified in the SC clear juice, and two more kinds of lignans, Schisandrin A and Schisandrin B, were identified in the seed extracts. The SR sample had the highest TPC and exhibited the best antioxidant capability. In conclusion, RI strengthened the liver protection effect of SC lignans effectively and safely, which was probably achieved by enhancing the antioxidant status and the positive effect of their combination was possibly attributed to both lignans and polyphenols. This study demonstrated that the combination of low-level SC lignans and RI might be used as a potential safe formula for beverage development to alleviate the adverse effects of long-term alcohol consumption.

Characterization of endocannabinoid-mediated induction of myeloid-derived suppressor cells involving mast cells and MCP-1

Endocannabinoids are lipid-signaling molecules found in the nervous system; however, their precise role in the periphery is unclear. In the current study, we observed that a single i.p. administration of AEA caused rapid induction of MDSCs. The MDSCs contained a mixture of granulocytic and monocytic subtypes and expressed Arg-1 and iNOS. The MDSCs suppressed T cell proliferation in vitro and used iNOS to mediate their effect. Moreover, adoptive transfer of MDSCs led to suppression of mBSA-induced DTH. Through the use of pharmacological inhibition, as well as genetic knockout mice, we found that the induction of MDSCs by AEA was CB1-dependent. The induction of MDSCs by AEA was reduced significantly in mast cell-deficient mice, while maintained in LPS-insensitive mice, showing that the induction of MDSCs by AEA was dependent, at least in part, on mast cells and independent of TLR4. Chemokine analysis of AEA- treated WT mice showed an early spike of MCP-1, which was decreased in Kit(W/W-sh) mice, showing a role of mast cells in the secretion of MCP-1 in response to AEA. Also, use of antibodies against MCP-1 or mice deficient in MCP-1 confirmed the role played by MCP-1. Interestingly, MCP-1 played a significant role in the induction of monocytic but not granulocytic MDSCs. Our studies demonstrate for the first time that endocannaboinids activate CB1 on mast cells to induce MCP-1, which facilitates recruitment of monocytic MDSCs.