Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 ^−/−) mice develop significant defects in the infiltration of Ly6C^hi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C^hi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C^int monocytes of Ifnar1 ^−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 ^−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C^hi monocytes. By using BM chimeric mice (WT BM into Ifnar1 ^−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C^hi monocytes. Of note, WT BM reconstituted Ifnar1 ^−/− chimeric mice with increased numbers of Ly6C^hi monocytes survived longer than influenza-infected Ifnar1 ^−/− mice. In contrast, WT mice that received Ifnar1 ^−/− BM cells with alternative Ly6C^int monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.     

The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS,Cigarette Smoke or Allergen Challenge

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4^−/− and Mrp4^+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4^−/− and Mrp4^+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4^+/+ or Mrp4^−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.     

The Matricellular Protein Periostin Is Required for Sost Inhibition and the Anabolic Response to Mechanical Loading and Physical Activity

Periostin (gene Postn) is a secreted extracellular matrix protein involved in cell recruitment and adhesion and plays an important role in odontogenesis. In bone, periostin is preferentially expressed in the periosteum, but its functional significance remains unclear. We investigated Postn^−/− mice and their wild type littermates to elucidate the role of periostin in the skeletal response to moderate physical activity and direct axial compression of the tibia. Furthermore, we administered a sclerostin-blocking antibody to these mice in order to demonstrate the influence of sustained Sost expression in their altered bone phenotypes. Cancellous and cortical bone microarchitecture as well as bending strength were altered in Postn^−/− compared with Postn^+/+ mice. Exercise and axial compression both significantly increased bone mineral density and trabecular and cortical microarchitecture as well as biomechanical properties of the long bones in Postn^+/+ mice by increasing the bone formation activity, particularly at the periosteum. These changes correlated with an increase of periostin expression and a consecutive decrease of Sost in the stimulated bones. In contrast, mechanical stimuli had no effect on the skeletal properties of Postn^−/− mice, where base-line expression of Sost levels were higher than Postn^+/+ and remained unchanged following axial compression. In turn, the concomitant injection of sclerostin-blocking antibody rescued the bone biomechanical response in Postn^−/− mice. Taken together, these results indicate that the matricellular periostin protein is required for Sost inhibition and thereby plays an important role in the determination of bone mass and microstructural in response to loading.     

Insulin receptor substrate 2 (IRS2)-deficient mice show sensorineural hearing loss that is delayed by concomitant protein tyrosine phosphatase 1B (PTP1B) loss of function

The insulin receptor substrate (IRS) proteins are key mediators of insulin and insulinlike growth factor 1 (IGF-1) signaling. Protein tyrosine phosphatase (PTP)-1B dephosphorylates and inactivates both insulin and IGF-1 receptors. IRS2-deficient mice present altered hepatic insulin signaling and β-cell failure and develop type 2–like diabetes. In addition, IRS2 deficiency leads to developmental defects in the nervous system. IGF1 gene mutations cause syndromic sensorineural hearing loss in humans and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. Our objective was to study the hearing function and cochlear morphology of Irs2-null mice and the impact of PTP1B deficiency. We have studied the auditory brainstem responses and the cochlear morphology of systemic Irs2^−/−Ptpn1^+/+, Irs2^+/+Ptpn1^−/−and Irs2^−/−Ptpn1^−/− mice at different postnatal ages. The results indicated that Irs2^−/−Ptpn1^+/+ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology with hypoinnervation of the cochlear ganglion and aberrant stria vascularis, compared with wild-type mice. Simultaneous PTP1B deficiency in Irs2^−/−Ptpn1^−/− mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for treating deafness associated with hyperglycemia and type 2 diabetes.     

Type I Interferons Promote Fatal Immunopathology by Regulating Inflammatory Monocytes and Neutrophils during Candida Infections

Invasive fungal infections by Candida albicans (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (Ifnar1^−/−), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6C^hi monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in Ifnar1^−/− mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.     

ABCA1 plays no role in the centripetal movement of cholesterol from peripheral tissues to the liver and intestine in the mouse

This study uses the mouse to explore the role of ABCA1 in the movement of this cholesterol from the peripheral organs to the endocrine glands for hormone synthesis and liver for excretion. The sterol pool in all peripheral organs was constant and equaled 2,218 and 2,269 mg/kg, respectively, in abca1^+/+ and abca1^−/− mice. Flux of cholesterol from these tissues equaled the rate of synthesis plus the rate of LDL-cholesterol uptake and was 49.9 mg/day/kg in control animals and 62.0 mg/day/kg in abca1^−/− mice. In the abca1^+/+ animals, this amount of cholesterol moved from HDL into the liver for excretion. In the abca1^−/− mice, the cholesterol from the periphery also reached the liver but did not use HDL. Fecal excretion of cholesterol was just as high in abac1^−/− mice (198 mg/day/kg) as in the abac1^+/+ animals (163 mg/day/kg), although the abac1^−/− mice excreted relatively more neutral than acidic sterols. This study established that ABCA1 plays essentially no role in the turnover of cholesterol in peripheral organs or in the centripetal movement of this sterol to the endocrine glands, liver, and intestinal tract for excretion.     

The reno-vascular A2B adenosine receptor protects the kidney from ischemia

Background

Acute renal failure from ischemia significantly contributes to morbidity and mortality in clinical settings, and strategies to improve renal resistance to ischemia are urgently needed. Here, we identified a novel pathway of renal protection from ischemia using ischemic preconditioning (IP).

Methods and Findings

For this purpose, we utilized a recently developed model of renal ischemia and IP via a hanging weight system that allows repeated and atraumatic occlusion of the renal artery in mice, followed by measurements of specific parameters or renal functions. Studies in gene-targeted mice for each individual adenosine receptor (AR) confirmed renal protection by IP in A1^−/−, A2A^−/−, or A3AR^−/− mice. In contrast, protection from ischemia was abolished in A2BAR^−/− mice. This protection was associated with corresponding changes in tissue inflammation and nitric oxide production. In accordance, the A2BAR-antagonist PSB1115 blocked renal protection by IP, while treatment with the selective A2BAR-agonist BAY 60–6583 dramatically improved renal function and histology following ischemia alone. Using an A2BAR-reporter model, we found exclusive expression of A2BARs within the reno-vasculature. Studies using A2BAR bone-marrow chimera conferred kidney protection selectively to renal A2BARs.

Conclusions

These results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia.     

A non‐redundant role for MKP5 in limiting ROS production and preventing LPS‐induced vascular injury

There are at least 11 mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and only 3 major groups of MAPKs, raising the question of whether these phosphatases have non-redundant functions in vivo. Using a modified mouse model of local Shwartzman reaction, we found that deletion of the MKP5 gene, but not the MKP1 gene, led to robust and accelerated vascular inflammatory responses to a single dose of LPS injection. Depletion of neutrophils significantly reduced the vascular injury in Mkp5^−/− mice, whereas adoptive transfer of Mkp5^−/− neutrophils replicated the LPS-induced skin lesions in wild-type recipients. Neutrophils isolated from Mkp5^−/− mice exhibited augmented p38 MAPK activation and increased superoxide generation on activation. The p38 MAPK inhibitor, SB203580, significantly reduced p47^phox phosphorylation and diminished superoxide production in neutrophils. p38 MAPK phosphorylated mouse p47^phox, and deletion of the p47^phox gene ablated the LPS-induced vascular injury in Mkp5^−/− mice. Collectively, these results show an earlier unrecognized and non-redundant function of MKP5 in restraining p38 MAPK-mediated neutrophil oxidant production, thereby preventing LPS-induced vascular injury.     

Neutrophil-Derived Myeloperoxidase Aggravates Non-Alcoholic Steatohepatitis in Low-Density Lipoprotein Receptor-Deficient Mice

Background

Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.

Methodology

Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR^−/−/MPO^−/−tp mice) were generated and compared with LDLR^−/−/MPO^+/+tp mice after induction of NASH by high-fat feeding.

Results

High-fat feeding caused a ∼4-fold induction of liver MPO in LDLR^−/−/MPO^+/+ mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR^−/−/MPO^−/−tp mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR^−/−/MPO^+/+tp mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR^−/−/MPO^−/−tp mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR^−/−/MPO^−/−tp mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.     

Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling

The early inflammatory response to influenza virus infection contributes to severe lung disease and continues to pose a serious threat to human health. The mechanisms by which neutrophils gain entry to the respiratory tract and their role during pathogenesis remain unclear. Here, we report that neutrophils significantly contributed to morbidity in a pathological mouse model of influenza virus infection. Using extensive immunohistochemistry, bone marrow transfers, and depletion studies, we identified neutrophils as the predominant pulmonary cellular source of the gelatinase matrix metalloprotease (MMP) 9, which is capable of digesting the extracellular matrix. Furthermore, infection of MMP9-deficient mice showed that MMP9 was functionally required for neutrophil migration and control of viral replication in the respiratory tract. Although MMP9 release was toll-like receptor (TLR) signaling-dependent, MyD88-mediated signals in non-hematopoietic cells, rather than neutrophil TLRs themselves, were important for neutrophil migration. These results were extended using multiplex analyses of inflammatory mediators to show that neutrophil chemotactic factor, CCL3, and TNFα were reduced in the Myd88 ^−/− airways. Furthermore, TNFα induced MMP9 secretion by neutrophils and blocking TNFα in vivo reduced neutrophil recruitment after infection. Innate recognition of influenza virus therefore provides the mechanisms to induce recruitment of neutrophils through chemokines and to enable their motility within the tissue via MMP9-mediated cleavage of the basement membrane. Our results demonstrate a previously unknown contribution of MMP9 to influenza virus pathogenesis by mediating excessive neutrophil migration into the respiratory tract in response to viral replication that could be exploited for therapeutic purposes.     

Group 1B phospholipase A₂ deficiency protects against diet-induced hyperlipidemia in mice

Excessive absorption of products of dietary fat digestion leads to type 2 diabetes and other obesity-related disorders. Mice deficient in the group 1B phospholipase A₂ (Pla2g1b), a gut digestive enzyme, are protected against diet-induced obesity and type 2 diabetes without displaying dietary lipid malabsorption. This study tested the hypothesis that inhibition of Pla2g1b protects against diet-induced hyperlipidemia. Results showed that the Pla2g1b^−/− mice had decreased plasma triglyceride and cholesterol levels compared with Pla2g1b^+/+ mice subsequent to feeding a high-fat, high-carbohydrate (hypercaloric) diet. These differences were evident before differences in body weight gains were observed. Injection of Poloxamer 407 to inhibit lipolysis revealed decreased VLDL production in Pla2g1b^−/− mice. Supplementation with lysophosphatidylcholine, the product of Pla2g1b hydrolysis, restored VLDL production rates in Pla2g1b^−/− mice and further elevated VLDL production in Pla2g1b^+/+ mice. The Pla2g1b^−/− mice also displayed decreased postprandial lipidemia compared with Pla2g1b^+/+ mice. These results show that, in addition to dietary fatty acids, gut-derived lysophospholipids derived from Pla2g1b hydrolysis of dietary and biliary phospholipids also promote hepatic VLDL production. Thus, the inhibition of lysophospholipid absorption via Pla2g1b inactivation may prove beneficial against diet-induced hyperlipidemia in addition to the protection against obesity and diabetes.     

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2^+/+, ACAT2^+/−, and ACAT2^−/− mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2^−/− mice was 16% compared with 46–47% in ACAT2^+/+ and ACAT2^+/− mice. Chylomicrons from ACAT2^+/+ and ACAT2^+/− mice carried ∼80% of total sterol mass as CE, whereas ACAT2^−/− chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2^−/− mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.     

Insufficient glucose supply is linked to hypothermia upon cold exposure in high-fat diet-fed mice lacking PEMT

Mice that lack phosphatidylethanolamine N-methyltransferase (Pemt^−/− mice) are protected from high-fat (HF) diet-induced obesity. HF-fed Pemt^−/− mice show higher oxygen consumption and heat production, indicating that more energy might be utilized for thermogenesis and might account for the resistance to diet-induced weight gain. To test this hypothesis, HF-fed Pemt^−/− and Pemt^+/+ mice were challenged with acute cold exposure at 4°C. Unexpectedly, HF-fed Pemt^−/− mice developed hypothermia within 3 h of cold exposure. In contrast, chow-fed Pemt^−/− mice, possessing similar body mass, maintained body temperature. Lack of PEMT did not impair the capacity for thermogenesis in skeletal muscle or brown adipose tissue. Plasma catecholamines were not altered by Pemt genotype, and stimulation of lipolysis was intact in brown and white adipose tissue of Pemt^−/− mice. HF-fed Pemt^−/− mice also developed higher systolic blood pressure, accompanied by reduced cardiac output. Choline supplementation reversed the cold-induced hypothermia in HF-fed Pemt^−/− mice with no effect on blood pressure. Plasma glucose levels were ∼50% lower in HF-fed Pemt^−/− mice compared with Pemt^+/+ mice. Choline supplementation normalized plasma hypoglycemia and the expression of proteins involved in gluconeogenesis. We propose that cold-induced hypothermia in HF-fed Pemt^−/− mice is linked to plasma hypoglycemia due to compromised hepatic glucose production.     

Heterozygosity for Fibrinogen Results in Efficient Resolution of Kidney Ischemia Reperfusion Injury

Fibrinogen (Fg) has been recognized to play a central role in coagulation, inflammation and tissue regeneration. Several studies have used Fg deficient mice (Fg^−/−) in comparison with heterozygous mice (Fg^+/−) to point the proinflammatory role of Fg in diverse pathological conditions and disease states. Although Fg^+/− mice are considered ‘normal’, plasma Fg is reduced to ∼75% of the normal circulating levels present in wild type mice (Fg^+/+). We report that this reduction in Fg protein production in the Fg^+/− mice is enough to protect them from kidney ischemia reperfusion injury (IRI) as assessed by tubular injury, kidney dysfunction, necrosis, apoptosis and inflammatory immune cell infiltration. Mechanistically, we observed binding of Fg to ICAM-1 in kidney tissues of Fg^+/+ mice at 24 h following IRI as compared to a complete absence of binding observed in the Fg^+/− and Fg^−/− mice. Raf-1 and ERK were highly activated as evident by significantly higher phosphorylation in the Fg^+/+ kidneys at 24 h following IRI as compared to Fg^+/− and Fg^−/− mice kidneys. On the other hand Cyclin D1 and pRb, indicating higher cell proliferation, were significantly increased in the Fg^+/− and Fg^−/− as compared to Fg^+/+ kidneys. These data suggest that Fg heterozygosity allows maintenance of a critical balance of Fg that enables regression of initial injury and promotes faster resolution of kidney damage.     

Calcineurin A‐β is required for hypertrophy but not matrix expansion in the diabetic kidney

Calcineurin is an important signalling protein that regulates a number of molecular and cellular processes. Previously, we found that inhibition of calcineurin with cyclosporine reduced renal hypertrophy and blocked glomerular matrix expansion in the diabetic kidney. Isoforms of the catalytic subunit of calcineurin are reported to have tissue specific expression and functions. In particular, the β isoform has been implicated in cardiac and skeletal muscle hypertrophy. Therefore, we examined the role of calcineurin β in diabetic renal hypertrophy and glomerular matrix expansion. Type I diabetes was induced in wild-type and β^−/− mice and then renal function, extracellular matrix expansion and hypertrophy were evaluated. The absence of β produced a significant decrease in total calcineurin activity in the inner medulla (IM) and reduced nuclear factor of activated T-cells (NFATc) activity. Loss of β did not alter diabetic renal dysfunction assessed by glomerular filtration rate, urine albumin excretion and blood urea nitrogen. Similarly, matrix expansion in the whole kidney and glomerulus was not different between diabetic wild-type and β^−/− mice. In contrast, whole kidney and glomerular hypertrophy were significantly reduced in diabetic β^−/− mice. Moreover, β^−/− renal fibroblasts demonstrated impaired phosphorylation of Erk1/Erk2, c-Jun N-terminal kinases (JNK) and mammalian target of rapamycin (mTOR) following stimulation with transforming growth factor-β and did not undergo hypertrophy with 48 hrs culture in high glucose. In conclusion, loss of the β isoform of calcineurin is sufficient to reproduce beneficial aspects of cyclosporine on diabetic renal hypertrophy but not matrix expansion. Therefore, while multiple signals appear to regulate matrix, calcineurin β appears to be a central mechanism involved in organ hypertrophy.     

The enteric nervous system of P2Y13 receptor null mice is resistant against high-fat-diet- and palmitic-acid-induced neuronal loss

Gastrointestinal symptoms have a major impact on the quality of life and are becoming more prevalent in the western population. The enteric nervous system (ENS) is pivotal in regulating gastrointestinal functions. Purinergic neurotransmission conveys a range of short and long-term cellular effects. This study investigated the role of the ADP-sensitive P2Y₁₃ receptor in lipid-induced enteric neuropathy. Littermate P2Y₁₃^+/+ and P2Y₁₃^−/− mice were fed with either a normal diet (ND) or high-fat diet (HFD) for 6 months. The intestines were analysed for morphological changes as well as neuronal numbers and relative numbers of vasoactive intestinal peptide (VIP)- and neuronal nitric oxide synthase (nNOS)-containing neurons. Primary cultures of myenteric neurons from the small intestine of P2Y₁₃^+/+ or P2Y₁₃^−/− mice were exposed to palmitic acid (PA), the P2Y₁₃ receptor agonist 2meSADP and the antagonist MRS2211. Neuronal survival and relative number of VIP-containing neurons were analysed. In P2Y₁₃^+/+, but not in P2Y₁₃^−/− mice, HFD caused a significant loss of myenteric neurons in both ileum and colon. In colon, the relative numbers of VIP-containing submucous neurons were significantly lower in the P2Y₁₃^−/− mice compared with P2Y₁₃^+/+ mice. The relative numbers of nNOS-containing submucous colonic neurons increased in P2Y₁₃^+/+ HFD mice. HFD also caused ileal mucosal thinning in P2Y₁₃^+/+ and P2Y₁₃^−/− mice, compared to ND fed mice. In vitro PA exposure caused loss of myenteric neurons from P2Y₁₃^+/+ mice while neurons from P2Y₁₃^−/− mice were unaffected. Presence of MRS2211 prevented PA-induced neuronal loss in cultures from P2Y₁₃^+/+ mice. 2meSADP caused no change in survival of cultured neurons. P2Y₁₃ receptor activation is of crucial importance in mediating the HFD- and PA-induced myenteric neuronal loss in mice. In addition, the results indicate a constitutive activation of enteric neuronal apoptosis by way of P2Y₁₃ receptor stimulation.     

Deletion of Nardilysin Prevents the Development of Steatohepatitis and Liver Fibrotic Changes

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1^+/+ and Nrd1^−/− mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1^+/+ and Nrd1^−/− mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1^−/− mice was lower than that in Nrd1^+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1^−/− mice than in Nrd1^+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1^+/+ and Nrd1^−/− mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1^−/− mice was significantly less than that in Nrd1^+/+ mice, indicating the decreased TNF-α shedding in Nrd1^−/− mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1^−/− mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.     

Neutrophil-Mediated Phagocytic Host Defense Defect in Myeloid Cftr-Inactivated Mice

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr−/−) mice and the non-inactivated control (Cftr^fl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr−/− lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.