CD4 Knockout Mice Research Articles

Mo1775 High Expression Level of PPARγ in CD24 Knockout Mice and Gender Specific Metabolic Changes: A Model of Insulin-Sensitive Obesity

Mo1775 High Expression Level of PPARγ in CD24 Knockout Mice and Gender Specific Metabolic Changes: A Model of Insulin-Sensitive Obesity

Chronic activation with a staphylococcal superantigen drives the expansion of CD4, CD8 double negative T cells and promotes multiorgan inflammation mimicking systemic lupus erythematosus in HLA class II transgenic mice. (HUM7P.306)

Abstract CD4 and CD8 coreceptors play an important role in the development and maturation of αβ TCR+ T cells. However, T cells lacking CD4 and CD8 (double negative T or DNT cells) are present in the periphery and are significantly expanded in systemic lupus erythematosus (SLE) and Sjogren’s syndrome. The antigens that cause expansion of these DNT cells are unknown. As T cell activation by superantigens does not require coreceptors, we hypothesized that superantigens could activate DNT cells and contribute to autoimmunity. HLA-DQ8 and HLA-DR3 transgenic mice lacking both CD4 and CD8 were generated by intercrossing CD4 and CD8 single knockout mice. The CD4.CD8 double knockout (DKO) mice harbored a diverse repertoire of DNT cells in the periphery, albeit at reduced numbers compared to wild type (WT) mice. DNT cells from DKO mice responded robustly to the superantigen, staphylococcal enterotoxin B, but not to HLA restricted conventional antigens. Implantation of mini-osmotic pumps delivering 10 or 50 μg of SEB continuously over 7 or 28 days induced a lupus-like disease in WT as well as DKO DR3 and DQ8 mice. However, the immunopathology was significantly more pronounced in DKO mice. Immunostaining revealed extensive infiltration of multiple organs including lungs, liver and kidneys with CD3+ T cells in DKO mice. Overall, our study unraveled the role of CD4 and CD8 coreceptors in T cell development/function and the role of superantigens and DNT cells in the pathogenesis of lupus.

Reply to “CD8 + T Cells Are Essential for Controlling Acute Friend Virus Infection in C57BL/6 Mice”

Reply to “CD8 ⁺ T Cells Are Essential for Controlling Acute Friend Virus Infection in C57BL/6 Mice”

The Requirement of CD8+ T Cells To Initiate and Augment Acute Cardiac Inflammatory Response to High Blood Pressure

Macrophage infiltration and activation in myocardium are hallmarks of acute cardiac inflammatory response to high blood pressure. However, the underlying mechanisms remain elusive. In this article, we report that CD8(+) T cells are required for cardiac recruitment and activation of macrophages. First, mice with CD8 gene-targeted (CD8 knockout) or CD8(+) T cells depleted by Ab showed significantly reduced cardiac inflammatory response to the elevation of blood pressure after angiotensin II (Ang II) infusion, whereas CD8 knockout mice reconstituted with CD8(+) T cells restored the sensitivity to Ang II. More importantly, CD8(+) T cells were required for macrophage infiltration in myocardium and subsequent activation to express proinflammatory cytokines and chemokines. Furthermore, macrophage activation required direct contact with activated CD8(+) T cells, but with TCR dispensable. TCR-independent activation of macrophages was further confirmed in MHC class I-restricted OVA-specific TCR transgenic mice, which showed a CD8(+) T cell activation and cardiac proinflammatory response to Ang II similar to that of wild-type mice. Finally, only myocardium-infiltrated, but not peripheral, CD8(+) T cells were specifically activated by Ang II, possibly by the cardiac IFN-γ that drove IFN-γR(+) CD8(+) T cell infiltration and activation. Thus, this work identified a TCR-independent innate nature of CD8(+) T cells that was critical in initiating the sterile immune response to acute elevation of blood pressure.

CD73 Expression Is Dynamically Regulated in the Germinal Center and Bone Marrow Plasma Cells Are Diminished in Its Absence

CD73 catalyzes the conversion of extracellular nucleosides to adenosine, modulating inflammatory and T cell responses. Elevated expression of CD73 marks subpopulations of murine memory B cells (MBC), but its role in memory development or function is unknown. Here, we demonstrate that CD73 is progressively upregulated on germinal center (GC) B cells following immunization, is expressed at even higher levels among T follicular helper cells, but is absent among plasma cells (PC) and plasmablasts (PB). We analyzed the T-dependent B cell response in CD73 knockout mice (CD73KO). During the early response, CD73KO and wild type (WT) mice formed GCs, MBCs and splenic PBs and PCs similarly, and MBCs functioned similarly in the early secondary response. Late in the primary response, however, bone marrow (BM) PCs were markedly decreased in CD73KO animals. Tracking this phenotype, we found that CD73 expression was required on BM-derived cells for optimal BM PC responses. However, deletion of CD73 from either B or T lymphocytes alone did not recapitulate the phenotype. This suggests that CD73 expression is sufficient on either cell type, consistent with its function as an ectoenzyme. Together, these findings suggest that CD73-dependent adenosine signaling is prominent in the mature GC and required for establishment of the long-lived PC compartment, thus identifying a novel role for CD73 in humoral immunity.

CD24 knockout prevents colorectal cancer in chemically induced colon carcinogenesis and in APCMin/CD24 double knockout transgenic mice

Increased expression of CD24 is seen in a large variety of solid tumors, including up to 90% of gastrointestinal (GI) tumors. Stable derivatives of SW480 colorectal cancer (CRC) cells that overexpress CD24 proliferate faster, and increase cell motility, saturation density, plating efficiency, and growth in soft agar. They also produce larger tumors in nude mice as compared to the parental SW480 cells. Most significantly, even depletion of one copy of the CD24 allele in the APC(Min/+) mice of a transgenic mouse model led to a dramatic reduction in tumor burden in all sections of the small intestine. Homozygous deletion of both CD24 alleles resulted in complete abolishment of tumor formation. Moreover, CD24 knockout mice exhibited resistance to chemically induced inflammation-associated CRC. Finally, a new signal transduction pathway is suggested: namely, CD24 expression downstream to COX2 and PGE2 synthesis, which is directly regulated by β-catenin. CD24 is shown in vitro and in vivo as being an important oncogene in the gut, and one that plays a critical role in the initiation and progression of carcinogenesis.

Comprehensive Behavioral Analysis of Cluster of Differentiation 47 Knockout Mice

Cluster of differentiation 47 (CD47) is a member of the immunoglobulin superfamily which functions as a ligand for the extracellular region of signal regulatory protein α (SIRPα), a protein which is abundantly expressed in the brain. Previous studies, including ours, have demonstrated that both CD47 and SIRPα fulfill various functions in the central nervous system (CNS), such as the modulation of synaptic transmission and neuronal cell survival. We previously reported that CD47 is involved in the regulation of depression-like behavior of mice in the forced swim test through its modulation of tyrosine phosphorylation of SIRPα. However, other potential behavioral functions of CD47 remain largely unknown. In this study, in an effort to further investigate functional roles of CD47 in the CNS, CD47 knockout (KO) mice and their wild-type littermates were subjected to a battery of behavioral tests. CD47 KO mice displayed decreased prepulse inhibition, while the startle response did not differ between genotypes. The mutants exhibited slightly but significantly decreased sociability and social novelty preference in Crawley’s three-chamber social approach test, whereas in social interaction tests in which experimental and stimulus mice have direct contact with each other in a freely moving setting in a novel environment or home cage, there were no significant differences between the genotypes. While previous studies suggested that CD47 regulates fear memory in the inhibitory avoidance test in rodents, our CD47 KO mice exhibited normal fear and spatial memory in the fear conditioning and the Barnes maze tests, respectively. These findings suggest that CD47 is potentially involved in the regulation of sensorimotor gating and social behavior in mice.

Chapter Seven - MHC Class I Recognition by Monocyte-/Macrophage-Specific Receptors

The most important transplantation antigens in the discrimination between "self" and "nonself" are encoded by genes in the major histocompatibility complex (MHC) locus (H-2 in mice). It has been assumed that T lymphocytes are the effector cells for allograft rejection, as athymic nude rodents fail to reject allografts. In 1988, we i.p. transplanted Meth A (H-2D(d)K(d)) tumor cells into C57BL/6 (H-2D(b)K(b)) mice and found macrophages to be cytotoxic against the allografts. In 1996, several groups using CD4 or CD8 knockout mice reported that non-T cells were the effector cells for the rejection of skin or organ allografts. In 1998, we ascertained that macrophages were the effector cells of skin allograft rejection. Recently, we isolated cDNA clones encoding monocyte/macrophage MHC receptors (MMRs) for H-2D(d) and H-2K(d); established H-2D(d)- and/or H-2K(d)-transgenic mice and lymphoma cells; and found, using MMR-deficient mice, that MMR and T-cell receptor were essential for the rejection of transgenic skin and lymphoma, respectively.

Phenotypic Identification of Spinal Cord-Infiltrating CD4+ T Lymphocytes in a Murine Model of Neuropathic Pain.

BackgroundNeuropathic pain is one of the most devastating kinds of chronic pain. Neuroinflammation has been shown to contribute to the development of neuropathic pain. We have previously demonstrated that lumbar spinal cord-infiltrating CD4+ T lymphocytes contribute to the maintenance of mechanical hypersensitivity in spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we further examined the phenotype of the CD4+ T lymphocytes involved in the maintenance of neuropathic pain-like behavior via intracellular flow cytometric analysis and explored potential interactions between infiltrating CD4+ T lymphocytes and spinal cord glial cells.ResultsWe consistently observed significantly higher numbers of T-Bet+, IFN-γ+, TNF-α+, and GM-CSF+, but not GATA3+ or IL-4+, lumbar spinal cord-infiltrating CD4+ T lymphocytes in the L5Tx group compared to the sham group at day 7 post-L5Tx. This suggests that the infiltrating CD4+ T lymphocytes expressed a pro-inflammatory type 1 phenotype (Th1). Despite the observation of CD4+ CD40 ligand (CD154)+ T lymphocytes in the lumbar spinal cord post-L5Tx, CD154 knockout (KO) mice did not display significant changes in L5Tx-induced mechanical hypersensitivity, indicating that T lymphocyte-microglial interaction through the CD154-CD40 pathway is not necessary for L5Tx-induced hypersensitivity. In addition, spinal cord astrocytic activation, represented by glial fibillary acidic protein (GFAP) expression, was significantly lower in CD4 KO mice compared to wild type (WT) mice at day 14 post-L5Tx, suggesting the involvement of astrocytes in the pronociceptive effects mediated by infiltrating CD4+ T lymphocytes.ConclusionsIn all, these data indicate that the maintenance of L5Tx-induced neuropathic pain is mostly mediated by Th1 cells in a CD154-independent manner via a mechanism that could involve multiple Th1 cytokines and astrocytic activation.

Rolofylline, an adenosine A1 receptor antagonist, inhibits osteoclast differentiation as an inverse agonist

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5'-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors (A1 R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1 R agonists neither affect basal osteoclastogenesis nor do they reverse A1 R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1 R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1 R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation. Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa. Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC50 = 20-70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold. Based on these findings, we hypothesize that the A1 R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A1 R antagonists act as inverse agonists to release A1 R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A1 R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.

Low-Bone-Mass Phenotype of Deficient Mice for the Cluster of Differentiation 36 (CD36)

Bone tissue is continuously remodeled by bone cells and maintenance of its mass relies on the balance between the processes of resorption and formation. We have reported the expression of numerous scavenger receptors, namely scavenger receptor (SR) class B type I and II (SR-BI and SR-BII), and CD36, in bone-forming osteoblasts but their physiological roles in bone metabolism are still unknown. To unravel the role of CD36 in bone metabolism, we determined the bone phenotype of CD36 knockout (CD36KO) mice and characterized the cell functions of osteoblasts lacking CD36. Weights of CD36KO mice were significantly lower than corresponding wild-type (WT) mice, yet no significant difference was found in femoral nor tibial length between CD36KO and WT mice. Analysis of bone architecture by micro-computed tomography revealed a low bone mass phenotype in CD36KO mice of both genders. Femoral trabecular bone from 1 to 6 month-old CD36KO mice showed lower bone volume, higher trabecular separation and reduced trabeculae number compared to WT mice; similar alterations were noticed for lumbar vertebrae. Plasma levels of osteocalcin (OCN) and N-terminal propeptide of type I procollagen (PINP), two known markers of bone formation, were significantly lower in CD36KO mice than in WT mice, whereas plasma levels of bone resorption markers were similar. Accordingly, histology highlighted lower osteoblast perimeter and reduced bone formation rate. In vitro functional characterization of bone marrow stromal cells and osteoblasts isolated from CD36KO mice showed reduced cell culture expansion and survival, lower gene expression of osteoblastic Runt-related transcription factor 2 (Runx2) and osterix (Osx), as well as bone sialoprotein (BSP) and osteocalcin (OCN). Our results indicate that CD36 is mandatory for adequate bone metabolism, playing a role in osteoblast functions ensuring adequate bone formation.

Thrombospondin-1/CD36 pathway contributes to bone marrow-derived angiogenic cell dysfunction in type 1 diabetes via Sonic hedgehog pathway suppression

Refractory wounds in diabetic patients present a significant clinical problem. Sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes. Regulation of SHH in wound healing is poorly understood. We hypothesize that thrombospondin-1 (TSP-1), through its receptor CD36, contributes to the SHH signaling defect in bone marrow-derived angiogenic cells (BMACs) in type 1 diabetic mice. Isolated BMACs from TSP-1-knockout mice demonstrated improved tube formation, migration, and adhesion in parallel with active SHH signaling. BMACs from STZ-induced type 1 diabetic mice showed significantly impaired Matrigel tube formation (n = 5; P < 0.05 vs. control), which was rescued by TSP-1 depletion (n = 5; P < 0.05 STZ-TSP-1(-/-) vs. STZ-WT) or exogenous SHH (20 mg/l, 24 h, n = 4; P < 0.05 vs. STZ-control). The expression of CD36 was elevated in BMACs from STZ mice (n = 4; P < 0.05). SHH signaling was significantly higher in BMACs from TSP-1(-/-) mice and TSP-1 receptor CD36-knockout mice (n = 6; P < 0.05 vs. WT) but not CD47-knockout mice (n = 3; P > 0.05 vs. WT). The impairment of recombinant human TSP-1 (2.2 nM, 24 h) on BMAC Matrigel tube formation was delayed significantly by CD36 deletion (n = 5; P < 0.05). CD36(-/-) BMACs demonstrated better tube formation under both normal and diabetic conditions with active SHH signaling (n = 4; P < 0.05 vs. WT BMACs). In conclusion, The TSP-1/CD36 pathway contributes to the SHH signaling defect, resulting in BMAC dysfunction in type 1 diabetic mice.

English

We have previously shown that CD36 is a membrane protein that facilitates long chain fatty acid (FA) transport by muscle tissues. We also documented the significant impact of muscle CD36 expression on heart function, skeletal muscle insulin sensitivity as well as on overall metabolism. To identify a comprehensive set of genes that are differentially regulated by CD36 expression in the heart, we used two microarray technologies (Affymetrix and Agilent) to compare gene expression in heart tissues from CD36 KnocK-Out (KO-CD36) versus wild type (WT-CD36) mice. The obtained results using the two technologies were similar with around 35 genes differentially expressed using both technologies. Absence of CD36 led to down-regulation of the expression of three groups of genes involved in pathways of FA metabolism, angiogenesis/apoptosis and structure. These data are consistent with the fact that the CD36 protein binds FA and thrombospondin 1 invoved respectively in lipid metabolism and anti-angiogenic activities. In conclusion, our findings led to validate our data analysis workflow and identify specific pathways, possibly underlying the phenotypic abnormalities in CD36 Knock -Out hearts.

CD38 in the nucleus accumbens and oxytocin are related to paternal behavior in mice

BackgroundMammalian sires participate in infant care. We previously demonstrated that sires of a strain of nonmonogamous laboratory mice initiate parental retrieval behavior in response to olfactory and auditory signals from the dam during isolation in a new environment. This behavior is rapidly lost in the absence of such signals when the sires are caged alone. The neural circuitry and hormones that control paternal behavior are not well-understood. CD38, a membrane glycoprotein, catalyzes synthesis of cyclic ADP-ribose and facilitates oxytocin (OT) secretion due to cyclic ADP-ribose-dependent increases in cytosolic free calcium concentrations in oxytocinergic neurons in the hypothalamus. In this paper, we studied CD38 in the nucleus accumbens (NAcc) and the role of OT on paternal pup retrieval behavior using CD38 knockout (CD38−/−) mice of the ICR strain.ResultsCD38−/− sires failed to retrieve when they were reunited with their pups after isolation together with the mate dams, but not with pup, in a novel cage for 10 min. CD38−/− sires treated with a single subcutaneous injection of OT exhibited recovery in the retrieval events when caged with CD38−/− dams treated with OT. We introduced human CD38 in the NAcc of CD38−/− sires using a lentiviral infection technique and examined the effects of local expression of CD38. Pairs of knockout dams treated with OT and sires expressing CD38 in the NAcc showed more retrieval (83% of wild-type sire levels). Complete recovery of retrieval was obtained in sires with the expression of CD38 in the NAcc in combination with OT administration. Other paternal behaviors, including pup grooming, crouching and huddling, were also more common in CD38−/− sires with CD38 expression in the NAcc compared with those in CD38−/− sires without CD38 expression in the NAcc.ConclusionsCD38 in the NAcc and OT are critical in paternal behavior.

Targeting antigens to CD180 rapidly induces antigen-specific IgG, affinity maturation, and immunological memory

Antigen (Ag) targeting is an efficient way to induce immune responses. Ag is usually coupled to an antibody (Ab) specific for a receptor expressed on dendritic cells (DCs), and then the Ag-anti-receptor is inoculated with an adjuvant. Here we report that targeting Ag to a receptor expressed on both B cells and DCs, the TLR orphan receptor CD180, in the absence of adjuvant rapidly induced IgG responses that were stronger than those induced by Ag in alum. Ag conjugated to anti-CD180 (Ag-αCD180) induced affinity maturation and Ab responses that were partially T cell independent, as Ag-specific IgGs were generated in CD40- and T cell-deficient mice. After preimmunization with Ag-αCD180 and boosting with soluble Ag, both WT and CD40 knockout (KO) mice rapidly produced Ag-specific IgG-forming cells, demonstrating that Ag-anti-CD180 induces immunological memory. The potent adjuvant effect of Ag-αCD180 required Ag to be coupled to anti-CD180 and the responsive B cells to express both CD180 and an Ag-specific B cell receptor. Surprisingly, CD180 Ag targeting also induced IgG Abs in BAFF-R KO mice lacking mature B cells and in mice deficient in interferon signaling. Targeting Ag to CD180 may be useful for therapeutic vaccination and for vaccinating the immune compromised.

CD38 Controls the Innate Immune Response against Listeria monocytogenes

CD38, adenosine-5'-diphosphate-ribosyl cyclase 1, is a multifunctional enzyme, expressed on a wide variety of cell types. CD38 has been assigned diverse functions, including generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Using a murine Listeria monocytogenes infection model, we found that CD38 knockout (KO) mice were highly susceptible to infection. Enhanced susceptibility was already evident within 3 days of infection, suggesting a function of CD38 in the innate immune response. CD38 was expressed on neutrophils and inflammatory monocytes, and especially inflammatory monocytes further upregulated CD38 during infection. Absence of CD38 caused alterations of the migration pattern of both cell types to sites of infection. We observed impaired accumulation of cells in the spleen but surprisingly similar or even higher accumulation of cells in the liver. CD38 KO and wild-type mice showed similar changes in the composition of neutrophils and inflammatory monocytes in blood and bone marrow, indicating that mobilization of these cells from the bone marrow was CD38 independent. In vitro, macrophages of CD38 KO mice were less efficient in uptake of listeria but still able to kill the bacteria. Dendritic cells also displayed enhanced CD38 expression following infection. However, absence of CD38 did not impair the capacity of mice to prime CD8(+) T cells against L. monocytogenes, and CD38 KO mice could efficiently control secondary listeria infection. In conclusion, our results demonstrate an essential role for CD38 in the innate immune response against L. monocytogenes.

Cardiomyocyte-specific ablation of CD36 improves post-ischemic functional recovery

Although pre-clinical evidence has suggested that partial inhibition of myocardial fatty acid oxidation (FAO) and subsequent switch to greater glucose oxidation for ATP production can prevent ischemia/reperfusion injury, controversy about this approach persists. For example, mice with germline deletion of the FA transporter CD36, exhibited either impaired or unchanged post-ischemic functional recovery despite a 40–60% reduction in FAO rates. Because there are limitations to cardiac studies utilizing whole body CD36 knockout (totalCD36KO) mice, we have now generated an inducible and cardiomyocyte-specific CD36 KO (icCD36KO) mouse to better address the role of cardiomyocyte CD36 and its regulation of FAO and post-ischemic functional recovery. Four to six weeks following CD36 ablation, hearts from icCD36KO mice had significantly decreased FA uptake compared to controls, which was paralleled by significant reductions in intramyocardial triacylglycerol content. Analysis of cardiac energy metabolism using ex vivo working heart perfusions showed that reduced FAO rates were compensated by enhanced glucose oxidation in the hearts from icCD36KO mice. In contrast to the totalCD36KO mice, hearts from icCD36KO mice exhibited significantly improved functional recovery following ischemia/reperfusion (18min of global no-flow ischemia followed by 40min of aerobic reperfusion). This improved recovery was associated with lower calculated proton production prior to and following ischemia compared to controls. Moreover, the amount of ATP generated relative to cardiac work was significantly lower in the hearts from icCD36KO mice compared to controls, indicating significantly increased cardiac efficiency in the hearts from icCD36KO mice. These data provide genetic evidence that reduced FAO as a result of diminished CD36-mediated FA uptake improves post-ischemic cardiac efficiency and functional recovery. As such, targeting cardiomyocyte FA uptake and FAO via inhibition of CD36 in the adult myocardium may provide therapeutic benefit during ischemia–reperfusion.

Inactivation of the immune receptor CD40 attenuates the development of cardiac hypertrophy in angiotensin-II induced hypertensive heart disease

Emerging evidence indicates that inflammatory activation is a crucial pathway in disease progression during HF. The co-stimulatory dyad CD40-CD40L is involved in pro-inflammatory activation of a wide range of immune and non-immune cells and plasma levels of soluble CD40L in HF patients are raised. Here we investigated whether CD40-CD40L interactions attenuate cardiac hypertrophy in hypertensive heart disease. CD40 knockout and wild type C57Bl6-J mice were treated with angiotensin II (AngII, 2.5 mg/kg/day) for 4 weeks. AngII-induced hypertension led to a significant increase in cardiac mass in Wt mice (+31%, P<0.01), but not in CD40 knockout mice (+10%, N.S.). Comparable changes were observed in diastolic wall thickness (echocardiography) and cardiomyocyte cross sectional area. To determine which CD40-TRAF interactions were involved, the effect of mutations in the intracellular TRAF binding domains of CD40 was investigated. Relative to mice with intact downstream CD40 signaling, disruption of TRAF2/3/5, TRAF6 or both binding sites was associated with reduced cardiac hypertrophy (cardiac mass -24% for CD40-TΔ2, -28% for CD40-TΔ6 and -26% in CD40-TΔ2/6; p<0.05). Reduced hypertrophy was not associated with a decline in immune cell infiltration (CD45+ cells) or collagen content. Although the exact mechanism remains to be elucidated the present findings indicate a role for the CD40 receptor in the development of cardiac hypertrophy.

Role of CD73 in renal sympathetic neurotransmission in the mouse kidney

Adenosine formed during renal sympathetic nerve stimulation (RSNS) enhances, by activating A1 receptors, the postjunctional effects of released norepinephrine and participates in renal sympathetic neurotransmission. Because in many cell types CD73 (ecto-5′-nucleotidase) is important for the conversion of 5′-AMP to adenosine, we investigated whether CD73 is necessary for normal renal sympathetic neurotransmission. In isolated kidneys from CD73 wild-type mice (CD73+/+; n = 17) perfused at a constant rate with Tyrode's solution, RSNS increased perfusion pressure by 17 ± 4, 36 ± 8, and 44 ± 10 mm Hg at 3, 5, and 7 Hz, respectively. Similar responses were elicited from kidneys isolated from CD73 knockout mice (CD73−/−; n = 13; 28 ± 11, 43 ± 10, and 44 ± 10 mm Hg at 3, 5, and 7 Hz, respectively); and a high concentration (100 μmol/L) of α,β-methyleneadenosine 5′-diphosphate (CD73 inhibitor) did not alter responses to RSNS in C57BL/6 mouse kidneys (n = 5; 21 ± 5, 36 ± 8, and 43 ± 9 at 3, 5, and 7 Hz, respectively). Measurements of renal venous adenosine and inosine (adenosine metabolite) by liquid chromatography-tandem mass spectrometry demonstrated that the metabolism of exogenous 5′-AMP to adenosine and inosine was similar in CD73−/− versus CD73+/+ kidneys. A1 receptor mRNA expression was increased in CD73−/− kidneys, and 2-chloro-N6-cyclopentyladenosine (0.1 μmol/L; A1 receptor agonist) enhanced renovascular responses to norepinephrine more in CD73−/− versus CD73+/+ kidneys. We conclude that CD73 is not essential for renal sympathetic neurotransmission because in the absence of renal CD73 other enzymes metabolize 5′-AMP to adenosine and because of compensatory upregulation of postjunctional coincident signaling between norepinephrine and adenosine.

FAT/CD36: A Major Regulator of Neuronal Fatty Acid Sensing and Energy Homeostasis in Rats and Mice

Hypothalamic “metabolic-sensing” neurons sense glucose and fatty acids (FAs) and play an integral role in the regulation of glucose, energy homeostasis, and the development of obesity and diabetes. Using pharmacologic agents, we previously found that ∼50% of these neurons responded to oleic acid (OA) by using the FA translocator/receptor FAT/CD36 (CD36). For further elucidation of the role of CD36 in neuronal FA sensing, ventromedial hypothalamus (VMH) CD36 was depleted using adeno-associated viral (AAV) vector expressing CD36 short hairpin RNA (shRNA) in rats. Whereas their neuronal glucosensing was unaffected by CD36 depletion, the percent of neurons that responded to OA was decreased specifically in glucosensing neurons. A similar effect was seen in total-body CD36-knockout mice. Next, weanling rats were injected in the VMH with CD36 AAV shRNA. Despite significant VMH CD36 depletion, there was no effect on food intake, body weight gain, or total carcass adiposity on chow or 45% fat diets. However, VMH CD36–depleted rats did have increased plasma leptin and subcutaneous fat deposition and markedly abnormal glucose tolerance. These results demonstrate that CD36 is a critical factor in both VMH neuronal FA sensing and the regulation of energy and glucose homeostasis.