Increasing evidence has shown that immune checkpoint molecules of the T-cell immunoglobulin and mucin domain (Tim) family are associated with diverse physiologic and pathologic processes. Previous... Show moreIncreasing evidence has shown that immune checkpoint molecules of the T-cell immunoglobulin and mucin domain (Tim) family are associated with diverse physiologic and pathologic processes. Previous studies of the role of Tim-1 in atherosclerosis using anti-Tim-1 antibodies have yielded contradictory results. We thus aimed to investigate atherosclerosis development in Tim-1 deficient mice.Mice with a specific loss of the Tim-1 mucin-domain (Tim-1Δmucin) and C57BL/6 (WT) mice received a single injection of a recombinant adeno-associated virus encoding murine Pcsk9 (rAAV2/8-D377Y-mPcsk9) and were fed a Western type diet for 13 weeks to introduce atherosclerosis.Tim-1Δmucin mice developed significantly larger lesions in the aortic root compared to WT mice, with significantly more macrophages and a trend towards a larger necrotic core. Furthermore, Tim-1Δmucin mice showed a significant loss of IL-10+ B cells and regulatory B cell subsets and increased pro-atherogenic splenic follicular B cells compared to WT mice. Moreover, Tim-1Δmucin mice displayed a dramatic reduction in Th2-associated immune response compared to controls but we did not observe any changes in humoral immunity.In summary, Tim-1Δmucin mice displayed a profound impairment in IL-10+ B cells and an imbalance in the Th1/Th2 ratio, which associated with exacerbated atherosclerosis. Show less
Mock, E.D.; Mustafa, M.; Gunduz-Cinar, O.; Cinar, R.; Petrie, G.N.; Kantae, V.; ... ; Stelt, M. van der 2020
N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis... Show moreN-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus–pituitary–adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior. Show less
Wilkerson, J.L.; Curry, Z.A.; Kinlow, P.D.; Mason, B.L.; Hsu, K.L.; Stelt, M. van der; ... ; Lichtman, A.H. 2018
A great need exists for the identification of new effective analgesics to treat sustained pain. However, most preclinical nociceptive assays measure behavioral responses evoked by noxious stimuli ... Show moreA great need exists for the identification of new effective analgesics to treat sustained pain. However, most preclinical nociceptive assays measure behavioral responses evoked by noxious stimuli (i.e., pain-stimulated behavior), which presents a challenge to distinguish between motor impairing and antinociceptive effects of drugs. Here, we demonstrate that chronic constriction injury of the sciatic nerve (CCI) elicits common pain-stimulated responses (i.e., mechanical allodynia and thermal hyperalgesia) as well as reduces marble burying/digging behaviors that occur during the early stages of the neuropathy and resolve within one week. Whereas drugs representing distinct classes of analgesics (i.e., morphine, valdecoxib, and gabapentin) reversed both CCI-induced and CCI-depressed nociceptive measures, diazepam lacked antinociceptive effects in all assays and the kappa opioid receptor agonist U69593 reversed pain-stimulated, but not pain-depressed behaviors. In addition, we tested drugs targeting distinct components of the endocannabinoid system, including agonists at cannabinoid receptors type 1 (CB1) and type 2 (CB2), as well as inhibitors of the endocannabinoid regulating enzymes fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL). Each of these drugs reversed all CCI-induced nociceptive measures, with the exception of the FAAH inhibitor that reversed pain-stimulated behaviors, only. These findings support the use of the mouse marble-burying assay as a model of pain-depressed behavior within the first week of sciatic nerve injury to examine candidate analgesics. These data also support existing preclinical research that cannabinoid receptor agonists and inhibitors of endocannabinoid regulating enzymes merit consideration for the treatment of pain. Show less
Soethoudt, M.; Grether, U.; Fingerle, J.; Grim, T.W.; Fezza, F.; Petrocellis, L. de; ... ; Stelt, M. van der 2017
Dendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including atherosclerosis. However, the molecular mechanisms by which DCs... Show moreDendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including atherosclerosis. However, the molecular mechanisms by which DCs mitigate or promote inflammatory pathogenesis are only partially understood. Previous studies have shown an important anti-inflammatory role for the transcription factor Krüppel-like factor 2 (KLF2) in regulating activation of various cell types that participate in atherosclerotic lesion development, including endothelial cells, macrophages, and T cells. We used a pan-DC, CD11c-specific cre-lox gene knockout mouse model to assess the role of KLF2 in DC activation, function, and control of inflammation in the context of hypercholesterolemia and atherosclerosis. We found that KLF2 deficiency enhanced surface expression of costimulatory molecules CD40 and CD86 in DCs and promoted increased T cell proliferation and apoptosis. Transplant of bone marrow from mice with KLF2-deficient DCs into Ldlr−/− mice aggravated atherosclerosis compared with control mice, most likely due to heightened vascular inflammation evidenced by increased DC presence within lesions, enhanced T cell activation and cytokine production, and increased cell death in atherosclerotic lesions. Taken together, these data indicate that KLF2 governs the degree of DC activation and hence the intensity of proatherogenic T cell responses. Show less
