Background: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain—an intracellular modulatory protease—interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. Methods: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. Results: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 subsets. This occurs via the proteolytic degradation of MEKK1 and the subsequent inhibition of TGF (transforming growth factor)-β1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-β type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. Conclusions: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.
Tissue-resident mast cells (MCs) have important roles in IgE-associated and -independent allergic reactions. Although microenvironmental alterations in MC phenotypes affect the susceptibility to allergy, understanding of the regulation of MC maturation is still incomplete. We previously reported that group III secreted phospholipase A2 (sPLA2-III) released from immature MCs is functionally coupled with lipocalin-type prostaglandin D2 (PGD2) synthase in neighboring fibroblasts to supply a microenvironmental pool of PGD2, which in turn acts on the PGD2 receptor DP1 on MCs to promote their proper maturation. In the present study, we reevaluated the role of sPLA2-III in MCs using a newly generated MC-specific Pla2g3-deficient mouse strain. Mice lacking sPLA2-III specifically in MCs, like those lacking the enzyme in all tissues, had immature MCs and displayed reduced local and systemic anaphylactic responses. Furthermore, MC-specific Pla2g3-deficient mice, as well as MC-deficient KitW-sh mice reconstituted with MCs prepared from global Pla2g3-null mice, displayed a significant reduction in irritant contact dermatitis (ICD) and an aggravation of contact hypersensitivity (CHS). The increased CHS response by Pla2g3 deficiency depended at least partly on the reduced expression of hematopoietic PGD2 synthase and thereby reduced production of PGD2 due to immaturity of MCs. Overall, our present study has confirmed that MC-secreted sPLA2-III promotes MC maturation, thereby facilitating acute anaphylactic and ICD reactions and limiting delayed CHS response.
PNPLA8, one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. However, little is known about its role in brain development. Here, we report 12 individuals from 10 unrelated families with biallelic ultra-rare variants inPNPLA8presenting with a wide spectrum of clinical features ranging from developmental and epileptic-dyskinetic encephalopathy (DEDE) to progressive movement disorders and no phenotype depending on the variants and their positions. Complete loss of PNPLA8 was associated with the severe end of the spectrum, showing DEDE manifestations and congenital or progressive microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 reduced the number of basal radial glial cells (bRGCs) and upper-layer neurons. By spatial transcriptomic analysis targeting apical radial glial cells (aRGCs), we found the downregulation of bRGC-related gene sets in patient-derived cerebral organoids. Lipidomic analysis revealed a decrease in the amount of lysophosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid, indicative of the disturbed phospholipid metabolism inPNPLA8knockout neural progenitor cells. Our data suggest that PNPLA8 has a critical role in the bRGC-mediated expansion of the developing human cortex by regulating the fate commitment of aRGCs.
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