The Activity of the Neutral Sphingomyelinase Is Important in T Cell Recruitment and Directional Migration

Abstract

Breakdown of sphingomyelin as catalyzed by the activity of sphingomyelinases profoundly affects biophysical properties of cellular membranes which is particularly important with regard to compartmentalization of surface receptors and their signaling relay. As it is activated both upon TCR ligation and co-stimulation in a spatiotemporally controlled manner, the neutral sphingomyelinase (NSM) has proven to be important in T cell activation, where it appears to play a particularly important role in cytoskeletal reorganization and cell polarization. Because these are important parameters in directional T cell migration and motility in tissues, we analyzed the role of the NSM in these processes. Pharmacological inhibition of NSM interfered with early lymph node homing of T cells in vivo indicating that the enzyme impacts on endothelial adhesion, transendothelial migration, sensing of chemokine gradients or, at a cellular level, acquisition of a polarized phenotype. NSM inhibition reduced adhesion of T cells to TNF-α/IFN-γ activated, but not resting endothelial cells, most likely via inhibiting high-affinity LFA-1 clustering. NSM activity proved to be highly important in directional T cell motility in response to SDF1-α, indicating that their ability to sense and translate chemokine gradients might be NSM dependent. In fact, pharmacological or genetic NSM ablation interfered with T cell polarization both at an overall morphological level and redistribution of CXCR4 and pERM proteins on endothelial cells or fibronectin, as well as with F-actin polymerization in response to SDF1-α stimulation, indicating that efficient directional perception and signaling relay depend on NSM activity. Altogether, these data support a central role of the NSM in T cell recruitment and migration both under homeostatic and inflamed conditions by regulating polarized redistribution of receptors and their coupling to the cytoskeleton.

Neutral Sphingomyelinase-2 (NSM 2) Controls T Cell Metabolic Homeostasis and Reprogramming During Activation

“…The nocodazole treatment did not affect total ATP levels in CTRL and NSM KD cells ( Figure 1C ). Next, we examined mitochondrial ATP transport channel functionality by measuring ATP levels in mitochondria isolated from primary T cells pretreated with a NSM2-specific inhibitor ES048 ( Collenburg et al, 2017 ) which reduced NSM2 activity about 40% ( Supplementary Figure S1B ) or nocodazole. Inhibition of NSM2 as well as cytosolic tubulin elevation significantly enhanced mitochondria ATP content, indicating that both NSM2 and tubulin regulate ATP accumulation in T-cell mitochondria ( Figure 1D ).…”

“…Neutral sphingomyelinase (NSM) pharmacological inhibition was achieved by incubating primary CD4 + T-cells with 1 μM GW4869 (SMPD2- and SMPD3-specific inhibitor, kindly provided by Cristoph Arenz) or with 1.5 μM ES048 ( Collenburg et al, 2017 ) 2 h before or 2 h after α-CD3/CD28 stimulation. Nucleofection of human T cells was performed according to the manufacturer’s protocol (Lonza).…”

“…When compared to those measured in brain or liver, expression levels of NSM2 in T-cells are rather low ( Hofmann et al, 2000 ). Nevertheless, NSM2 activity proved to have a substantial impact on T-cell cytoskeleton dynamics, morphological polarization, and migration toward chemotactic signals, and, most importantly, for the optimal performance of TCR signaling ( Gassert et al, 2009 ; Collenburg et al, 2017 ; Börtlein et al, 2018 ). Our more recent studies identified the TCR/NSM2/PKCζ pathway as crucial for TCR signal amplification and sustainment especially at low doses of stimulation ( Börtlein et al, 2018 ).…”

The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling

“…A central role of PKCζ in formation of the polarity complex in epithelial cells is well documented ( 68 , 69 ). This atypical PKC also regulates chemokine-induced integrin activation and polarization in effector T cells ( 70 ), and we have recently shown that SDF-1α driven physical T cell polarization on fibronectin and inflamed endothelial cells is also NSM dependent ( 71 ). The ability of PKCζ to bind ceramide and its role in cilia formation in stem cells and neural progenitors and in polarization of primitive ectodermal cells has been previously established ( 54 , 56 , 72 , 73 ).…”

Ceramide is a Potential Activator of Immune Responses Against Tumors

“…Intriguingly, recruitment and migration of T cells regulated by ceramide/sphingomyelinases under both homeostatic and inflammtory conditions is recently noted. 8 The promotion of immune cells infiltration and chemotaxis into tumor will shed light on efficacious treatment and perpetual eradication of tumors. At this end, ceramide-driven immune cell migration would be another novel topic of immune therapies for human tumors.…”