Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses

Acid sphingomyelinase (ASM), a lipid hydrolase enzyme, has the potential to modulate various cellular activation responses via the generation of ceramide and by interaction with cellular receptors. We have hypothesized that ASM modulates CD4⁺ T-cell receptor activation and impacts immune responses. We first observed interactions of ASM with the intracellular domains of both CD3 and CD28. ASM further mediates T-cell proliferation after anti-CD3/CD28 antibody stimulation and alters CD4⁺ T-cell activation signals by generating ceramide. We noted that various pharmacological inhibitors of ASM or knockdown of ASM using small hairpin RNA inhibit CD3/CD28-mediated CD4⁺ T-cell proliferation and activation. Furthermore, such blockade of ASM bioactivity by biochemical inhibitors and/or molecular-targeted knockdown of ASM broadly abrogate T-helper cell responses. In conclusion, we detail immune, pivotal roles of ASM in adaptive immune T-cell responses, and propose that these pathways might provide novel targets for the therapy of autoimmune and inflammatory diseases.Acid sphingomyelinase (ASM), a lipid hydrolase enzyme localized to lysosomes and cell membranes, converts sphingomyelin to ceramide,¹ an important lipid messenger mediating cell signaling.^{2, 3} Through the generation of ceramide, ASM appears to have an important role in regulating cell differentiation, proliferation, and apoptosis.^{1, 4} Abnormalities in ASM bioactivity result in multiple system disorders. As an example, patients with Niemann–Pick disease, who have mutations in the ASM gene, exhibit neurological symptoms at early age, and develop visceral organ abnormalities in later life.⁴ Patients with Niemann–Pick disease are at risk of infections,⁵ as can be modeled in ASM-deficient mice.^{6, 7} This phenotype has been attributed to phagocyte dysfunction.⁸ Recently, however, ASM function has also been described and noted in various other non-phagocytic immune cells, for example, regulating cytotoxic granule secretion by CD8⁺ T cells.⁹ASM has been reported to modulate T-cell receptor (TCR) signaling initiated by TNF,¹⁰ mediate CD28 signals,¹¹ and induce or rescue CD4⁺ T cells from apoptosis under certain circumstances.^{12, 13} By generating ceramide, ASM serves as a regulator of intracellular downstream signaling. However, the exact manner whereby ASM participates in TCR/CD3 or/and CD28 signaling remains controversial.^{10, 11, 14} Furthermore, the molecular mechanisms as to how ASM regulates CD4⁺ T-cell activation are still largely unexplored.Adaptive immune responses are important in the maintenance of human immune homeostasis. Imbalances in T-helper cell (Th) responses associated with aberrant CD4⁺ T-cell activation contribute to the development of inflammation as in human autoimmune diseases.^{15, 16} It remains unclear whether or how ASM might dictate Th responses during the progression of inflammatory diseases.In the present study, we confirm that ASM interacts with CD3 and CD28, and mediates intracellular signals that control CD4⁺ T-cell activation. ASM inhibition either by pharmacological inhibitors of ASM or knockdown of ASM results in decreased ceramide production. This leads to non-responsiveness of CD4⁺ T-cell to CD3/CD28 engagement, and causes globally diminished Th responses. These data suggest the pivotal role of ASM in CD3/CD28 intracellular signaling and adaptive immune responses, and also provide a potential target for the therapy of immune disease.