Novel Mechanism That Regulates Innate immunity

Abstract

BackgroundWe have recently identified Niemann‐Pick type C2 (NPC2) protein as important regulator of somatic cell plasticity and inflammation (1,2). The latter unfolds in the setting of NPC2‐deficiency and is characterized by significant increase in production and secretion of inflammatory mediators and immune modulators including IL‐1β, IL‐6 and prostaglandin E2 (1–3). Here we probed involvement of NPC2 in regulation of innate immunity by examining its role in macrophage differentiation, polarization and in the response of human fibroblasts to the immunogenic stimuli.MethodsNPC2‐null human skin fibroblasts were derived from patients with NPC2 disease (2). The human monocytic cell line THP‐1 and human cryopreserved peripheral blood mononuclear cells (PBMC) were obtained respectively from ATCC and C.T.L. sources. THP‐1 monocyte‐derived macrophages were obtained through PMA stimulation (4). PBMC macrophage differentiation was performed according to C.T.L. protocol. PBMC macrophage polarization was achieved according to (5). Real‐time RT‐PCR analysis was conducted as described (1).ResultsImmunostaining of the lesional area of human atherosclerotic aorta (provided by Dr. N. Weintraub) revealed increased expression of NPC2 protein and its strong association with inflammatory cells. Real‐time RT‐PCR data demonstrated robust and gradual upregulation of NPC2 expression during macrophage differentiation reaching 4.2±0.3 at 24 h and 7.1±1.0 fold at 48 h after PMA stimulation. Silencing NPC2 gene in THP‐1 monocytes with shRNA triggered their spontaneous differentiation into macrophages as evidenced by the upregulated expression of specific markers CD11b (3.2±0.6), MSR1 (2.0±0.05) and IL‐6 (62.6±5.0). NPC2 protein (gift from Dr. C. Heegaard), when it was added to M2 macrophage polarization medium at 6 or 60 nM, skewed macrophage polarization efficiently and dose‐dependently toward M1. Finally, NPC2‐null fibroblasts demonstrated dramatically muted response to IFN‐γ (~ 100‐fold) and to the TLR2, TLR3, TLR4 and MDA‐5 specific ligands (~ 15‐fold) despite significant (up to 5‐fold) increase in the TLRs gene and protein expression.ConclusionThis work identifies a novel, NPC2‐mediated multifaceted mechanism that regulates innate immunity at the levels of macrophage differentiation, polarization, and cell response to immunogenic stimuli.Support or Funding InformationNIH RO1AR049010 and American Heart Association Grant SDG 023017N