Systematic Evaluation of the Cellular Innate Immune Response During the Process of Human Atherosclerosis

Abstract

BackgroundThe concept of innate immunity is well recognized within the spectrum of atherosclerosis, which is primarily dictated by macrophages. Although current insights to this process are largely based on murine models, there are fundamental differences in the atherosclerotic microenvironment and associated inflammatory response relative to humans. In this light, we characterized the cellular aspects of innate immune response in normal, nonprogressive, and progressive human atherosclerotic plaques.Methods and ResultsA systematic analysis of innate immune response was performed on 110 well‐characterized human perirenal aortic plaques with immunostaining for specific macrophage subtypes (M1 and M2 lineage) and their activation markers, neopterin and human leukocyte antigen–antigen D related (HLA‐DR), together with dendritic cells (DCs), natural killer (NK) cells, mast cells, neutrophils, and eosinophils. Normal aortae were devoid of low‐density lipoprotein, macrophages, DCs, NK cells, mast cells, eosinophils, and neutrophils. Early, atherosclerotic lesions exhibited heterogeneous populations of (CD68+) macrophages, whereby 25% were double positive “M1” (CD68+/ inducible nitric oxide synthase [iNOS]+/CD163−), 13% “M2” double positive (CD68+/iNOS −/CD163+), and 17% triple positive for (M1) iNOS (M2)/CD163 and CD68, with the remaining (≈40%) only stained for CD68. Progressive fibroatheromatous lesions, including vulnerable plaques, showed increasing numbers of NK cells and fascin‐positive cells mainly localized to the media and adventitia whereas the M1/M2 ratio and level of macrophage activation (HLA‐DR and neopterin) remained unchanged. On the contrary, stabilized (fibrotic) plaques showed a marked reduction in macrophages and cell activation with a concomitant decrease in NK cells, DCs, and neutrophils.ConclusionsMacrophage “M1” and “M2” subsets, together with fascin‐positive DCs, are strongly associated with progressive and vulnerable atherosclerotic disease of human aorta. The observations here support a more complex theory of macrophage heterogeneity than the existing paradigm predicated on murine data and further indicate the involvement of (poorly defined) macrophage subtypes or greater dynamic range of macrophage plasticity than previously considered.

Extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time‐dependent manner during atherothrombosis

“…In thrombosed coronary atherosclerotic plaques (be they ruptured or eroded), we found only a relatively small number of MCETs and EETs, in line with the usually small number of mast cells and eosinophils, respectively, that have been reported in plaques, including the lesions of MI patients [41,42]. Still, despite their low number, mast cells attract interest because of the role that they play in the process of plaque destabilisation via their release of pro-inflammatory cytokines and vasoactive mediators (histamine, chymase, and tryptase).…”

Type-I interferons in atherosclerosis

“…Neutrophils are infiltrating leukocytes present in both mouse and human plaques (van Dijk et al, 2016;van Leeuwen et al, 2008;Zernecke et al, 2008). During human atherosclerosis development, their number significantly increases in the vulnerable lesions (van Dijk et al, 2016).…”

“…Though the direct role of eosinophils in atherogenesis remains unclear, clinical studies reveal that absolute eosinophil count, intraplaque eosinophil markers, and eosinophil-specific chemokines are associated with increased risk of CVDs (Haley et al, 2000;Kitano et al, 2017;Lee et al, 2001;Niccoli et al, 2010;Tanaka et al, 2012). While EG2 + eosinophils are considered to be absent during the atherosclerosis development according to immunohistochemistry (IHC) staining of human plaques (van Dijk et al, 2016), the occurrence of eosinophil extracellular trap was identified in human thrombosed atherosclerotic specimen by double staining of eosinophil major basic protein and citrullinated histone H3 (Pertiwi et al, 2019), which might trigger IFN-α production in pDCs. Circulating eosinophils can affect atherogenesis through their cytokine production.…”

Human C-reactive protein aggravates osteoarthritis development in mice on a high-fat diet

“…Knee joints from both groups were stained with chromogenic triple-labelling immunohistochemistry (IHC) for M1 and M2 macrophage subsets, based on a previously described method for human tissue 23 . Primary antibodies targeted murine macrophages (F4/80, 1:100, MF48000 (BM8), Invitrogen), M1 macrophages (iNOS, 1:400, ab136918 (K13-A), Abcam) and M2 macrophages (CD206, 1:100, AF2535, R&D Systems).…”