Stress-induced cholinergic signaling promotes inflammation-associated thrombopoiesis

Abstract

To study the role of the stress-induced "readthrough" acetylcholinesterase splice variant, AChE-R, in thrombopoiesis, we used transgenic mice overexpressing human AChE-R (TgR). Increased AChE hydrolytic activity in the peripheral blood of TgR mice was associated with increased thrombopoietin levels and platelet counts. Bone marrow (BM) progenitor cells from TgR mice presented an elevated capacity to produce mixed (GEMM) and megakaryocyte (Mk) colonies, which showed intensified labeling of AChE-R and its interacting proteins RACK1 and PKC. When injected with bacterial lipopolysaccharide (LPS), parent strain FVB/N mice, but not TgR mice, showed reduced platelet counts. Therefore, we primed human CD34 ؉ cells with the synthetic ARP 26 peptide, derived from the cleavable C-terminus of AChE-R prior to transplantation, into sublethally irradiated NOD/SCID mice. Engraftment of human cells (both CD45 ؉ and CD41 ؉ Mk) was significantly increased in mice that received ARP 26 IntroductionThe number of circulating blood cells is tightly regulated by cytokines and chemokines capable of immediate response to various stimuli. 1 Adjustment to changing needs involves rapid mobilization of cells from the bone marrow (BM) and the vascular marginal pool in response to inflammation, stress, or injury. 2 An example is inflammation-inducible hematopoiesis, which was thoroughly studied in murine models using bacterial lipopolysaccharide (LPS), the main cell-wall component of gram-negative bacteria. 3 LPS is an endotoxin that stimulates an acute inflammatory response via the CD14 receptor and the Toll-like receptor-4 (TLR4) found on monocytes and tissue macrophages. 4 LPS-TLR4 interaction initiates a signal transduction cascade that leads to the release of pro-inflammatory cytokines, 3 including tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤, -6, and -8, and others. These cytokines activate the mobilization of hematopoietic cells from the BM 5 and set in motion the migration of leukocytes from blood vessel walls, increasing their numbers in the circulation. 6 The net result of this process is an immediate and dramatic increase in the number of circulating peripheral blood (PB) cells, needed to mount the immune response, accompanied by a corresponding decrease in BM cell numbers, which in turn induces a compensatory increase in their production. 7 Many factors are involved in abating the inflammatory response and allowing the recovery of hemostasis. Acetylcholine (ACh), one of these factors, acts by attenuating the secretion of proinflammatory cytokines at the posttranscriptional level via activation of nicotinic receptors on tissue macrophages. 8 Circulating acetylcholinesterase (AChE) controls the levels of ACh, suggesting promotion of the inflammatory process under AChE excess. 9 There are 3 C-terminally variant forms of AChE: synaptic (S), erythrocytic (E), and readthrough (R). All are ubiquitously expressed in hematopoietic cell lineages, especially in megakaryocytes (Mks) and erythrocytes. [10][11][12] Importantly, AChE contri...

Acetylcholinesterase/C terminal binding protein interactions modify Ikaros functions, causing T lymphopenia

“…Marrow cells were harvested as detailed. 13 In situ hybridization and intracellular localization of AChE-S mRNA were performed as detailed. 9,18 AChE catalytic activity was determined as detailed.…”

“…This unusual blood cell profile was opposite to the stress-induced enhancement of granulopoiesis and thrombocytosis, under AChE-R overexpression. 12 We conclude that AChE-S excess, likely through alleviating the suppressive effects of CtBP with which it interacts, suppresses T lymphopoiesis and thrombopoiesis, in contrast to AChE-R, which promotes granulopoiesis and thrombopoiesis 9,13 (Figure 7b). …”

“…Fluorescent detectors sensitivity, adjustment, isotype controls and analysis were all as detailed. 9,13 Antibodies used for flow cytometry are listed in Supplementary information.…”

“…CFU-GEMM, CFU-GM and CFU-Mk were grown as previously described. 13 BFU-E were cultured in Alpha-MEM (Biological Industries) supplemented with 0.8% methyl cellulose, 10% FCS, 10% bovine serum albumin (BSA, Boehringer Ingelheim GmbH, Biberach an der Riss, Germany) 2-mercaptoethanol, 3 U/ml recombinant human (r-hu)-erythropoietin and 10 ng/ml r-mouse-CSF.…”

“…9 Also, transgenic mice overexpressing AChE-R display elevated lymphopoiesis and platelet counts. 12,13 Nevertheless, it remained unclear whether the gain of AChE-R function by itself suffices to induce the myelopoiesis and thrombocytosis that follow stress insults, or if the loss of AChE-S function(s) is also causally involved.…”