Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3-kinase signaling
August
Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3- kinase signaling
Supreet Khanal 1 2
Hameeda Sultana 0 1 2
John D. Catravas 2
Jason A. Carlyon 2
Girish Neelakanta 0 1 2
0 Center for Molecular Medicine, College of Sciences, Old Dominion University, Norfolk, VA, United States of America, 3 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, United States of America, 4 School of Medical Diagnostic and Translational Sciences, College of Health Sciences, Old Dominion University, Norfolk, VA, United States of America, 5 Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine , Richmond, VA , United States of America
1 Department of Biological Sciences, Old Dominion University , Norfolk, VA , United States of America
2 Editor: Ulrike Gertrud Munderloh, University of Minnesota , UNITED STATES
Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.
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Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This study was supported by Old
Dominion University Start-up funds to GN and HS
and by NIH R01 AI072683 to JAC. The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
Introduction
In the United States, human granulocytic anaplasmosis (HGA) is one of the most common
tick-borne diseases [
1, 2
]. Previous studies have shown that up to 30% of human population in
endemic areas may have been exposed to A. phagocytophilum infections [
3, 4
]. At least 15, 952
HGA cases have been reported since 1995 with a 12-fold increased rate in 2001±2011 [5].
Infections in many cases are asymptomatic [2, 3, 5, 6]. However, HGA infections could lead to
Competing interests: The authors have declared
that no competing interests exist.
severe illness and death in many individuals particularly that are immunocompromised [
5
].
The common clinical manifestations of HGA include fever, malaise, headache, and/or myalgia.
However, arthralgia, nausea, vomiting or cough may occur in some severely infected persons.
In addition, thrombocytopenia (reduced platelet numbers), leucopenia, anemia and/or ele
vated levels of liver enzymes are often evident in HGA cases [
2, 5, 6
].
In the mammalian hosts, A. phagocytophilum survives primarily in the neutrophils, where it
enters membrane-bound vacuoles that do not fuse with lysosome, thereby protecting itself
from host toxic components and degradation [
7, 8
]. In addition, A. phagocytophilum delays
apoptosis of the neutrophils by modulation of multiple apoptotic pathways [
9, 10
]. Several
studies have shown that A. phagocytophilum alters host gene expression for its survival and
replication [10±15]. A. phagocytophilum is closely related to Ehrlichia species [
6, 16, 17
].
Ehrlichia chaffeensis is reported to alter cell cycle genes for its survival in human monocytic cell line
[18]. A. phagocytophilum also infects and survives in other hematopoietic cells [
5, 19, 20
].
While much is known about the interactions of A. phagocytophilum with neutrophils, very lit
tle is known whether this bacterium alters cell cycle gene expression for its survival in
hematopoietic cells.
Megakaryocytes are the precursor cells for the production of platelets [
21
]. Initially,
megakaryocytes mature and differentiate in bone marrow [
21
]. Upon differentiation,
megakaryocytes extend their cytoplasmic structures to form proplatelets that later form segments leading
to the formation of platelets [
21
]. Due to difficulty in the isolation of homogenous populations
of bone marrow megakaryoblast cells, the use of in vitro cell lines has greatly facili (...truncated)