AUTHOR=Dumler J. Stephen , Lichay Marguerite , Chen Wan-Hsin , Rennoll-Bankert Kristen E. , Park Jin-ho 

TITLE=Anaplasma phagocytophilum Activates NF-κB Signaling via Redundant Pathways

JOURNAL=Frontiers in Public Health

VOLUME=8

YEAR=2020

URL=https://www.frontiersin.org/journals/public-health/articles/10.3389/fpubh.2020.558283

DOI=10.3389/fpubh.2020.558283

ISSN=2296-2565

ABSTRACT=<p><italic>Anaplasma phagocytophilum</italic> subverts neutrophil function permitting intracellular survival, propagation and transmission. Sustained pro-inflammatory response, recruitment of new host cells for population expansion, and delayed apoptosis are associated with prolonged nuclear presence of NF-κB. We investigated NF-κB signaling and transcriptional activity with <italic>A. phagocytophilum</italic> infection using inhibitors of NF-κB signaling pathways, and through silencing of signaling pathway genes. How inhibitors or silencing affected <italic>A. phagocytophilum</italic> growth, inflammatory response (transcription of the κB-enhanced genes <italic>CXCL8</italic> and <italic>MMP9</italic>), and NF-κB signaling pathway gene expression were tested. Among <italic>A. phagocytophilum</italic>-infected HL-60 cells, nuclear NF-κB p50, p65, and p52 were detected by immunoblots or iTRAQ proteomics. <italic>A. phagocytophilum</italic> growth was affected most by the IKKαβ inhibitor wedelolactone (reductions of 96 to 99%) as compared with SC-514 that selectively inhibits IKKβ, illustrating a role for the non-canonical pathway. Wedelolactone inhibited transcription of both <italic>CXCL8</italic> (<italic>p</italic> = 0.001) and <italic>MMP9</italic> (<italic>p</italic> = 0.002) in infected cells. Compared to uninfected THP-1 cells, <italic>A. phagocytophilum</italic> infection led to &gt;2-fold down regulation of 64 of 92 NF-κB signaling pathway genes, and &gt;2-fold increased expression in only 4. Wedelolactone and SC-514 reversed downregulation in all 64 and 45, respectively, of the genes down-regulated by infection, but decreased expression in 1 gene with SC-514 only. Silencing of 20 NF-κB signal pathway genes increased bacterial growth in 12 (<italic>IRAK1, MAP3K1, NFKB1B, MAP3K7, TICAM2, TLR3, TRADD, TRAF3, CHUK, IRAK2, LTBR</italic>, and <italic>MALT1</italic>). Most findings support canonical pathway activation; however, the presence of NFKB2 in infected cell nuclei, selective non-canonical pathway inhibitors that dampen <italic>CXCL8</italic> and <italic>MMP9</italic> transcription with infection, upregulation of non-canonical pathway target genes <italic>CCL13</italic> and <italic>CCL19</italic>, enhanced bacterial growth with <italic>TRAF3</italic> and <italic>LTBR</italic> silencing provide evidence for non-canonical pathway signaling. Whether this impacts distinct inflammatory processes that underlie disease, and whether and how <italic>A. phagocytophilum</italic> subverts NF-κB signaling via these pathways, need to be investigated.</p>