Abstract
Anaplasma phagocytophilum 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 A. phagocytophilum infection using inhibitors of NF-κB signaling pathways, and through silencing of signaling pathway genes. How inhibitors or silencing affected A. phagocytophilum growth, inflammatory response (transcription of the κB-enhanced genes CXCL8 and MMP9), and NF-κB signaling pathway gene expression were tested. Among A. phagocytophilum-infected HL-60 cells, nuclear NF-κB p50, p65, and p52 were detected by immunoblots or iTRAQ proteomics. A. phagocytophilum 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 CXCL8 (p = 0.001) and MMP9 (p = 0.002) in infected cells. Compared to uninfected THP-1 cells, A. phagocytophilum infection led to >2-fold down regulation of 64 of 92 NF-κB signaling pathway genes, and >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 (IRAK1, MAP3K1, NFKB1B, MAP3K7, TICAM2, TLR3, TRADD, TRAF3, CHUK, IRAK2, LTBR, and MALT1). Most findings support canonical pathway activation; however, the presence of NFKB2 in infected cell nuclei, selective non-canonical pathway inhibitors that dampen CXCL8 and MMP9 transcription with infection, upregulation of non-canonical pathway target genes CCL13 and CCL19, enhanced bacterial growth with TRAF3 and LTBR silencing provide evidence for non-canonical pathway signaling. Whether this impacts distinct inflammatory processes that underlie disease, and whether and how A. phagocytophilum subverts NF-κB signaling via these pathways, need to be investigated.
Highlights
Anaplasma phagocytophilum is an Ixodes spp. tick-transmitted zoonotic rickettsia of small mammals, cervids, and ruminants [1]
When transcriptional profiles of A. phagocytophilum-infected myeloid cells were queried for differential expression of a set of 104 NF-κB signaling pathway genes, 9 were upregulated >2-fold and none were downregulated more than 2-fold in 3 of 4 datasets evaluated (Supplementary Table 1C)
A. phagocytophilum has a remarkable capacity to survive within the harsh niche of the primary innate immune antimicrobial cell, the neutrophil
Summary
Anaplasma phagocytophilum is an Ixodes spp. tick-transmitted zoonotic rickettsia of small mammals, cervids, and ruminants [1]. Granulocytic anaplasmosis is a mild to severe disease of humans, horses, and dogs, characterized in humans by fever, headache, myalgias, thrombocytopenia, leukopenia, and liver injury [1]. This obligate intracellular rickettsial bacterium infects and propagates within granulocytes, largely neutrophils, in blood and bone marrow after it is acquired by tick bite and disseminated into the blood. A. phagocytophilum evolved to survive within the antimicrobial confines of neutrophils by co-opting a number of pathways that regulate endosomal entry, vacuolar trafficking, and signals that lead to delayed apoptosis and reduced antimicrobial activities, while promoting an inflammatory environment that recruits new host cells to expand bacterial populations and inducing inflammatory cell injury as a mechanism for disease pathogenesis [3,4,5]. A key for understanding how disease occurs with A. phagocytophilum is the observation that clinical signs of illness in humans and animal models are not closely linked to bacterial loads, but are more closely associated with stimulation of host immune and inflammatory signaling such as with upregulation of the genes for interleukin 1 (IL1), C-X-C motif chemokine ligand 8 (CXCL8), matrix metalloprotease−9 (MMP9), or innate immune responses, and subversion of regulatory aspects of cytotoxic lymphocytes [6,7,8,9,10,11,12]
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