Repression ofrac2mRNA Expression byAnaplasma phagocytophilaIs Essential to the Inhibition of Superoxide Production and Bacterial Proliferation

Abstract

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O2−), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91phox, an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91phox from the CMV immediate early promoter maintained the ability to generate O2− 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91phox transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.

Insights into Pathogen Immune Evasion Mechanisms: Anaplasma phagocytophilum Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils

“…In contrast, there are immediate (within 30 min) and dramatic increases in neutrophil Second, our data clearly demonstrate that genes encoding components of NADPH oxidase, including gp91 phox and Rac2, remain unchanged or are up-regulated in human neutrophils during A. phagocytophilum infection (Table I). These results are at variance with previous studies that suggest the inability of PMNs to produce ROS production following uptake of A. phagocytophilum is due to decreased gp91 phox and/or Rac2 transcript levels (12,14). However, those studies were limited in scope and/or used HL60 cells rather than human PMNs (12,14).…”

“…Moreover, none of the genes encoding NADPH oxidase components, including CYBB (gp91 phox ) and CYBA (p22 phox ), were significantly down-regulated after A. phagocytophilum infection (Table I). These results clearly indicate that failure of A. phagocytophilum to elicit production of ROS by PMNs, or to block ROS production by a second stimulus, is not due to repression of genes encoding NADPH oxidase proteins, as previously suggested (12)(13)(14)(15).…”

“…Previous studies in promyelocytic HL60 cells suggest that the ability of A. phagocytophilum to inhibit ROS generation is linked to down-regulation of NADPH oxidase components (12)(13)(14)(15). However, this mechanism has not been demonstrated in human neutrophils.…”

“…Studies evaluating functional alterations in A. phagocytophilum-infected PMNs suggest that the pathogen delays PMN apoptosis (6,7), minimizes proinflammatory cytokine release (8,9), and inhibits and/or fails to activate ROS production (10 -13). Mechanisms underlying the ability of A. phagocytophilum to inhibit production of PMN ROS are unclear (12)(13)(14)(15).…”

ASC/PYCARD and Caspase-1 Regulate the IL-18/IFN-γ Axis during Anaplasma phagocytophilum Infection

“…We previously demonstrated that IL-1␤ and IL-18 were up-regulated when A. phagocytophilum infects neutrophils and promyelocytic cells (3,7,8). Some Nod-like receptors (NLR) 3 have been shown to regulate IL-1␤ and IL-18 secretion (9 -13).…”