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The Journal of Immunology, 2000, 164: 3946-3949.
Copyright © 2000 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Infection by the Agent of Human Granulocytic Ehrlichiosis Prevents the Respiratory Burst by Down-Regulating gp91phox1

Rila Banerjee*, Juan Anguita*, Dirk Roos{dagger} and Erol Fikrig2,*

* Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; and {dagger} Central Laboratory of the Netherlands Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Human granulocytic ehrlichiosis (HGE)3 is a newly described tick-borne disease that is caused by an obligate intracellular pathogen with a tropism for neutrophils (1, 2). Infection is often accompanied by fever, myalgia, and leukopenia and can sometimes result in death (1, 2). Morulae containing HGE bacteria can be found within the cytoplasm of bloodstream polymorphonuclear neutrophils (PMNs) during acute disease (1). Bone marrow progenitors (3), HL-60 cells (a promyelocytic tumor cell line), and C3H/HeN mice can become infected with the HGE agent, facilitating the in vitro and in vivo study of this pathogen (4, 5).

Neutrophils are primary effector cells in host defenses (6), and the respiratory burst that is initiated by NADPH oxidase plays a major role in microbial eradication (7). In resting cells, the four components of the inactive oxidase are unassembled: p47phox and p67phox are present in the cytosol and gp91phox and p22phox are in the plasma membrane (8, 9, 10). During activation, p47phox and p67phox, along with Rac2, translocate to the plasma membrane, where they associate with flavocytochrome b558, the key membrane-bound component that is composed of gp91phox and p22phox (7, 11). Formation of the complex is essential for superoxide anion (O2-) generation. Defects in oxidase activity, as demonstrated in chronic granulomatous disease, result in increased susceptibility to various infectious agents (12, 13). To survive, the agent of HGE must have evolved strategies to persist in this hostile environment. Indeed, HGE organisms reside in vacuoles that do not fuse with lysosomes, providing insight into one such tactic (14, 15). We now investigate the effect of HGE bacteria on the respiratory burst because of the paradox that this organism preferentially persists within neutrophils.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cultivation of the HGE agent and superoxide release

HL-60 cells were cultured in Dulbecco’s medium with 20% FCS at 37°C in 5% CO2 and infected with the HGE agent (4, 5). At 5 days, >90% of the cells contained morulae. In some assays, HL-60 cells were exposed to heat-killed HGE bacteria for 24 h or to supernatant (10 ml of supernatant from Ehrlichia-infected HL-60 cells) for 5 days. HL-60 cells (2 x 105/ml) were incubated at 37°C in 5% CO2 with 1 µM retinoic acid and cultured for 6 days for maximum differentiation (3, 16, 17). Superoxide anion was measured in both control and infected (both uninduced and retinoic acid-induced) HL-60 cells. In some assays, cells were also treated with IFN-{gamma} (1000 U/2 x 105 cells) for 48 h before the assay and then centrifuged at 500 x g for 10 min at 4°C to harvest the cells (18). In all assays, PMA (200 ng/ml) was used as a stimulating agent along with luminol and an enhancer of chemiluminescence, and superoxide anion was expressed in relative luminometer units (RLU). For the studies with HL-60 cells induced with IFN-{gamma}, a Lumat LB 9501 luminometer (Wallac, Gaithersburg, MD) was used, and for the retinoic acid-differentiated HL-60 cells, a TD-20/20 luminometer (Promega, Madison, WI) was used. The RLU for the two machines are different and should not be directly compared.

RT-PCR detection of NADPH oxidase subunits and HGE bacteria in HL-60 cells

cDNA was prepared from 5 µg of total RNA using random primers, and PCR amplification was then performed (19). The reaction mixture contained 5 µl of 10x PCR buffer with MgCl2, 1 µl of 10 mM dNTP, 4 µl of 20 µM primers, 0.5 µl of Taq polymerase (5 U/µl), and 2 µl of cDNA. For semiquantitative PCR, serial dilutions of the template were used. The primers were gp91phox (403 bp, 5'-GCTGTTCAATGCTTGTGGCT-3', 5'-TCTCCTCATCATGGTGCACA-3'), p22phox (325 bp, 5'-GT TTGTTTTGTGCCTGCTGGAGT-3', 5'-TGGGCGGCTGCTTGATGGT-3'), p67phox (726 bp, 5'-CGAGGGAACCAGCTGATAGA-3', 5'-CATGGGAACACTGAGCTTCA-3'), p47phox (767 bp, 5'-ACCCAGCCAGCACTATGTGT-3', 5'-AGTAGCCTGTGACGTCGTCT-3'), HGE 16S rRNA (4) (250 bp, 5'-TGTAGGCGGTTCGGTAAGTTAAAG-3', 5'-GCACTCATCGTTTACAGCGTG-3'), and ß-actin (300 bp, 5'-AGCGGGAAATCGTGCGTG-3', 5'-CAGGGTACATGGTGGTGCC-3').

Flow cytometric analysis of plasma membrane-associated gp91phox protein

Plasma membrane-associated gp91phox protein was determined using mAb 7D5. HL-60 cells (107/ml), both control and infected (treated with or without IFN-{gamma}), were resuspended in PBS/1% FCS, and gp91phox protein was detected with mAb 7D5 and a fluorescein-conjugated goat-anti-mouse-IgG Ab (20). HL-60 cells stained with a control IgG1 mAb of the same isotype as mAb 7D5 were used for comparison and did not demonstrate binding (data not shown).

Infection of C3H mice with the HGE agent

Six-week-old C3H/HeN mice were housed in filter-framed cages. A volume of 0.1 ml of blood from an Ehrlichia-infected SCID mouse was used to inoculate groups of five C3H mice (4, 21). Mice were sacrificed at 2 and 8 days, and splenic neutrophils were used to examine gp91phox expression. Spleen cells from five mice were pooled and plated in flasks in RPMI with 10% FBS at 37°C, 5% CO2. Nonadherent cells were removed after 1 h and subjected to negative selection using mouse anti-CD4, anti-CD8a, anti-B220, and anti-Pan-NK cells (PharMingen, San Diego, CA) and goat-anti-mouse-IgG bound to magnetic beads (Perspective Biosystems, Cambridge, MA). A total of 2 x 106 neutrophils were used to isolate RNA that was then reverse transcribed to obtain cDNA. The primers for murine gp91phox were 5'-GTCAAGTGCCCCAAGGTATCCA-3' and 5'-TTGTAGCTGAGGAAGTTGGC-3'.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The production of O2- in HL-60 cells infected with the HGE agent was first examined (Fig. 1Go). As expected, some O2- was detected in HL-60 cells activated with PMA (13). In contrast, HL-60 cells infected with Ehrlichia failed to produce O2- (Fig. 1Go). As a control, Escherichia coli did not inhibit O2- production (not shown). Cells were then stimulated with IFN-{gamma} to increase NADPH oxidase activity. IFN-{gamma} induced O2- levels in uninfected HL-60 cells but not in the Ehrlichia-infected cells (Fig. 1Go). Similar results were observed with HL-60 cells terminally differentiated into neutrophils with retinoic acid (Fig. 2Go). These data show that HGE bacteria inhibit the respiratory burst under a variety of conditions.



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  		FIGURE 1. Superoxide anion formation in uninfected and Ehrlichia-infected HL-60 cells using a chemiluminescence assay. PMA was used as an activating agent in all assays. Data are presented in RLU. Results are the mean ± SDs of three experiments.

 


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  		FIGURE 2. Time-course of superoxide anion formation in uninfected and Ehrlichia-infected HL-60 cells differentiated to neutrophils using retinoic acid. Cells were incubated with PMA for different time periods (0.5, 1, 2, 3, and 4 h), and O2- was expressed in RLU. Results are the mean ± SDs of three studies. {circ}, Infected cells; •, uninfected cells.

 
To explore the mechanism by which O2- was suppressed, the influence of HGE bacteria on the expression of genes encoding the multicomponent NADPH oxidase complex was examined by RT-PCR (Fig. 3Go). Similar levels of p22phox, p47phox, and p67phox mRNA were apparent in control and infected cells (Fig. 3Goa, lanes 1 and 2). In contrast, gp91phox mRNA was not present in HL-60 cells infected with live Ehrlichia. Infection with HGE bacteria also inhibited the expression of gp91phox mRNA in HL-60 cells that were differentiated into neutrophils using retinoic acid (Fig. 3Goa, lanes 3 and 4). gp91phox mRNA expression was not affected when HL-60 cells were exposed to heat-killed organisms, or incubated with medium from Ehrlichia-infected HL-60 cells (Fig. 3Gob), demonstrating that dead bacteria or a soluble factor secreted by the HGE agent were not capable of down-regulating gp91phox. Cells were then induced with IFN-{gamma} to determine whether HGE bacteria could alter gp91phox mRNA levels under conditions of maximal stimulation (18). As expected, IFN-{gamma} markedly increased gp91phox mRNA expression (Fig. 4Goa) in uninfected HL-60 cells. Lower levels of gp91phox mRNA were detected in the Ehrlichia-infected cells in response to IFN-{gamma} than in uninfected cells. Serial dilution PCR analysis indicated that gp91phox mRNA was evident in Ehrlichia-infected cells when the cDNA template was used at a 1:4 dilution and in control cells at a 1:32 dilution (not shown). Ehrlichia mRNA was detected in these cells, verifying that the bacteria persisted (Fig. 4Gob). However, this lower level of gp91phox transcription could not reverse the respiratory burst arrest by Ehrlichia (Fig. 1Go). Therefore, additional Ehrlichia-induced effects must also contribute to respiratory burst inhibition.



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  		FIGURE 3. Effect of infection with the HGE agent on the expression of mRNA for NADPH oxidase components. a, RT-PCR using primers for p22phox, p47phox, p67phox, and gp91phox with uninfected HL-60 cells (lane 1), HGE bacteria-infected HL-60 cells (lane 2), uninfected retinoic acid-differentiated HL-60 cells (lane 3), and HGE bacteria-infected retinoic acid-differentiated HL-60 cells (lane 4). b, Expression of gp91phox mRNA in HL-60 cells (lane 1), HL-60 cells treated with heat-killed HGE organisms (lane 2), and HL-60 cells grown in medium from Ehrlichia-infected HL-60 cells (lane 3). ß-actin levels were measured as a control. One of five experiments with similar results is shown.

 


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  		FIGURE 4. Effect of IFN-{gamma} on the expression of gp91phox mRNA in uninfected and Ehrlichia-infected HL-60 cells. a, RT-PCR analysis of gp91phox mRNA expression in HL-60 cells (lane 1), IFN-{gamma}-stimulated HL-60 cells (lane 2), Ehrlichia-infected HL-60 cells (lane 3), and IFN-{gamma}-stimulated Ehrlichia-infected HL-60 cells (lane 4). b, RT-PCR showing HGE bacterial mRNA (16S rRNA primers) load in unstimulated (lane 1) and IFN-{gamma}-stimulated HL-60 cells (lane 2). One of four experiments with similar results is shown.

 
gp91phox protein expression was then examined to determine whether HGE bacteria influenced formation of the NADPH oxidase complex on the plasma membrane. HL-60 cells were analyzed by FACS using mAb 7D5, which recognizes an extracytoplasmic epitope of the gp91phox protein (22). mAb 7D5 bound to uninfected but not to Ehrlichia-infected HL-60 cells (Fig. 5Go). HL-60 cells further stimulated with IFN-{gamma} demonstrated a large increase in mAb 7D5 binding, and only very weak mAb 7D5 reactivity was observed in Ehrlichia-infected, IFN-{gamma}-induced cells (Fig. 5Go). Therefore, infection with the HGE agent reduced gp91phox protein on the plasma membrane.



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  		FIGURE 5. Flow cytometric analysis of plasma membrane associated gp91phox protein expression in control and HGE-infected HL-60 cells. HL-60 cells (control or infected) were treated with or without IFN-{gamma} and then probed with mAb 7D5. One of four experiments with similar results is shown.

 
Inhibition of gp91phox mRNA expression by the HGE agent was then assessed in a murine model of granulocytic ehrlichiosis (4). As expected, morulae were evident during the first weeks of infection (4) and observed in 12% of the splenic neutrophils on 8 days. At 2 and 8 days, splenic neutrophils were examined for gp91phox expression (Fig. 6Go). gp91phox mRNA levels were lower in the infected tissue than in uninfected controls. gp91phox mRNA could be detected when the cDNA template was used at a dilution of 1:100 (barely visible) and 1:400 in mice infected with Ehrlichia for 2 and 8 days, respectively, and at a dilution of 1:1600 in uninfected mice. The disproportionate degree of suppression may be due to an Ehrlichia burden in some neutrophils that is too low for direct visualization. These data demonstrate that down-regulation of gp91phox mRNA levels also occurs in vivo.



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  		FIGURE 6. The influence on HGE bacteria on gp91phox mRNA expression in vivo. RT-PCR analysis of expression of gp91phox mRNA in the splenic neutrophils of groups of five uninfected C3H mice, and mice infected with Ehrlichia for 2 days and 8 days are shown. ß-actin mRNA levels were measured as a control. cDNAs from the neutrophils were serially diluted, and PCR was performed. Dilutions: lane 1, 1:1; lane 2, 1:10; lane 3, 1:100; lane 4, 1:200; lane 5, 1:400; lane 6, 1:800; lane 7, 1:1600; and lane 8, 1:3200.

 
Diverse pathogens, including Legionella pneumophila, Toxoplasma gondii, Chlamydia, Ehrlichia risticii (which infects macrophages), Entamoeba histolytica, and Leishmania, have been shown to inhibit the respiratory burst; however, the mechanism(s) is (are) not known (23, 24, 25, 26, 27, 28). Suppression of NADPH oxidase activity by down-regulating expression of a critical subunit of the enzyme complex by HGE bacteria represents a new mechanism by which microbes circumvent the oxidant-generating respiratory burst. It is intriguing that Ehrlichia targets the gene, gp91phox, which is associated with chronic granulomatous disease (12), and suggests that HGE bacteria induces a transient state in which the host may be more susceptible to secondary infections. Understanding the biological basis of respiratory burst arrest by pathogens should facilitate the development of new strategies to prevent infectious diseases and modify inflammatory responses.


   Acknowledgments
 
We thank Sankar Ghosh for help with the luminometric assays and Debbie Beck for technical assistance.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health, Brown-Coxe Fellowship Program, and a gift from SmithKline Beecham Biologicals. E.F. is the recipient of a Clinical-Scientist Award in Translational Research from the Burroughs Wellcome Fund. Back

2 Address correspondence and reprint requests to Dr. Erol Fikrig, 608 Laboratory of Clinical Investigation, Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208031, New Haven, CT 06520-8031. E-mail address: Back

3 Abbreviations used in this paper: HGE, human granulocytic ehrlichiosis; PMN, polymorphonuclear neutrophil; RLU, relative luminometer unit. Back

Received for publication December 27, 1999. Accepted for publication February 17, 2000.


   References
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 Introduction
 Materials and Methods
 Results and Discussion
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Divergent Interactions of Ehrlichia chaffeensis- and Anaplasma phagocytophilum-Infected Leukocytes with Endothelial Cell Barriers
Infect. Immun., December 1, 2003; 71(12): 6728 - 6733.
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J. A. Carlyon, M. Akkoyunlu, L. Xia, T. Yago, T. Wang, R. D. Cummings, R. P. McEver, and E. Fikrig
Murine neutrophils require {alpha}1,3-fucosylation but not PSGL-1 for productive infection with Anaplasma phagocytophilum
Blood, November 1, 2003; 102(9): 3387 - 3395.
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M. Lin and Y. Rikihisa
Ehrlichia chaffeensis and Anaplasma phagocytophilum Lack Genes for Lipid A Biosynthesis and Incorporate Cholesterol for Their Survival
Infect. Immun., September 1, 2003; 71(9): 5324 - 5331.
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K.-S. Choi, J. Garyu, J. Park, and J. S. Dumler
Diminished Adhesion of Anaplasma phagocytophilum-Infected Neutrophils to Endothelial Cells Is Associated with Reduced Expression of Leukocyte Surface Selectin
Infect. Immun., August 1, 2003; 71(8): 4586 - 4594.
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H. Scaife, Z. Woldehiwet, C. A. Hart, and S. W. Edwards
Anaplasma phagocytophilum Reduces Neutrophil Apoptosis In Vivo
Infect. Immun., April 1, 2003; 71(4): 1995 - 2001.
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J. A. Carlyon, W.-T. Chan, J. Galan, D. Roos, and E. Fikrig
Repression of rac2 mRNA Expression by Anaplasma phagocytophila Is Essential to the Inhibition of Superoxide Production and Bacterial Proliferation
J. Immunol., December 15, 2002; 169(12): 7009 - 7018.
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J. W. IJdo, C. Wu, S. R. Telford III, and E. Fikrig
Differential Expression of the p44 Gene Family in the Agent of Human Granulocytic Ehrlichiosis
Infect. Immun., September 1, 2002; 70(9): 5295 - 5298.
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J. Mott, Y. Rikihisa, and S. Tsunawaki
Effects of Anaplasma phagocytophila on NADPH Oxidase Components in Human Neutrophils and HL-60 Cells
Infect. Immun., March 1, 2002; 70(3): 1359 - 1366.
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R. R. Ganta, M. J. Wilkerson, C. Cheng, A. M. Rokey, and S. K. Chapes
Persistent Ehrlichia chaffeensis Infection Occurs in the Absence of Functional Major Histocompatibility Complex Class II Genes
Infect. Immun., January 1, 2002; 70(1): 380 - 388.
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M. Akkoyunlu, S. E. Malawista, J. Anguita, and E. Fikrig
Exploitation of Interleukin-8-Induced Neutrophil Chemotaxis by the Agent of Human Granulocytic Ehrlichiosis
Infect. Immun., September 1, 2001; 69(9): 5577 - 5588.
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J. Mott and Y. Rikihisa
Human Granulocytic Ehrlichiosis Agent Inhibits Superoxide Anion Generation by Human Neutrophils
Infect. Immun., December 1, 2000; 68(12): 6697 - 6703.
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R. Banerjee, J. Anguita, and E. Fikrig
Granulocytic Ehrlichiosis in Mice Deficient in Phagocyte Oxidase or Inducible Nitric Oxide Synthase
Infect. Immun., July 1, 2000; 68(7): 4361 - 4362.
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