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Infection and Immunity, July 2000, p. 4361-4362, Vol. 68, No. 7
Section of Rheumatology, Department of
Internal Medicine, Yale University School of Medicine, New Haven,
Connecticut 06520
Received 22 February 2000/Returned for modification 24 March
2000/Accepted 28 March 2000
Mice deficient in phox (gp91phox Human granulocytic ehrlichiosis
(HGE) is a newly recognized vector-borne infectious disease of
increasing importance in the United States and Europe (3, 6, 16,
22). Prominent clinical manifestations of disease include fever,
headache, and myalgias (23). HGE bacteria primarily infect
neutrophils and survive within membrane-bound vacuoles known as morulae
(23). A promyelocytic cell line (HL-60) has been used to
culture HGE organisms in vitro, and bone marrow precursors have been
infected with the HGE agent, stimulating further research (9, 10,
14). Mice can also be infected with HGE bacteria, facilitating in
vivo studies of pathogenesis and immunity (2, 11, 21).
Immunocompetent mice develop an infection in which the HGE agent is
usually detected during the first 10 days of infection and is then
generally cleared from the bloodstream (2, 20, 21).
Sometimes, however, ehrlichiae can be detected by PCR at later
intervals after challenge (2, 20, 21).
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Granulocytic Ehrlichiosis in Mice Deficient in
Phagocyte Oxidase or Inducible Nitric Oxide Synthase
ABSTRACT
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/) or NOS2
(NOS2/) were infected with the agent of human
granulocytic ehrlichiosis (HGE) to evaluate the importance of these
pathways in the eradication of HGE bacteria. NOS2/ mice
had delayed clearance of the HGE agent in comparison to control or
gp91phox/ mice, suggesting that reactive nitrogen
intermediates play a role in the early control of HGE.
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FIG. 1.
HGE infection in gp91phox /
mice. At 8 and 12 days, splenocytes from HGE-infected
gp91phox/ and control mice (five animals per
group) were isolated and pooled, and RT-PCR was performed using
HGE-specific primers. One of three studies with similar results is
shown.
Two important microbicidal pathways of phagocytes are the production of
reactive oxygen intermediates (ROI) by respiratory burst oxidase (phox)
and reactive nitrogen intermediates (RNI) by inducible nitric oxide
synthase (NOS2) (4, 7). Mice deficient in phox
(gp91phox/) or NOS2 (NOS2/)
have also demonstrated the importance of these enzymes in host defense
against a variety of pathogens (5, 18, 19). Recent data
suggest that HGE bacteria use several strategies to survive within the
hostile environment of the neutrophil. Morulae do not fuse with
lysosomes, providing one mechanism of persistence (17, 24).
HGE bacteria also inhibit the formation of ROI through selective
downregulation of the gp91phox component of the
NADPH oxidase complex (1). HGE in mice deficient in phox or
NOS2 was investigated to understand the role of ROI and RNI in
granulocytic ehrlichiosis.
Granulocytic ehrlichiosis in gp91phox/ or
NOS2/ mice.
Mice were infected with HGE
bacteria and then examined for infection at intervals up to 20 days.
For infection, blood (0.1 ml) from C.B.17 SCID mice infected with
ehrlichiae (donors) was intraperitoneally injected into wild-type
(control), gp91phox/, or
NOS2/ mice (20). SCID mice had morulae in
10% of the neutrophils, thereby ensuring challenge with a constant
number of organisms to each experimental mouse. Reverse
transcription-PCR (RT-PCR) was then used to assess levels of viable
ehrlichiae in the splenocytes. cDNA was prepared from total RNA
isolated from pooled splenocytes of groups of five control and
experimental mice, and PCR was performed using 16S rRNA primers (5'-TGT
AGG CGG TTC GGT AAG TTA AAG-3' and 5'-GCA CTC ATC GTT TAC AGC GTG-3')
that amplify a 250-bp fragment specific for HGE (20). In the
first experiment, both gp91phox/
(5) and control mice had detectable bacteria on day 8 (Fig. 1). Ehrlichiae were no longer detectable by PCR in either the control
or gp91phox/ animals on day 12, demonstrating
substantial clearance of the pathogen at this point. In a second study,
NOS2/ mice (15) had appreciable HGE on day
12 and then cleared the HGE agent on day 20, indicating a delay in
bacterial clearance (Fig. 2). Since
signaling of gamma interferon (IFN-
) through its receptor results in
induction of NOS2 (12), HGE infection in mice lacking the
IFN- receptor (IFN-R/) was also evaluated
(13). At 12 days ehrlichiae were detected in
NOS2/ and IFN-R/ mice but not in
control animals (Fig. 3). Moreover, NOS2
mRNA levels were markedly reduced in the IFN-R/
mice, suggesting that NOS2-mediated early clearance of HGE bacteria is
dependent upon IFN-.
|
|
/ mice
demonstrate that the course of granulocytic ehrlichiosis is unchanged
in the absence of phox. This finding is in agreement with the in vitro
observation that HGE bacteria actively inhibit the respiratory burst by
downregulating gp91phox, thereby developing a
local environment that has reduced levels of ROI (1).
gp91phox/ and control mice can, however, both
clear the HGE bacteria after 12 days, suggesting that alternative
mechanisms are responsible for the control of progressive infection.
Our studies also demonstrate that NOS2 is important for the control of
early infection because HGE can be readily detected at 12 days in
NOS2/ mice. Furthermore, IFN- is likely to play a
role in the NOS2-mediated clearance of HGE bacteria because NOS2 levels
were lower in IFN-R/ mice. Studies with
Trypanosoma cruzi- and Listeria
monocytogenes-infected IFN-R/ mice have shown
similar reductions in NOS2 expression (8, 12). Nevertheless,
in both gp91phox/ and NOS2/
mice, HGE bacteria were cleared at 12 or 20 days. This demonstrates that neither pathway is necessary for the eradication of persistent infection, perhaps because humoral and cellular responses to HGE can
aid bacterial clearance. Indeed, antibodies to HGE bacteria are
sufficient to partially protect mice from infection (20). Understanding the host immune response to HGE should enhance our understanding of the pathways that facilitate bacterial clearance and
the evolution of HGE infection in mice.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant 51873, the Brown-Coxe Fellowship Program, and a gift from SmithKline Beecham Biologicals. E. Fikrig is the recipient of a Clinical-Scientist Award in Translational Research from the Burroughs Wellcome Fund.
We thank C. Nathan (Cornell University Medical College) and J. S. Mudgett (Merck Research Laboratories) for providing us with the
NOS2/ mice and Debbie Beck for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: 608 Laboratory of Clinical Investigation, Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St., P.O. Box 208031, New Haven, CT 06520-8031. Phone: (203) 785-2453. Fax: (203) 785-7053. E-mail: erol.fikrig{at}yale.edu.
Editor: R. N. Moore
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Infection and Immunity, July 2000, p. 4361-4362, Vol. 68, No. 7
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