Cutting Edge: Antibody-Mediated Cessation of Hemotropic Infection by the Intraerythrocytic Mouse Pathogen grahamii This information is current as of September 27, 2021. Jan Koesling, Toni Aebischer, Christine Falch, Ralf Schülein and Christoph Dehio J Immunol 2001; 167:11-14; ; doi: 10.4049/jimmunol.167.1.11 http://www.jimmunol.org/content/167/1/11 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: Antibody-Mediated Cessation of Hemotropic Infection by the Intraerythrocytic Mouse Pathogen Bartonella grahamii1

Jan Koesling,* Toni Aebischer,* Christine Falch,† Ralf Schu¨lein,†‡ and Christoph Dehio2†‡

studied in most detail in rats experimentally infected by Bar- The genus Bartonella includes important human-specific and tonella tribocorum (6). After residing in an unknown primary zoonotic pathogens which cause intraerythrocytic bacteremia niche, the onset of bacteremia in this model occurs 5–6 days Downloaded from in their mammalian reservoir host(s). It is accepted that cel- postinfection by a synchronous wave of bacterial adhesion and lular immunity plays a decisive role in the host’s defense invasion into mature erythrocytes. The intracellular against most intracellular bacteria. Bartonella sp. infection in replicate until reaching a steady number, which is sustained for the immunocompetent host typically leads to immunity against the remaining life span of the infected erythrocytes (eventually homologous challenge. The basis of this immunity, be it cellu- exceeding several weeks). The initial wave of erythrocyte in- lar or humoral, is unclear. In this study, the course of Bar- fection is followed by reinfection waves occurring in intervals tonella grahamii bacteremia in immunocompetent and immu- of 3–6 days. As found similarly in other animal models of Bar- http://www.jimmunol.org/ nocompromised mice was compared. In immunocompetent tonella infection (7–10), the phase of intraerythrocytic bactere- hosts, the bacteremia is transient and induces a strong hu- mia subsides spontaneously after a few months (typically 8–10 wk). moral immune response. In contrast, bacteremia persists in The infection triggers an immune response which confers protective immunocompromised B and T cell-deficient mice. Immune se- immunity as challenging convalescent animals with the same Bar- rum transfer beginning with day 6 postinfection to B cell-de- tonella strain does not result in reinfection (7, 10), whereas challeng- ficient mice unable to produce Igs converted the persistent bac- ing with a different Bartonella strain may again cause bacteremia (10). teremia to a transient course indistinguishable from that of However, the immune effector mechanism(s) mediating termination immunocompetent animals. These data demonstrate an essen- of Bartonella bacteremia are not yet elucidated. Both a humoral im- by guest on September 27, 2021 tial role for specific Abs in abrogating the intraerythrocytic mune response with high IgG titers (10) as well as a cellular immune response by T cell activation (8, 11–13) can be triggered by Bar- bacteremia of B. grahamii in mice. The Journal of Immunol- tonella infection. Classical studies of intracellular pathogens such as ogy, 2001, 167: 11–14. Listeria monocytogenes and Mycobacterium tuberculosis have pro- vided evidence for a critical role for cellular immunity in host defense wo Bartonella species, and (14, 15). Therefore, cellular immune responses have long been con- , cause hemotropic infection in hu- sidered to be a hallmark of immunity to intracellular bacterial patho- T mans known as oroya fever (1) and (2), gens (16). However, erythrocytes cannot present Ags to the immune respectively. In the last decade, a number of Bartonella spp. system in a MHC-dependent way due to the lack of MHC on their naturally causing hemotropic infection in various mammals surface. Intraerythrocytic bartonellae should thus be hidden from a (i.e., cats, dogs, rats, and mice) have been associated with an respective cellular immune response. A growing body of evidence expanding spectrum of human diseases (3–5). The intraeryth- suggests that Ab can contribute to immunity against several other rocytic lifestyle appears to be the common parasitic strategy of intracellular pathogens (reviewed in Ref. 17), such as Salmonella (18, all Bartonella spp. in their respective mammalian reservoirs. 19), Mycobacterium (20), Legionella (21), Brucella (22), and Plas- The process of erythrocyte parasitism by Bartonella has been modium (23). A better understanding of the host immune response(s) interfering with Bartonella infections may facilitate the design of strategies to control these emerging pathogens. To this end, we have *Department of Molecular Biology, Max Planck Institute for Infection Biology, Ber- established a mouse model of transient Bartonella bacteremia in wild- lin, Germany; †Department of Infection Biology, Max Planck Institute for Biology, type and persistent bacteremia in B cell-deficient strains. Further by ‡ Tubingen, Germany; and Department of Molecular Microbiology, Biozentrum of the immune serum transfer we formally demonstrate that Abs are re- University of Basel, Basel, Switzerland quired for immune control of intraerythrocytic Bartonella infection Received for publication February 7, 2001. Accepted for publication May 7, 2001. in mice. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Deutsche Forschungsgemeinschaft Grant De 539/4-1 Materials and Methods and Swiss National Science Foundation Grant 3100-061777.00/1 (both to C.D.). Mice, bacteria, and infection 2 Address correspondence and reprint requests to Dr. Christoph Dehio, Department of Molecular Microbiology, Biozentrum of the University of Basel, Klingelbergstrasse Six- to 8-wk-old female BALB/c mice were purchased from Harlan and 70, CH-4056 Basel, Switzerland. E-mail address: [email protected] Winkelmann (Borchen, Germany), C57BL/6 from Bundesinstitut fu¨r

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

● 12 CUTTING EDGE: Ab-MEDIATED CESSATION OF Bartonella INFECTION gesundheitlichen Verbraucherschutz und Veterina¨rmedizin (Berlin, Ger- many), C57BL/6-IghϪ/Ϫ (IghϪ/Ϫ) and C57BL/6-Rag1Ϫ/Ϫ (RagϪ/Ϫ) mice from The Jackson Laboratory (Bar Harbor, ME). A Bartonella isolate from field-vole (strain IBS 376, generously provided by Yves Pie´mont, Stras- bourg, France) was identified as B. grahamii by sequence analysis of the citrate synthase gene (gltA) as previously described (24). Bacteria were cultivated on Columbia blood agar with 5% defibrinated sheep blood 3 ϫ 9 (CB-agar) at 35°C and 5% CO2 for 7 days, and 2 10 CFU suspended in 200 ␮l PBS were injected i.v. Bacteremia was analyzed by plating citrate blood samples previously frozen at Ϫ80°C on CB-agar and CFU enumeration. Serum transfer experiments Serum was obtained from blood pooled from 20 C57BL/6 mice. Normal serum was collected from naive mice and immune serum from mice 40–82 days postinfection. To increase the Ig concentration, serum precipitation was performed with 45% ammonium sulfate (25). The precipitate was di- alyzed against PBS. On days 6, 11, 18, and 32 postinfection, 1 mg protein of normal or immune serum precipitate within 300 ␮l PBS or PBS alone was injected i.v. into B. grahamii-infected mice. ELISA Downloaded from Ab titers were determined colorimetrically by solid-phase ELISA essen- tially as described (26), except that B. grahamii whole cell lysate was used for Ag coating of microtiter plates. In brief, dilutions of serum samples were added into Ag-coated microtiter plates. For detection, HRP-conju- gated rat anti-mouse IgG1 or IgG2a (Nordic, Tilburg, The Netherlands) and orthophenyldiamine-supplemented peroxide were used. Absorbance

was measured at 492 nm. http://www.jimmunol.org/ Intraeythrocytic bacteria The gentamicin protection assay was performed as described previously (6). Blood smears were stained by SpotTest acridine orange stain as specified by the manufacturer (Difco, Detroit, MI) and fluorescent in- traerythrocytic bacteria were visualized by a Leica DM IRBE inverted fluorescence microscope (Leica, Deerfield, IL) using filter block GFP. Results and Discussion B. grahamii has been isolated from the blood of various small by guest on September 27, 2021 woodland mammals including wild mice (27, 28). To establish a murine infection model for immunological studies, we injected B. grahamii i.v. into wild-type C57BL/6 or BALB/c mice and deter- mined the course of bacteremia by CFU enumeration. The course FIGURE 1. B. grahamii infection of mice and their humoral immune response. Infection of immunocompetent C57BL/6 mice (n ϭ 4) was per- of B. grahamii bacteremia was indistinguishable for both mouse formed by i.v. injection of 2 ϫ 109 CFU suspended in 200 ␮l PBS. Serum strains and is illustrated for C57BL/6 in Fig. 1A (data for BALB/c and citrate blood samples were collected on days 5, 10, 17, 31, 45, 59, and not shown). All four inoculated animals were bacteremic by 1 wk 77 postinfection. A, Time course of the bacteremia. CFU per ml of citrate postinfection, and bacteremia peaked at 2 wk before dropping be- blood were determined by culture on CB-agar plates performed in tripli- low detectable levels between 8 and 11 wk postinfection. The ob- cates. Averages were plotted for individual animals (C57BL/6 #1–4). B, served characteristics of B. grahamii bacteremia in experimentally IgG1 and IgG2a Abs in pooled serum of infected C57BL/6 mice (n ϭ 4) infected wild-type mice are consistent with the well-characterized at day 45 postinfection. Titers were determined by serial dilution in an B. tribocorum rat model (6) as well as other animal models of ELISA of B. grahamii whole-cell lysates, and the averages of the chro- Bartonella infection (7–10). mogenic readout (A492) performed in triplicates were plotted. The plot Ͼ The intracellular localization of B. grahamii was confirmed by illustrates the linear range of the test ( 1:30 for IgG1 and 1:1,000–1: 10,000 for IgG2a). C, Time course of the IgG response. Individual serum gentamicin protection assays (6), which permit the distinction be- samples diluted within the linear test range (1:15 for IgG1 and 1:2,000 for tween extracellular and intracellular bacteria. Furthermore, fluo- IgG2a) were examined by ELISA. For each time point, averages of the rescence microscopic examination of acridine orange-stained chromogenic readout (OD492) performed in triplicates for all four animals blood smears indicated an intraerythrocytic localization of bacteria (C57BL/6 #1–4) were plotted and SDs are indicated as bars. (data not shown). The humoral response triggered by B. grahamii infection in C57BL/6 was measured by a solid-phase ELISA. IgG1 and IgG2a by high relative titers of IgG2a compared with a moderate IgG1 titers (Fig. 1B) were determined by serial dilution of serum from response (Fig. 1C). day 45 postinfection. Appropriate dilutions of all serum samples To evaluate the specific Igs produced by B cells and eventually were prepared (1:15 for IgG1 and 1:2000 for IgG2a) to measure an additional contribution of T cells for abrogating infection, the the time course of Ab titers in the linear range of the ELISA (Fig. bacteremic course in infected immunocompetent C57BL/6 mice 1C). These data clearly demonstrate that B. grahamii infection of was compared with that in congenic immunocompromised ani- wild-type C57BL/6 mice triggers a strong Ab response dominated mals. In IghϪ/Ϫ mice lacking Ig-producing B cells and in RagϪ/Ϫ mice devoid of both an intact B and T cell compartment, the he- 3 Abbreviation used in this paper: CB-agar, Columbia blood agar with 5% defi- motropic infection increased for at least 4 wk and persisted at a brinated sheep blood. steady level (Fig. 2). These data clearly demonstrate that B cells or The Journal of Immunology 13

FIGURE 2. Course of B. grahamii bacteremia in immunodeficient (T and/or B cell-deficient) mice. C57BL/6-Rag1Ϫ/Ϫ (n ϭ 5), C57BL/6-IghϪ/Ϫ (n ϭ 5), and C57BL/6 wild type (n ϭ 4) were B. grahamii infected by i.v. injection of 2 ϫ 109 CFU/200 ␮l PBS. Citrate blood samples were col- lected on days 5, 10, 17, 31, 45, 59, and 77 postinfection. CFU per milliliter of citrate blood were determined by culture on CB-agar plates performed in triplicates. Average values were plotted and SDs indicated as bars. Downloaded from

Abs were necessary to abrogate an infection of B. grahamii. The additional absence of T cells in RagϪ/Ϫmice did not result in higher CFUs/ml blood than in IghϪ/Ϫ mice. Interestingly, the per-

sistent bacteremia in both immunodeficient mouse strains is not http://www.jimmunol.org/ fatal, indicating a high degree of adaptation of B. grahamii to cause long-lasting infections in its reservoir host organism. FIGURE 3. Effect of serum transfer on B. grahamii infection of C57BL/ To evaluate the role of Abs specific for B. grahamii to control 6-IghϪ/Ϫ (B cell-deficient) mice. A, IgG1 and IgG2a titers in serum pre- bacteremia, we adoptively transferred serum Igs to infected recip- cipitates. Serum pooled from 20 naive or B. grahamii-infected C57BL/6 ients. Igs from serum pools from naive (normal serum) or B. gra- mice were precipitated, dialyzed, and adjusted to 1 mg protein/300 ␮l PBS. hamii infected C57BL/6 mice (immune serum) were precipitated B. grahamii-reactive immunoglobulins within serial dilutions of serum pre- by ammonium sulfate and dialyzed against PBS. Ig obtained from cipitates were determined by ELISA and the average values of the chro- mogenic readout (OD492) performed in triplicates were plotted. B, Time immune serum contained relatively high titers of IgG2a and mod- Ϫ Ϫ course of the bacteremia. C57BL/6-Igh / mice (n ϭ 5) were infected with by guest on September 27, 2021 erate titers for IgG1 directed against B. grahamii Ags (Fig. 3A). B. grahamii followed on days 6, 11, 18, and 32 postinfection by i.v. in- This composition agreed with the relative titers of IgG isotypes in jection of either PBS or 1-mg protein doses of normal or infected serum sera from infected mice (Fig. 1B). On days 6, 11, 18, and 32 precipitate. Citrate blood samples were collected on days 5, 10, 17, 31, 45, postinfection, Igs were injected i.v. into infected immunodeficient 59, and 77 postinfection. CFU per mililiter of citrate blood were deter- recipients. The success of the transfer of specific Abs was con- mined by culture on CB-agar plates performed in triplicates. Average val- firmed by determining anti-B. grahamii IgG titers in serum from ues were plotted and SDs are indicated as bars. the recipient mice (data not shown). Fig. 3B illustrates that transfer of the PBS carrier or Ig from normal serum had no measurable effect on the persistence of bacteremia in IghϪ/Ϫ mice. In contrast, transfer of immune serum Ig resulted in a transient bacteremia (except for B. bacilliformis in humans) and persists the life span indistinguishable from the course of Bartonella bacteremia ob- of this terminally differentiated cell type (6), the course of the served in immunocompetent C57BL/6 mice (cf Fig. 2). These data hemotropic Bartonella infection should reflect the average life- conclusively demonstrate an essential role for Abs in terminating time of erythrocytes. The characteristic prolonged course of the B. grahamii infection in mice. bacteremia in immunocompetent mice or IghϪ/Ϫ mice after im- There appear to be multiple mechanisms by which Ab can mune serum transfer is indeed consistent with this idea. influence the course of infections with intracellular pathogens In summary, we have established a mouse model for transient (17). For example, specific Abs bind to Plasmodium spp. Ag and persistent B. grahamii infection. By immune serum transfer to exposed on the surface of infected erythrocytes (29). Such bind- infected immunocompromised mice, we provide convincing evi- ing may cause complement-mediated host cell lysis and opso- dence that a specific Ab response is required to abrogate the in- nization. Opsonized erythrocytes are more prone to macrophage traerythrocytic bacteremia. For the design of vaccination strategies phagocytosis (30). If a similar mechanism would operate against zoonotic Bartonella in their mammalian reservoirs, this against Bartonella-infected erythrocytes, infections should be model should allow testing if transferred Abs alone can provide rapidly cleared after immune serum transfer. Instead, we favor immunity to primary challenge infection and may aid identifying the idea that Ab mediate killing of extracellular Bartonella or Ags that elicit neutralizing Ab. prevent their invasion into erythrocytes. We have shown that in rats invasion of erythrocytes occurs in periodical waves every Acknowledgments 3–6 days, seeded from a yet uncharacterized primary niche (6). We thank Dr. Yves Pie´mont (University Louis Pasteur, Strasbourg, France) If these reinfection waves are terminated by neutralizing Ab, for providing bacterial strains. We are grateful to Robert Hurwitz and An- the intraerythrocytic bacteremia should remain until the in- nette Dietrich for their excellent technical assistance. We also thank Chris- fected, aging erythrocyte population is cleared. Since the infec- topher Snyder for critically reading this manuscript. We thank Dr. Thomas tion of erythrocytes by bartonellae is typically nonhemolytic F. Meyer for his interest and continuous support. 14 CUTTING EDGE: Ab-MEDIATED CESSATION OF Bartonella INFECTION

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