Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 is online at: average * The Journal of Immunology Anaplasma phagocytophilum , 20 of which you can access for free at: 2005; 174:6364-6372; ; from submission to initial decision 4 weeks from acceptance to publication J Immunol doi: 10.4049/jimmunol.174.10.6364 http://www.jimmunol.org/content/174/10/6364 Insights into Pathogen Immune Evasion Mechanisms: Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils Dori L. Borjesson, Scott D. Kobayashi, Adeline R. Whitney, Jovanka M. Voyich, Cynthia M. Argue and Frank R. DeLeo cites 28 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2005/05/03/174.10.6364.DC1 This article http://www.jimmunol.org/content/174/10/6364.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* • Why • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Insights into Pathogen Immune Evasion Mechanisms: Anaplasma phagocytophilum Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils1 Dori L. Borjesson,2* Scott D. Kobayashi,† Adeline R. Whitney,† Jovanka M. Voyich,† Cynthia M. Argue,* and Frank R. DeLeo† Polymorphonuclear leukocytes (PMNs or neutrophils) are essential to human innate host defense. However, some bacterial patho- gens circumvent destruction by PMNs and thereby cause disease. Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, survives within PMNs in part by altering normal host cell processes, such as production of reactive oxygen species (ROS) and apoptosis. To investigate the molecular basis of A. phagocytophilum survival within neutrophils, we used Affymetrix microarrays to measure global changes in human PMN gene expression following infection with A. phagocytophilum. Notably, A. Downloaded from phagocytophilum uptake induced fewer perturbations in host cell gene regulation compared with phagocytosis of Staphylococcus aureus. Although ingestion of A. phagocytophilum did not elicit significant PMN ROS, proinflammatory genes were gradually up-regulated, indicating delayed PMN activation rather than loss of proinflammatory capacity normally observed during phago- cytosis-induced apoptosis. Importantly, ingestion of A. phagocytophilum failed to trigger the neutrophil apoptosis differentiation program that typically follows phagocytosis and ROS production. Heat-killed A. phagocytophilum caused some similar initial alterations in neutrophil gene expression and function, which included delaying normal PMN apoptosis and blocking Fas-induced http://www.jimmunol.org/ programmed cell death. However, at 24 h, down-regulation of PMN gene transcription may be more reliant on active infection. Taken together, these findings suggest two separate antiapoptotic processes may work concomitantly to promote bacterial sur- vival: 1) uptake of A. phagocytophilum fails to trigger the apoptosis differentiation program usually induced by bacteria, and 2) a protein or molecule on the pathogen surface can mediate an early delay in spontaneous neutrophil apoptosis. The Journal of Immunology, 2005, 174: 6364–6372. olymorphonuclear leukocytes (PMNs3 or neutrophils) are lum-infected PMNs suggest that the pathogen delays PMN apo- a first line of defense in the human innate immune re- ptosis (6, 7), minimizes proinflammatory cytokine release (8, 9), by guest on September 25, 2021 P sponse to bacterial pathogens. Most ingested bacteria are and inhibits and/or fails to activate ROS production (10–13). killed by the combined effects of PMN reactive oxygen species Mechanisms underlying the ability of A. phagocytophilum to in- (ROS) and cytotoxic granule components (1). However, some hibit production of PMN ROS are unclear (12–15). pathogens have evolved means to circumvent killing by PMNs and Recent studies indicate bacterial phagocytosis induces an apo- cause disease. The mechanisms used by pathogens to evade de- ptosis differentiation program in human PMNs that most likely struction by the innate immune system are incompletely contributes to the resolution of infections (16, 17). The apoptosis characterized. differentiation program is characterized by changes in a common Anaplasma phagocytophilum, the agent of human granulocytic set of genes triggered by uptake of bacteria or other particles that anaplasmosis, is an obligate intracellular bacterium known to sur- accelerate normal PMN apoptosis (16, 17). In contrast, A. phago- vive within PMNs (2, 3). A. phagocytophilum enters neutrophils cytophilum delays normal PMN apoptosis to facilitate productive primarily through a receptor-mediated endocytic pathway (4), and intracellular infection (6, 7). Inasmuch as induction of PMN apo- thus may ultimately reside in a modified endosomal compartment ptosis is regulated at the level of transcription (16–18), a compre- (5). Studies evaluating functional alterations in A. phagocytophi- hensive view of gene expression patterns in A. phagocytophilum- infected PMNs is critical to understanding processes that permit *Department of Veterinary Population Medicine, College of Veterinary Medicine, the pathogen to reside within PMNs. To that end, we studied University of Minnesota, St. Paul, MN 55108; and †Laboratory of Human Bacterial global gene expression in human PMNs during infection with A. Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infec- phagocytophilum. Our results suggest failure of A. phagocytophi- tious Diseases, Hamilton, MT 59840 lum to trigger the PMN apoptosis differentiation program and abil- Received for publication August 19, 2004. Accepted for publication March 2, 2005. ity of the organism to inhibit spontaneous neutrophil apoptosis are The costs of publication of this article were defrayed in part by the payment of page distinct processes that contribute to pathogen survival. 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 in part by Grant AI-51529 (to D.L.B.) from the National Materials and Methods Institutes of Health. Materials 2 Address correspondence and reprint requests to Dr. Dori L. Borjesson, Department PLUS of Veterinary Population Medicine, College of Veterinary Medicine, University of Dextran T-500 and Ficoll-Paque (1.077 g/L) were purchased from Minnesota, 1365 Gortner Avenue, St. Paul, MN 55108. E-mail address: Amersham Biosciences. Sterile water ((Irrigation, United States Pharma- [email protected] copoeia (USP)) and 0.9% sodium chloride (Irrigation, USP) were obtained 3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; BAX, B cell from Baxter Healthcare. PE-conjugated mAb specific for IL-1R antagonist line 2-associated X-protein; IL-1RN, IL-1R antagonist; ROS, reactive oxygen spe- (IL-1RN) (clone AS17) and isotype control mAb were purchased from BD cies; RPMI/H, RPMI 1640 medium buffered with 10 mM HEPES. Biosciences. RPMI 1640 medium was purchased from Invitrogen Life Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 6365 Technologies. Unless indicated, all other reagents were obtained from incubator with 5% CO2 for up to 24 h. At the indicated times, medium was Sigma-Aldrich. aspirated and cells were washed with 500 ␮l of cold PBS and fixed with 4% paraformaldehyde on ice for 30 min. Fixative was aspirated, and cells Neutrophil isolation were washed with 500 ␮l of PBS. Uningested bacteria were then counter- Human PMNs were isolated from heparinized venous blood of healthy stained with anti-Alexa Fluor 488 Ab conjugated to Alexa Fluor 594 in 500 ␮ ␮ individuals with a widely used method (18). All studies with human blood l of PBS (7.5 g/ml final Ab concentration) (Molecular Probes) for 15 ␮ were performed in accordance with protocols approved by the Institutional min. Coverslips/Cells were washed twice in 500 l of PBS and mounted Review Board for Human Subjects at the University of Minnesota and the onto microscope slides with mounting medium (Aqua Polymount; Poly- National Institute of Allergy and Infectious Diseases. Blood was mixed 1:1 sciences). One hundred PMNs from random fields of view were evaluated with 0.9% sodium chloride (Baxter Healthcare) containing 3.0% Dextran at each time point, and ingested bacteria were scored using a fluorescence T-500 (Amersham) for 20 min at room temperature to sediment eryth- microscope (Nikon E800 microscope; Nikon). Bacteria stained with Alexa rocytes. The leukocyte-enriched supernatant was centrifuged at 670 ϫ g for Flour 488 (green only) were scored as ingested, and those stained with 10 min and resuspended in 35 ml of 0.9% sodium chloride. Ficoll- Alexa Fluor 488 and Alexa Fluor 594 were counted as uningested/surface PaquePLUS (10 ml) was pipetted carefully beneath the cell suspension