Impaired Colonic B-Cell Responses by Gastrointestinal Bacillus anthracis Infection

MAJOR ARTICLE Impaired Colonic B-Cell Responses by Gastrointestinal Bacillus anthracis Infection Bikash Sahay,1,2,a Jennifer L. Owen,3,a Mojgan Zadeh,1,2 Tao Yang,1,2 Yaíma L. Lightfoot,1,2 Firas Abed,1,2 and Mansour Mohamadzadeh1,2 1 Department of Infectious Diseases and Pathology, 2Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, and 3Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville Keywords. anthrax toxin; Bacillus anthracis; B-1 cells; type 2 innate lymphoid cells. Anthrax is a zoonotic disease caused by the introduction of Bacillus anthracis endospores either by respiratory, oral, or cutaneous routes. With the gastrointestinal (GI) form of the disease, symptoms begin as nausea, vomiting, mild diarrhea, fever, and headaches, which soon progress into hemorrhagic diarrhea, hematemesis, ascites, and, eventually, septic shock and death. Virulent B. anthracis contains 2 plasmids ( pXO1 and pXO2), for toxin production and capsule formation, respectively [1]. The pXO1 encodes protective antigen (PA), lethal factor (LF), and edema factor (EF); lethal factor cleaves mitogen-activated protein kinases (MAPKs) to subvert immune cells, while EF increases cellular levels of cyclic adenosine monophosphate (cAMP), causing edema [2]. PA binds to its receptors expressed on host cells and facilitates cellular entry of Received 7 March 2014; accepted 30 April 2014; electronically published 14 May 2014. a B. S. and J. L. O. contributed equally to this work. Correspondence: Mansour Mohamadzadeh, PhD, Department of Infectious Diseases and Pathology, University of Florida, 2015 SW 16th Ave, Bldg 1017, Rm V3-149, Gainesville, FL 32608 (m.zadeh@ufl.edu). The Journal of Infectious Diseases® 2014;210:1499–507 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jiu280 LF and EF. The nonphagocytic capsule that protects the bacteria from innate cells is encoded by pXO2 [3]. Both the respiratory and GI tracts are lined by mucosae; however, the presence of digestive enzymes and a greater microbial load differentiates these 2 locations. Commensal gut microbes and the immune system have coevolved over several million years [4]. One of the most common and effective responses of the mammalian host against bacteria is secretion of immunoglobulin A (IgA) at mucosal surfaces [5]. Gut mucosae secrete massive amounts of IgA, the lack of which causes dysbiosis [6]. Currently, B cells are grouped into 2 major classes, B-1 cells and B-2 cells. B-1 cells, which include CD5+ B-1a and CD5− B-1b subsets, differ from conventional B-2 cells in that they develop from fetal liver progenitors [7], represent the major Bcell subpopulation in the peritoneal and pleural cavities [8], and in steady state, produce germ line–encoded immunoglobulin M (IgM) and IgA to maintain commensals and resist common pathogens [9, 10]. B-1 cells are instrumental in producing antibodies without T-cell help, allowing for rapid antibody responses against microbial gut residents, including Bacillus species [11]. Several bacilli compose the gut microbiota [11]; therefore, IgA may play a critical role in controlling microbial infection, including GI B. anthracis B Cells in GI Anthrax • JID 2014:210 (1 November) • 1499 Ingestion of Bacillus anthracis spores causes gastrointestinal (GI) anthrax. Humoral immune responses, particularly immunoglobulin A (IgA)–secreting B-1 cells, play a critical role in the clearance of GI pathogens. Here, we investigated whether B. anthracis impacts the function of colonic B-1 cells to establish active infection. GI anthrax led to significant inhibition of immunoglobulins (eg, IgA) and increased expression of program death 1 on B-1 cells. Furthermore, infection also diminished type 2 innate lymphoid cells (ILC2) and their ability to enhance differentiation and immunoglobulin production by secreting interleukin 5 (IL-5). Such B-1–cell and ILC2 dysfunction is potentially due to cleavage of p38 and Erk1/2 mitogen-activated protein kinases in these cells. Conversely, mice that survived infection generated neutralizing antibodies via the formation of robust germinal center B cells in Peyer’s patches and had restored B-1–cell and ILC2 function. These data may provide additional insight for designing efficacious vaccines and therapeutics against this deadly pathogen. infection. Hence, we hypothesized that B. anthracis interferes with B-1–cell function to establish active infection. Herein, we report that toxins secreted by B. anthracis impair immunoglobulin secretion and surface receptor expression on B-1 cells. Additionally, type 2 innate lymphoid cells (ILC2) that support the local expansion of B-1 cells [12] are compromised. These data strongly indicate that survival from infection necessitates neutralizing antibodies by expansion of germinal center B cells in the Peyer’s patches and uncompromised function of B-1 cells and ILC2 in the gut. were incubated with 1 multiplicity of infection of B. anthracis Sterne spores or left untreated for 1, 3, and 6 hours. Treated and untreated cells were stained for B-1 and ILC2 cell markers along with MAPK phospho-specific fluorescently tagged antibodies and analyzed. Sera Analyses Immunoglobulins in the sera of Sterne-infected and uninfected A/J mice were measured, as described previously [14]. Lethal Toxin Neutralization Assay MATERIALS AND METHODS Sera of Sterne-infected A/J mice were analyzed for the presence of anti-LT antibodies, as previously described [15]. Mice and Ethics Statement B. anthracis Spore Preparation and Mouse Infections Spores were prepared from a toxigenic nonencapsulated strain of B. anthracis (Sterne), as described previously [13]. To calculate final concentrations, serial dilutions (1:10) were grown in triplicate on lysogeny broth agar plates (Sterne), and colonies were counted. Mice were orally infected with Sterne spores (109 spores/100 µL of phosphate-buffered saline [PBS] per mouse). Real-Time Polymerase Chain Reaction (PCR) Analysis RNA isolated from colons was subjected to quantitative real-time PCR analysis, as described earlier [14]. A list of primers used and their sequences can be found in Supplementary Table 1. Flow Cytometry and Antibodies Colonic lamina propria (LP) cells were isolated as previously described [14], with minor modifications. Digestion buffer consisted of Dulbecco’s modified Eagle’s medium (DMEM; (Gibco, Life Technologies) containing 0.25 mg/mL collagenase type VII (Sigma-Aldrich), 0.125 U/mL Liberase TM Research Grade (Roche Applied Science, Indianapolis, IN), 10 mM HEPES, 0.1 M CaCl2 (Sigma-Aldrich), and 5% fetal bovine serum (FBS; 3 × 10-minute digestions). Flow cytometric analyses were performed using a BD LSRFortessa (BD Biosciences). Data were analyzed with FlowJo software (Tree Star, Ashlan (...truncated)