Changes in transcription patterns in BoMac cells in response to PDD spirochetes

Papillomatous digital dermatitis (PDD) is a polymicrobial infection in soft tissue adjacent to the hoof and is the leading cause of lameness in dairy cattle. Treponema phagedenis-like (TPL) spirochetes are a constant feature of PDD lesions and are localized deep in infected tissue. Host-cell response mechanisms to TPL bacteria are poorly understood. To assess how bovine macrophages respond to cellular constituents of TPL spirochetes, changes in transcription were analyzed using serial analysis of gene expression (SAGE) and real time RT-PCR. This analysis revealed that bovine macrophages responded to stimulation with TPL-lysates by induction of several pro-apoptotic genes. Although several proinflammatory cytokines (eg. IL-6 and IL-8) are induced in treated macrophages, receptors and their accessory proteins for IL-1, IL-6, and IL-11 are either down regulated or unchanged. Surprisingly, IL-1beta is not induced and IL-18 is suppressed in treated macrophages. Two genes encoding proteins having negative effects on NF?B, I?B and Siva-1, are significantly induced in stimulated cells. Combined, these data suggest that the innate immune response of bovine macrophages exposed to TPL cellular constituents are impaired thereby enabling bacteria to resist clearance and induce lesion formation. Keywords: Bovine macrophage, transcription response, spirochete bacteria Overall design: An immortalized bovine macrophage cell line (BoMac) was used throughout this study (supplied by Dr. Judith Stabel, USDA, ARS, NADC, Ames, IA). Cells were propagated in RPMI 1640 medium (GIBCO, Gaithersburg, MD) containing 10% fetal calf serum (Colorado Serum Company), 100µg/ml streptomycin, and 100U/ml penicillin (Sigma, St. Louis, MO) at 37C with 5% CO2. PDD spirochetes (Treponema phagedenis-like strain 1A) in the exponential phase of growth were harvested by centrifugation (10,000g for 15 min.) and washed twice with PBS. Cell lysates were prepared by sonication using short-wave pulses of 30s each (Vibra Cell, Sonics and Materials Inc, Danbury, Connecticut) until no intact cells were observed by dark field microscopy. Protein concentrations of whole cell sonicates (WCS) were determined using the Bradford assay (Bio-Rad, Hercules, CA) following the manufacturers instructions. Bovine macrophages grown to about 90% confluence were stimulated with WCS lysate of TPL strain 1A (1 µg/ml) suspended in complete RPMI 1040. Cells were incubated at 37oC for 4, 8, and 18 hrs. Cell monolayers were disrupted with RLT+ lysis solution and total cellular RNA isolated with RNeasy kits (Qiagen) according to the manufacturer’s instructions. Where appropriate cell suspensions were further disrupted by homogenization using Qiashredder columns (Qiagen). Residual DNA was removed by DNaseI treatment, followed by RNA purification (Qiagen). Cellular mRNA was enriched from total RNA using PolyATract mRNA isolation system II (Promega, Madison, WI) and used as starting material for preparation of SAGE libraries. Two SAGE libraries were prepared from 5 ug ds-cDNA, one each from non-stimulated and WCS-stimulated macrophages (4 hr treatment) following established protocols using NlaIII as the anchoring enzyme. Concatermerized ditags were size selected by electrophoresis through polyacrylamide gels, then ligated with SphI-digested pZERO-1 (Invitrogen Corp.) and used to transform E. coli to zeocin resistance. Transformed colonies were picked at random to 96-well plates and plasmid DNA prepared by alkaline lysis (Qiagen). Plasmid DNA was sequenced unidirectionally from the T7 primer region using fluorescent dideoxy chain-termination reactions separated in a 3700-sequence analyzer (Applied Biosystems, Inc.). Sequence data interpreted using Phred and Sequencher (Gene Codes Corporation, Ann Arbor, MI) were combined and edited manually. Inserts from a total of 2155 and 2131 plasmids were sequenced with average error rates of 0.00435 and 0.00977 from control and stimulated cell libraries, respectively. Data files for each library were processed with eSAGE software to extract ditags, remove duplicate sequences, and compare the two libraries to determine statistically significant differences in tag frequency. SAGE data were validated by quantifying transcription levels of selected genes using RNA isolated from three distinct biological replicates each of treated and control samples. Semiquantitative RT-PCR was done in a DNA Engine Opticon 2 detection system (MJ Research, Inc) using QuantiTect™ SYBR Green PCR Kit (Qiagen), gene specific primers (Table 1), with 200 ng/µl cDNA template. PCR primers were designed using Primer Design 5 software (Sci Ed Central) from sequences obtained from GenBank. All reactions were performed in triplicate and qRT-PCR data were analyzed by using the 2-(??Ct) method. The ß-actin gene was used as the control gene (?Ct) and non-stimulated samples were used as the calibrator (??Ct).