Mary M. Petzke

Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-β

Phagocytosed Borrelia burgdorferi (Bb) induces inflammatory signals that differ both quantitatively and qualitatively from those generated by spirochetal lipoproteins interacting with Toll-like receptor (TLR) 1/2 on the surface of human monocytes. Of particular significance, and in contrast to lipoproteins, internalized spirochetes induce transcription of IFN-β. Using inhibitory immunoregulatory DNA sequences (IRSs) specific to TLR7, TLR8, and TLR9, we show that the TLR8 inhibitor IRS957 significantly diminishes production of TNF-α, IL-6, and IL-10 and completely abrogates transcription of IFN-β in Bb-stimulated monocytes. We demonstrate that live Bb induces transcription of TLR2 and TLR8, whereas IRS957 interferes with their transcriptional regulation. Using confocal and epifluorescence microscopy, we show that baseline TLR expression in unstimulated monocytes is greater for TLR2 than for TLR8, whereas expression of both TLRs increases significantly upon stimulation with live spirochetes. By confocal microscopy, we show that TLR2 colocalization with Bb coincides with binding, uptake, and formation of the phagosomal vacuole, whereas recruitment of both TLR2 and TLR8 overlaps with degradation of the spirochete. We provide evidence that IFN regulatory factor (IRF) 7 is translocated into the nucleus of Bb-infected monocytes, suggesting its activation through phosphorylation. Taken together, these findings indicate that the phagosome is an efficient platform for the recognition of diverse ligands; in the case of Bb, phagosomal signaling involves a cooperative interaction between TLR2 and TLR8 in pro- and antiinflammatory cytokine responses, whereas TLR8 is solely responsible for IRF7-mediated induction of IFN-β.

Recognition of Borrelia burgdorferi, the Lyme Disease Spirochete, by TLR7 and TLR9 Induces a Type I IFN Response by Human Immune Cells

Borrelia burgdorferi is the spirochetal agent of Lyme disease, a multisystemic disorder characterized by inflammation. Using global transcriptional profiling, we characterized the response of human PBMCs exposed to B. burgdorferi in an ex vivo coculture system. The expression profiles induced by B. burgdorferi were marked by the intense up-regulation of IFN-responsive transcripts and transcripts involved in the JAK/STAT signaling pathway. Transcript levels of IFN-α, IFN-β, and IRF7, and protein concentrations of IFN-α, were significantly elevated relative to those in unstimulated PBMCs. The induction of IFN-α was completely dependent upon phagocytosis of B. burgdorferi. Addition of a soluble type I IFN receptor, B18R, did not abolish the induction of IFN-inducible genes, indicating that B. burgdorferi directly elicits enhanced expression of these genes independently of type I IFN feedback signaling. Inhibitors of either TLR7 or TLR9 significantly reduced B. burgdorferi-stimulated IFN-α protein expression and transcription of IFN-induced genes. Simultaneous inhibition of both TLR7 and TLR9 completely abrogated IFN-α induction. The IFN-α-producing populations in PBMCs were identified as plasmacytoid dendritic and CD14+CD11c+ cells. These results reveal a TLR7/9-dependent signaling pathway used by human PBMCs to initiate a type I IFN response to the extracellular bacterium B. burgdorferi.

Borrelia burgdorferi Requires Glycerol for Maximum Fitness During The Tick Phase of the Enzootic Cycle

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is a vector-borne pathogen that cycles between a mammalian host and tick vector. This complex life cycle requires that the spirochete modulate its gene expression program to facilitate growth and maintenance in these diverse milieus. B. burgdorferi contains an operon that is predicted to encode proteins that would mediate the uptake and conversion of glycerol to dihydroxyacetone phosphate. Previous studies indicated that expression of the operon is elevated at 23°C and is repressed in the presence of the alternative sigma factor RpoS, suggesting that glycerol utilization may play an important role during the tick phase. This possibility was further explored in the current study by expression analysis and mutagenesis of glpD, a gene predicted to encode glycerol 3-phosphate dehydrogenase. Transcript levels for glpD were significantly lower in mouse joints relative to their levels in ticks. Expression of GlpD protein was repressed in an RpoS-dependent manner during growth of spirochetes within dialysis membrane chambers implanted in rat peritoneal cavities. In medium supplemented with glycerol as the principal carbohydrate, wild-type B. burgdorferi grew to a significantly higher cell density than glpD mutant spirochetes during growth in vitro at 25°C. glpD mutant spirochetes were fully infectious in mice by either needle or tick inoculation. In contrast, glpD mutants grew to significantly lower densities than wild-type B. burgdorferi in nymphal ticks and displayed a replication defect in feeding nymphs. The findings suggest that B. burgdorferi undergoes a switch in carbohydrate utilization during the mammal to tick transition. Further, the results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.

Borrelia burgdorferi bba74 is expressed exclusively during tick feeding and is regulated by both arthropod- and mammalian host-specific signals.

Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in the Borrelia burgdorferi tick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA, ospC, and ospE) and their proteins. In vitro, transcripts for bba74 were expressed at 23 degrees C and further enhanced by a temperature shift (37 degrees C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-type B. burgdorferi grown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type and rpoS mutant spirochetes, revealed that, like ospA, bba74 is transcribed by sigma(70) and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type and rpoS mutant spirochetes was conducted to determine the transcriptional and translational profiles of bba74 during the tick-mouse cycle. Results from these studies revealed (i) that bba74 is transcribed by sigma(70) exclusively during the larval and nymphal blood meals and (ii) that transcription of bba74 is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of B. burgdorferi to complete its infectious life cycle, the temporal compartmentalization of this genes transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation of ospA and ospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue as B. burgdorferi transitions between its arthropod vector and mammalian host.

Borrelia burgdorferi RNA Induces Type I and III Interferons via Toll-Like Receptor 7 and Contributes to Production of NF-κB-Dependent Cytokines

ABSTRACT Borrelia burgdorferi elicits a potent cytokine response through activation of multiple signaling receptors on innate immune cells. Spirochetal lipoproteins initiate expression of NF-κB-dependent cytokines primarily via TLR2, whereas type I interferon (IFN) production is induced through the endosomal receptors TLR7 and TLR9 in human dendritic cells and TLR8 in monocytes. We demonstrate that DNA and RNA are the B. burgdorferi components that initiate a type I IFN response by human peripheral blood mononuclear cells (PBMCs). IFN-α protein and transcripts for IRF7, MX1, and OAS1 were induced by endosomal delivery of B. burgdorferi DNA, RNA, or whole-cell lysate, but not by lysate that had been treated with DNase and RNase. Induction of IFN-α and IFN-λ1, a type III IFN, by B. burgdorferi RNA or live spirochetes required TLR7-dependent signaling and correlated with significantly enhanced transcription and expression of IRF7 but not IRF3. Induction of type I and type III IFNs by B. burgdorferi RNA could be completely abrogated by a TLR7 inhibitor, IRS661. In addition to type I and type III IFNs, B. burgdorferi RNA contributed to the production of the NF-κB-dependent cytokines, IFN-γ, interleukin-10 (IL-10), IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α), by human PBMCs. Collectively, these data indicate that TLR7-dependent recognition of RNA is pivotal for IFN-α and IFN-λ1 production by human PBMCs, and that RNA-initiated signaling contributes to full potentiation of the cytokine response generated during B. burgdorferi infection.

Pattern of Proinflammatory Cytokine Induction in RAW264.7 Mouse Macrophages Is Identical for Virulent and Attenuated Borrelia burgdorferi

Lyme disease pathogenesis results from a complex interaction between Borrelia burgdorferi and the host immune system. The intensity and nature of the inflammatory response of host immune cells to B. burgdorferi may be a determining factor in disease progression. Gene array analysis was used to examine the expression of genes encoding cytokines, chemokines, and related factors in the joint tissue of infected C3H/HeJ mice and in a murine macrophage-like cell line in response to a disseminating or attenuated clinical isolate of B. burgdorferi. Both isolates elicited a robust proinflammatory response in RAW264.7 cells characterized by an increase in transcript levels of genes encoding CC and CXC chemokines, proinflammatory cytokines, and TNF superfamily members. Transcription of genes encoding IL-1β, IL-6, MCP-1, MIP-1α, CXCR4, and TLR2 induced in RAW264.7 cells by either live or heat-killed spirochetes did not differ significantly at any time point over a 24-h period, nor was there a difference in the protein levels of IL-10, TNF-α, IL-6, and IL-12p70 in culture supernatants. Thus, induction of host macrophage expression of proinflammatory mediators by host macrophages does not contribute to the differential pathogenicity of different B. burgdorferi strains.

Borrelia burgdorferi Pathogenesis and the Immune Response.

Borrelia burgdorferi is the tick-borne etiologic agent of Lyme disease. The spirochete must negotiate numerous barriers in order to establish a disseminated infection in a mammalian host. These barriers include migration from the feeding tick midgut to the salivary glands, deposition in skin, manipulation or evasion of the localized host immune response, adhesion to and extravasation through an endothelial barrier, hematogenous dissemination, and establishment of infection in distal tissue sites. Borrelia burgdorferi proteins that mediate many of these processes and the nature of the host response to infection are described.

Induction of type I and type III interferons by Borrelia burgdorferi correlates with pathogenesis and requires linear plasmid 36.

The capacity for Borrelia burgdorferi to cause disseminated infection in humans or mice is associated with the genotype of the infecting strain. The cytokine profiles elicited by B. burgdorferi clinical isolates of different genotype (ribosomal spacer type) groups were assessed in a human PBMC co-incubation model. RST1 isolates, which are more frequently associated with disseminated Lyme disease in humans and mice, induced significantly higher levels of IFN-α and IFN-λ1/IL29 relative to RST3 isolates, which are less frequently associated with disseminated infection. No differences in the protein concentrations of IFN-γ, IL-1β, IL-6, IL-8, IL-10 or TNF-α were observed between isolates of differing genotype. The ability of B. burgdorferi to induce type I and type III IFNs was completely dependent on the presence of linear plasmid (lp) 36. An lp36-deficient B. burgdorferi mutant adhered to, and was internalized by, PBMCs and specific dendritic cell (DC) subsets less efficiently than its isogenic B31 parent strain. The association defect with mDC1s and pDCs could be restored by complementation of the mutant with the complete lp36. The RST1 clinical isolates studied were found to contain a 2.5-kB region, located in the distal one-third of lp36, which was not present in any of the RST3 isolates tested. This divergent region of lp36 may encode one or more factors required for optimal spirochetal recognition and the production of type I and type III IFNs by human DCs, thus suggesting a potential role for DCs in the pathogenesis of B. burgdorferi infection.

Identification of OppA2 linear epitopes as serodiagnostic markers for Lyme disease.

ABSTRACT Laboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agent Borrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number of B. burgdorferi antigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique to B. burgdorferi as well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique to B. burgdorferi as antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during early B. burgdorferi infection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenic Borrelia species responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease.

Borrelia burgdorferi induces a type I interferon response during early stages of disseminated infection in mice.

BackgroundLyme borrelia genotypes differ in their capacity to cause disseminated disease. Gene array analysis was employed to profile the host transcriptome induced by Borrelia burgdorferi strains with different capacities for causing disseminated disease in the blood of C3H/HeJ mice during early infection.ResultsB. burgdorferi B515, a clinical isolate that causes disseminated infection in mice, differentially regulated 236 transcripts (P < 0.05 by ANOVA, with fold change of at least 2). The 216 significantly induced transcripts included interferon (IFN)-responsive genes and genes involved in immunity and inflammation. In contrast, B. burgdorferi B331, a clinical isolate that causes transient skin infection but does not disseminate in C3H/HeJ mice, stimulated changes in only a few genes (1 induced, 4 repressed). Transcriptional regulation of type I IFN and IFN-related genes was measured by quantitative RT-PCR in mouse skin biopsies collected from the site of infection 24 h after inoculation with B. burgdorferi. The mean values for transcripts of Ifnb, Cxcl10, Gbp1, Ifit1, Ifit3, Irf7, Mx1, and Stat2 were found to be significantly increased in B. burgdorferi strain B515-infected mice relative to the control group. In contrast, transcription of these genes was not significantly changed in response to B. burgdorferi strain B331 or B31-4, a mutant that is unable to disseminate.ConclusionsThese results establish a positive association between the disseminating capacity of B. burgdorferi and early type I IFN induction in a murine model of Lyme disease.