Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and -independent responses which include induction of IFN-beta

Abstract

It is widely believed that innate immune responses to Borrelia burgdorferi (Bb) are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs) elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-beta and a number of interferon-stimulated genes (ISGs), which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-alpha, IL-6, IL-10 and IL-1beta in monocytes than did lysates. Secreted IL-18, which, like IL-1beta, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-beta and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs.

Figure 1. Distinct gene expression profiles obtained from human PBMCs stimulated with either live or lysed Borrelia burgdorferi.

Genes whose normalized intensity values were deemed to be statistically significant in the PBMC array, and had a known biologic function, were used to determine the total number of genes whose intensity values were proportionately higher or lower in PBMCs in response to either live Bb and/or lysed Bb vs. unstimulated conditions. The top half of the Venn diagrams show the total number of gene transcripts that were more intensely or exclusively regulated by live and/or lysed Bb vs. unstimulated conditions (N = 213 genes) (A), or less intensely or exclusively down-regulated by live and/or lysed Bb vs. unstimulated cells (N = 187) (B). For similarly up-regulated (N = 177) or down-regulated (N = 170) genes, the arrows point down to a direct comparative analysis between live or lysed Bb generated gene intensity values. Genes in this category were further stratified depending on whether or not they were proportionately higher (live to lysed ratio >1.5), lower (live to lysed ratio <1.5) or equal between live vs lysed Bb stimulated cells.

Figure 2. Live Borrelia burgdorferi elicits greater cytokine transcription and secretion from isolated human monocytes than borrelial lysates.

Monocytes were incubated with live or lysed Bb at various MOIs (1∶10∶100), 100 ng/ml of lipopolysacharide (LPS) and 10 µg/ml the TLR2 synthetic ligand MMP (Mitogenic pentapeptide). Transcription (4 hour stimulation experiments) and secretion (8 hour stimulation experiments) of IL1-β, TNF-α and IL-6 was determined by either qRT-PCR (fold increased in transcript copies compared to unstimulated cells) or bead array cytokine concentrations (pg/ml) in supernatants. Bars depict the means+/−standard error of the mean from a minimum of four independent experiments. P-values for transcriptional and translational comparisons between live and lysed Bb for each of the equivalent MOIs (1, 10 and 100) are shown above the corresponding bar.

Figure 3. IL-18 is secreted in response to live Bb but not lysates.

Monocytes were either unstimulated or incubated with live or lysed Bb at various MOIs (1∶10∶100) and 100 ng/ml lipopolysacharide (LPS). Secreted IL-18 was determined by ELISA as described in the Methods. Bars depict the mean value (pg/ml)+/−standard error of the mean from three independent experiments. P-values calculated for comparative cytokine production between cells stimulated by live and lysed Bb at equivalent MOI are shown above the corresponding bars. NS = not significant.

Figure 4. Live Bb, but not borrelial lysates or synthetic TLR2 ligands, induce IFN-β transcription in isolated human monocytes.

Monocytes were incubated with live or lysed Bb MOI (10), 100 ng/ml of lipopolysacharide (LPS) and the 10 µg/ml of the TLR2 ligand MMP (Mitogenic pentapeptide). Transcription of IFN-β was determined by qRT-PCR. Bars depict the means+/−standard error of the mean from a minimum of four independent experiments. P value shown corresponds to the statistical comparison between live and lysed Bb (MOI 10∶1) induced IFN-β transcripts.

Figure 5. Differentially regulated type I interferons genes monocytes were incubated with live or lysed Bb MOI (10∶1) and gene intensity values were determined by a type I interferon.

RT² Profiler Array system. (A) The figure shows comparative analysis between gene transcript values generated by monocytes stimulated with live Bb vs unstimulated cells. Genes depicted outside the lines were 4 fold higher or lower and deemed to be statistically significantly differentially regulated (p<0.01) in the array. (B) Comparative analysis between gene transcript values generated by bacterial lysates vs unstimulated cells.

Figure 6. Isolated monocytes contain phagocytosed and degraded B. burgdorferi but not intact spirochetes.

(A) Epifluorescence image (100×) acquired from human monocytes incubated for 4-hours with Bb-GFP (MOI 100∶1) and labeled with the cell membrane marker FM4-64 (red) and the nuclear dye DAPI (blue). Phagocytosed (both coiled and degraded) Bb-GFP and fragmented nuclei were observed. (B–F) Orthogonal view (y,z and x,z axes) of optical sections (x,y axes) through a confocal stack of isolated monocytes incubated with Bb-GFP (MOI 100∶1) and labeled with lysotracker (red). (B) Digital enlargement of an extracellular spirochete (labeled # 1) in close proximity to a monocyte. Panels C–F are presented in 2 µm increments [(C) 6 µm, (D) 8 µm (E) 10 µm and (F) 12 µm] through the depth of the monocytes (total depth = 14 µm). Arrows and asterisks point to several internalized and degraded Bb-GFP, whereas colocalized phagolysosomes and fluorescent spirochetes shown in yellow are indicated by the arrowhead. The numbers 1 and 2 are placed next to individual extracellular Bb-GFP. Shapes within x,y axes are represented with their corresponding position in the x,z and y,z axes.

Figure 7. TLR2 deficient and wild type murine macrophages (peritoneal macrophages or bone marrow derived macrophages) internalize Borrelia burgdorferi (Bb), and generate similar cytokine responses.

(A) Wild type (WT) or TLR2-deficient (TLR2^−/−) mouse derived peritoneal macrophages were incubated for six hours with Bb-GFP (MOI 100∶1) and analyzed for GFP expression by flow cytometry. Individual macrophage populations shown in the cytograms were selectively gated for analysis based on F4/80 PE expression and Bb-GFP signal. (B) Representative confocal image of internalized Bb-GFP also labeled with lysotracker in TLR2^−/− bone marrow derived macrophages incubated for six-hours with live Bb (MOI 100∶1). Inset shows a bar graph depicting the percentage of WT or TLR2^−/− macrophages that had spirochetes contained within phagosomal vacuoles. (C) Murine peritoneal macrophages were stimulated for six-hours with: 100 ng/ml of LPS, 10 µg/ml of Mitogenic Pentapeptide (MMP), live Bb-GFP or spirochetal lysates (MOI 10∶1) and compared to unstimulated (UN) cells. The bars represent the average TNF-α concentration (pg/ml) and standard error of the mean calculated from five independent experiments. (D) RNA from similarly stimulated bone marrow derived macrophages (MOI 10∶1) was extracted for qRT-PCR pro-IL-1β quantitation. Fold increase in transcript copies between each of the stimuli is compared to unstimulated cells. Bars depict the means+/−standard error of the mean from a minimum of three independent experiments. P values shown in both C and D correspond to the statistical comparison between WT and TLR2 −/− stimulated macrophages.

Figure 8. Wild type (WT) and TLR2 deficient bone marrow derived macrophages (BMDMs) generate similar IFN-β responses to Bb.

WT or TLR2−/− mouse BMDMs were incubated for six hours with Bb (MOI 10∶1). Fold increase in IFN-β transcript copies measured from Bb stimulated BMDMs are compared to control values from unstimulated cells. Bars depict the means+/−standard error of the mean. Values were not statistically significantly different.

Figure 9. Flagellin deficient Bb induces NF-κB mediated cytokine responses in human monocytes.

Isolated human monocytes were incubated with live flagellin deficient Bb at various MOIs (1∶10∶100), 100 ng/ml of lipopolysacharide (LPS) and 10 µg/ml of the synthetic TLR2 ligand MMP (Mitogenic pentapeptide). IL-1β, TNF-α, IL-6 and IL-10 protein concentrations (pg/ml) were quantitated (see Methods) in supernatants obtained from 8 hour stimulation experiments. Bars depict the means (pg/ml)+/−standard error of the mean for each cytokine measured from two independent experiments.