B. burgdorferi burden in C57BL/6 WT and SLPI-/- mice.

WT and SLPI-/- mice were infected with 105 spirochetes by subcutaneous injection. (A-C) Spirochete burden in skin was assessed by ear punch biopsies at 7d (A) 14 d (B), and 21-28 d (C) post infection. (D and E) Spirochete burden in tibiotarsal joint and heart tissues was assessed at 21-28 d (D, heart, E, joint) post infection. At least n = 6 mice were infected in each group. The spirochete burden was measured by qPCR detecting flaB and normalized to mouse β-actin. Each data point represents an individual animal. Representative data were shown from three separate experiments. The error bars represent mean ± SEM and p-values are calculated using the non-parametric Mann-Whitney test.

Assessment of ankle inflammation in WT and SLPI-/- mice infected with B. burgdorferi at 21-28 dpi.

(A) Representative images are shown of the tibiotarsal joints of WT and SLPI-/- mice with/without B. burgdorferi infection at 21-28 dpi. The swelling is indicated by the red arrow. (B) Swelling of the tibiotarsal joints of individual mice was scored visually by an observer blinded to the experimental groups (scale of 0 (negative) to 3 (severe)). (C) The tibiotarsal joint of each mouse was dissected, fixed, sectioned, and stained with H&E. Representative images from B. burgdorferi-infected C57BL/6 WT and SLPI-/- mice are shown. Lower magnification (left panels, Scale bar: 100 μm) and higher magnification (right panels, Scale bar: 50 μm) of selected areas (black rectangle) are shown. (D) The severity of periarticular inflammation was scored blindly by the pathologist on a scale of 0 (negative) to 3 (severe). Infected animals are shown in red. None of the mice exhibited synovial inflammation. Results from two independent experiments were pooled and shown here. The error bars represent mean ± SEM and p-values are calculated using the non-parametric Mann-Whitney test.

Immune profile analysis of infected WT and SLPI-/- mice.

(A, B) Infiltrating cell population analysis of tibiotarsal joint tissues of infected WT and SLPI-/- mice. (A) The neutrophil population was gated on the CD11bLY6G double positive cells among the CD45 positive cells. (B) The macrophage population was gated on the CD64 positive cells among the CX3CR1 positive myeloid cells. Results from two independent experiments were pooled and shown here. (C-E) Expression levels of CXCR2 (C), MCP-1 (D), and CCR2 (E) were assessed in the tibiotarsal tissue using RT-qPCR. (F) The serum cytokine profile was assess using mouse cytokine/chemokine 32-plex array. An increase in IL-6 was observed in the infected SLPI-/- mice. (G, H) The serum level of neutrophil elastase (NE) was measured using an ELISA kit. (I) Serum levels of MMPs were assessed using a mouse MMP 5-Plex Discovery Assay. An increase in MMP-8 was observed in the infected SLPI-/- mice. Serum was obtained by cardiac puncture of WT and SLPI-/- C57BL/6 mice with/without infection at 21-28 dpi (F, G, and I) and of infected C3H/HeN mice at 21 dpi (H). black, PBS-sham infection; red, B. burgdorferi infection. Each data point represents an individual animal. The error bar represents mean ± SEM and p-values are calculated using the non-parametric Mann-Whitney test.

Serum SLPI levels in Lyme disease subjects versus healthy controls.

The serum level of SLPI was measured by ELISA. Sera samples were from 5 adult healthy controls (HC). 18 samples were from people with Lyme disease (LD) including 5 samples from 3 subjects presenting with Lyme arthritis (red) and 13 samples from 4 subjects with erythema migrans (black). The error bar represents mean ± SEM and p- values are calculated using the non-parametric Mann-Whitney test.

B. burgdorferi interaction with human and murine SLPI.

(A) Sandwich ELISA results show the interaction of B. burgdorferi whole cell lysates with human SLPI. ELISA plates were coated with B. burgdorferi whole cell lysates and probed with increasing amount of human SLPI (blue) or human Fc proteins (black) as the negative control. The values plotted represent the mean ± SEM of duplicates from two experiments. P-value is displayed in the graph and determined using the non-parametric Mann-Whitney test. (B and C) Flow cytometry histograms show binding of human (B) and murine (C) SLPI to B. burgdorferi cultured at 33°C (solid line) and 37°C (dash line). B. burgdorferi was cultured to a density of 106/ml. The same volume of cultures was incubated at 33°C or 37°C for 24 hrs before adding 10 nM (green) or 1 μM (red) human or murine SLPI. The binding was detected with goat anti human or murine SLPI and donkey anti goat AF647 or AF488. B. burgdorferi alone (grey) and antibody control (blue) were used as negative controls. (D) Immuno-fluorescent microscopy was used to directly observe the binding of B. burgdorferi with human and murine SLPI. Merged and single-color images are shown. Scale bar: 10 μm.