hslpi  (R&D Systems)

Journal: bioRxiv

Article Title: Secretory leukocyte protease inhibitor influences periarticular joint inflammation in B. burgdorferi -infected mice

doi: 10.1101/2024.11.24.625079

Figure Lengend Snippet: (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 10 6 /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.

Article Snippet: The cells were pelleted, rinsed, resuspended, and incubated with 10 nM and 1 µM human SLPI (R&D Systems, #1274-PI-100) and murine SLPI (produced in lab as described above) at 4°C for 2 h. The binding was detected with goat anti human or murine SLPI (R&D Systems, #AF1274 and AF1735) and Alexa Fluor 488 or Alexa Fluor 647 donkey anti goat IgG (H+L) (Invitrogen, #A32814, A-21447).

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Negative Control, MANN-WHITNEY, Flow Cytometry, Binding Assay, Cell Culture, Incubation, Control, Microscopy

Journal: mBio

Article Title: Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice

doi: 10.1128/mbio.02554-23

Figure Lengend Snippet: SLPI is increased in mouse urinary tract following UTI. ( A ) Urine SLPI levels in C57BL/6J mock-infected ( n = 3–5) and UTI89-infected ( n = 9–10) mice following log base 10 transformation. A time series ANOVA was performed on all time points after 0 including imputed values for missing samples (two mock and five infected). A post hoc Student’s t test was performed on imputed data with FDR correction. ( B ) qRT-PCR of Slpi in whole bladder homogenates at 3 hpi ( n = 12), 7 hpi ( n = 8), and 3 dpi ( n = 3) represented as fold change to mock-infected mice ( n = 3–4) (Student’s t test). ( C ) Log base 10 CFU/mL of UTI89 in urine of infected mice over time. Dashed line represents limit of detection (LOD). ( D ) ROC curve analysis using SLPI levels in panel A to classify bacteriuria shown in panel C with area under the curve (AUC) values listed for each timepoint. Time points 7 hpi and 2 dpi have been offset slightly to aid visualization.

Article Snippet: The culture was then diluted 1:50 in 1× PBS + 1% LB broth and incubated statically at 37°C with recombinant human SLPI protein (rSLPI) (CAS: 1274-PI-100, R&D Systems) at these final concentrations: 20, 10, and 1 μM.

Techniques: Infection, Transformation Assay, Quantitative RT-PCR

Journal: mBio

Article Title: Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice

doi: 10.1128/mbio.02554-23

Figure Lengend Snippet: SLPI is expressed in the bladder epithelium. ( A and B ) Immunofluorescence staining of SLPI protein expression (green) and DNA using Hoechst stain (blue) in mock-infected ( A ) and infected ( B ) bladders from Slpi +/+ mice. Left panels (5× magnification, 200 µm scale bars) are merged images of SLPI and DNA shown as smaller panels on the right. ( C–E ) Mean fluorescence intensity (MFI) of mock and infected bladder epithelium for DNA ( C ) and SLPI ( D ) with MFI ratio of SLPI/DNA ( E ) (Student’s t test). ( F ) Comparison of supernatant SLPI levels in control (PBS) or infected (UTI89) 5637 bladder epithelial cells represented as percent of starting SLPI in each well. ( G ) Fold change in Slpi transcription as measured by qRT-PCR in 5637 cells at 2 hpi. For panels F and G , shapes denote separate biological replicate experiments, each point represents the average of 4 or more individual wells (paired Student’s t test).

Article Snippet: The culture was then diluted 1:50 in 1× PBS + 1% LB broth and incubated statically at 37°C with recombinant human SLPI protein (rSLPI) (CAS: 1274-PI-100, R&D Systems) at these final concentrations: 20, 10, and 1 μM.

Techniques: Immunofluorescence, Staining, Expressing, Infection, Fluorescence, Comparison, Control, Quantitative RT-PCR

Journal: mBio

Article Title: Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice

doi: 10.1128/mbio.02554-23

Figure Lengend Snippet: Slpi −/− mice have prolonged bacteriuria compared to Slpi +/+ mice. ( A ) Urine bacterial titers from Slpi +/+ (blue) and Slpi −/− mice (green) during 1 week of infection with UTI89. Data are log base 10 transformed CFU/mL and are combined from seven experiments with 5–37 mice per genotype per time point (Student’s t test). ( B ) Averaged cytospin cell counts of neutrophils in urine from uninfected ( n = 5–8), mock-infected ( n = 3–8), and UTI89-infected ( n = 6–26) Slpi +/+ (blue) and Slpi −/ − mice (green) from 0 hpi to 2 dpi. Data are combined from four separate experiments (Student’s t test). ( C ) Bladder bacterial titers in log base 10 CFU/g. Data are combined from four experiments with 5–14 mice per genotype per time point. ( D ) Kidney bacterial titers in log base 10 CFU/g. Data are combined from seven experiments with 5–19 mice per genotype per time point. ( E ) Ex vivo gentamicin protection assay on infected bladders from Slpi +/+ (blue) and Slpi −/− mice (green) showing log CFU/g of tissue for recovered UTI89 from luminal or intracellular samples. ( F ) In vitro recombinant SLPI (rSLPI) antimicrobial activity against UTI89 and DH5α in PBS + 1% LB. All samples were normalized to PBS control group (green) and 1 µM LL-37 antimicrobial protein was used as a positive control (purple). The final concentrations of rSLPI in each culture were: 20 µM (blue), 10 µM (yellow), and 1 µM (orange). For panels A–E , gray dashed lines on each plot represent average limit of detection (LOD) for that tissue type.

Article Snippet: The culture was then diluted 1:50 in 1× PBS + 1% LB broth and incubated statically at 37°C with recombinant human SLPI protein (rSLPI) (CAS: 1274-PI-100, R&D Systems) at these final concentrations: 20, 10, and 1 μM.

Techniques: Infection, Transformation Assay, Ex Vivo, In Vitro, Recombinant, Activity Assay, Control, Positive Control

Journal: mBio

Article Title: Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice

doi: 10.1128/mbio.02554-23

Figure Lengend Snippet: Slpi −/− mice demonstrate a dysregulated immune response in the bladder at 1 dpi. ( A ) Schematic of experimental approach to assess differences in whole bladder transcriptome of mock-infected Slpi +/+ (M- wt , n = 4) and Slpi −/− (M- ko , n = 3) and their UTI89-infected Slpi +/+ (I- wt , n = 14) and Slpi −/− (I- ko , n = 11) counterparts. ( B ) Volcano plot showing genes significantly upregulated in infected Slpi +/+ (I- wt , blue) and infected Slpi −/− mice (I- ko , green) above the dotted line. ( C ) Bubble volcano plot showing gene set enrichment analysis of GO pathways between I- wt and I- ko mice. Differentially expressed child pathways are shown above the dotted line and colored by the following categories: immune regulation (blue), DNA replication and repair (yellow), wound healing (orange), small molecule biosynthesis (pink), muscle growth (purple), neuronal regulation (green), or other (gray). GO pathways listed below the plot are representative parent pathways for each color group. Pathway size is shown as bubble size (inset key). ( D ) Enrichment plots (left) and count heatmaps (right) of inset labeled pathways from panel C . Log base 2 fold change between I- wt and I- ko is shown in blue to green on the left, while normalized read counts for each group are shown as two columns to the right in purple. Significantly enriched genes as determined by DESeq2 are boxed. Five leading edge genes of each pathway that are either significantly enriched (boxed) and/or show the greatest enrichment are shown. Significantly enriched genes shown here are also labeled in panel B . Note that gene Lep is listed two times as part of two different pathways. Dotted lines in all plots represent P value cutoff of 0.05.

Article Snippet: The culture was then diluted 1:50 in 1× PBS + 1% LB broth and incubated statically at 37°C with recombinant human SLPI protein (rSLPI) (CAS: 1274-PI-100, R&D Systems) at these final concentrations: 20, 10, and 1 μM.

Techniques: Infection, Labeling

Journal: mBio

Article Title: Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice

doi: 10.1128/mbio.02554-23

Figure Lengend Snippet: Slpi -/- mice experience prolonged bladder inflammation after UTI. ( A ) Representative hematoxylin and eosin staining of bladders from mock-infected Slpi +/+ (blue, top row), UTI89-infected Slpi +/+ (blue, second row), and UTI89-infected Slpi −/− mice (green, last row) at 7 hpi, 1, 2, 3, and 7 dpi. Images are 20× magnification with 1 mm scale bars. ( B ) Bladder inflammation scores ( n = 4–14) mice per group, combined from five experiments (one-sided Student’s t test). Outlined points correspond to bladder images shown in panel A. ( C ) Correlation of bladder inflammation score to urine log base 10 CFU/mL for all time points ( n = 2–12 per genotype per time point combined from five experiments; Spearman rank). ( D ) Log base 10 transformed neutrophil elastase (NE) levels in Slpi +/+ (blue) and Slpi −/− mice (green) following infection. These data are combined from six experiments with 10–30 mice per genotype per timepoint (Student’s t -test).

Article Snippet: The culture was then diluted 1:50 in 1× PBS + 1% LB broth and incubated statically at 37°C with recombinant human SLPI protein (rSLPI) (CAS: 1274-PI-100, R&D Systems) at these final concentrations: 20, 10, and 1 μM.

Techniques: Staining, Infection, Transformation Assay

Journal: mBio

Article Title: Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice

doi: 10.1128/mbio.02554-23

Figure Lengend Snippet: Levels of urine SLPI are changed in women with bacteriuria. ( A ) Flowchart describing the filtering process for clinical study patient samples. ( B ) SLPI protein measured in urine samples (one-sided Student’s t test). ( C ) Total protein in urine corresponding to samples in panel B (Student’s t test). Shapes represent urine culture status: negative culture with no UTI symptoms (circles), culture positive for uropathogen (triangle). All positive uropathogen cultures were positive for Escherichia coli .

Article Snippet: The culture was then diluted 1:50 in 1× PBS + 1% LB broth and incubated statically at 37°C with recombinant human SLPI protein (rSLPI) (CAS: 1274-PI-100, R&D Systems) at these final concentrations: 20, 10, and 1 μM.

Techniques: