Journal: Frontiers in Pain Research

Article Title: Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia

doi: 10.3389/fpain.2023.1274811

Figure Lengend Snippet: Markers for non-neuronal cells in TG.

Article Snippet: The following antibodies were used: anti-Iba1 rabbit polyclonal (Thermo Fisher Scientific, San Diego, CA, USA; Cat: PA5-27436; 1:300); anti-s100a8 rat IgG2B monoclonal (R&D Systems; clone # 335806; Cat: MAB3059-SP 1:200); and anti-NFH chicken polyclonal (Novus Biologicals; Cat: NB300-217; 1:2000).

Techniques:

Journal: Frontiers in Pain Research

Article Title: Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia

doi: 10.3389/fpain.2023.1274811

Figure Lengend Snippet: Representations of non-neuronal cells in TG. ( A – A ”’) Representative microphotographs of Ccr2 RFP /Cx3cr1 GFP reporter mouse TG sections show relative expressions of macrophage markers Cx3cr1 (green), Ccr2 (red), and Iba1 (blue). The yellow arrows on panel A show Cx3cr1 + /Iba1 + /Ccr2 − macrophages in TG. The yellow arrows on panels ( A ”) and ( A ”’) show Cx3cr1 − /Iba1 + /Ccr2 − macrophages in TG. ( B–B ’) Representative microphotographs of Ccr2 RFP /Cx3cr1 GFP reporter mouse TG sections show expression patterns of a macrophage marker Cx3cr1 (green) and a neutrophil/monocyte marker S100a8 (red). ( C ) A representative microphotograph of a Col1a2 cre /Ai14 fl/ reporter mouse TG sections shows the expression of a fibroblast marker Col1a2. ( D ) A representative microphotograph of a Col1a2 cre /Ai14 fl/ reporter mouse TG section shows relative expressions of a fibroblast marker Col1a2 and an A-fiber neuronal marker NFH. The scales are presented in each microphotograph.

Article Snippet: The following antibodies were used: anti-Iba1 rabbit polyclonal (Thermo Fisher Scientific, San Diego, CA, USA; Cat: PA5-27436; 1:300); anti-s100a8 rat IgG2B monoclonal (R&D Systems; clone # 335806; Cat: MAB3059-SP 1:200); and anti-NFH chicken polyclonal (Novus Biologicals; Cat: NB300-217; 1:2000).

Techniques: Expressing, Marker

Journal: Frontiers in immunology

Article Title: S100a9 Protects Male Lupus-Prone NZBWF1 Mice From Disease Development.

doi: 10.3389/fimmu.2021.681503

Figure Lengend Snippet: FIGURE 1 | S100a9 expression is elevated within Gr1highCD11b+ cells of male lupus-prone mice and regulates B cell differentiation in vitro and antibody production in vivo. Gr1highCD11b+ and Gr1lowCD11b+ cells were isolated from 9 week old male and female NZBWF1 lupus-prone mice by flow cytometry and levels of S100a9 (A), S100a8 (B), and S100a4 (C) mRNA were determined by RT-PCR analysis (n = 4). Graphs show mean +/- SEM. (D) Supernatants from overnight cultured Gr1highCD11b+ cells from 4 wk old NZBWF1 male mice suppress IFNa/CD40L-driven B cell differentiation. Shown is the average (± SEM) of 4 independent experiments. (E) S100A8/A9 heterodimer levels in serum, spleen and bone marrow from 9 wk old NZBWF1 mice. Each symbol represents one mouse. Male vs. female: p = 0.07; two-way ANOVA. (F) Gr1highCD11b+ cells from 9 wk old S100a9-deficienct male NZBWF1 mice fail to suppress cytokine-driven B cell differentiation in vitro. IgM/IgG-secreting cells were enumerated by ELISPOT and are presented as number of cells per 105 plated B cells. Data shown represent the mean ± SEM of 4 independent assays. *P < 0.05; **P < 0.01, Student’s unpaired t test.

Article Snippet: For identification of Mac2, S100a8 and S100a9, sections were blocked in 2% fetal bovine serum (FBS) in Hank’s Balanced Salt Solution (HBSS) for 30 min. Primary rat antimouse MAC2 (clone 125401)(BioLegend), rat anti-mouse S100a8 (clone MAB3059) or mouse S100A9 clone AF2065) (both from (R & D Systems, Minneapolis, MN) antibodies were added at 1:1500, 1:250 and 1:250 dilution, respectively, and sections were left to incubate for 1 hour in a humidified chamber at room temperature.

Techniques: Expressing, Cell Differentiation, In Vitro, In Vivo, Isolation, Cytometry, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunospot

Journal: eLife

Article Title: A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection

doi: 10.7554/eLife.65762

Figure Lengend Snippet: ( A ) Neutrophils were isolated from the bone marrow of naive C57BL/6 female mice and stimulated with heat-killed HSV-2 at a multiplicity of infection (MOI) of 1 for 4 hr (bottom row) or left unstimulated (top row). NETs are identified by areas of diffuse 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) that overlap with citrullinated histone H3 (green). Data are representative of two independent experiments. ( B-C ) Padi4 fl/fl x S100a8 -Cre (PAD4 CKO) or Cre- littermate controls were infected with HSV-2 as described in . Inflammation scores were monitored for 7 d.p.i. (Ctrl: n = 10, KO: n = 9) ( B ), and infectious virus was measured by plaque assay in vaginal washes collected on the indicated d.p.i. (Ctrl: n = 8, CKO: n = 8) ( C ). Data in ( B-C ) are pooled from two independent experiments. Statistical significance was analyzed by repeated measures two-way ANOVA with ( B ) or without ( C ) Geisser-Greenhouse correction and Bonferroni's multiple comparisons test; all comparisons were not significant. Raw values for each biological replicate, epsilon values, and specific p values are provided in .

Article Snippet: S100A8 was detected with a rat anti-mouse S100A8 primary antibody (clone 63N13G5, Novus Biologicals) and a goat anti-rat IgG conjugated to AlexaFluor 568 (Life Technologies) in a similar manner.

Techniques: Isolation, Infection, Staining, Virus, Plaque Assay

Journal: eLife

Article Title: A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection

doi: 10.7554/eLife.65762

Figure Lengend Snippet: ( A ) Neutrophils were isolated from the bone marrow using a Histopaque gradient and stimulated with heat-killedHSV-2 at an MOI of 5 for 16 hr (n = 4) of left unstimulated (n = 4) and then incubated with DCFDA. Plots show gating for leukocytes (left) and CD11b + Ly6G + neutrophils (middle). DCFDA fluorescence was measured by flow cytometry in unstimulated (shaded histogram) or HSV-2 stimulated (open histogram) neutrophils. Graph shows mean fluorescence intensity (MFI) of DCFDA (reactive oxygen species [ROS]). Ncf2 KO (n = 10) or littermate controls (n = 8) ( B–C ) or Stim1 fl/fl x Stim2 fl/fl x S100a8 -Cre (STIM1/STIM2 DKO) (n = 4) or littermate controls (n = 5) ( D–E ) were infected as described in . Mice were monitored for genital inflammation for 1 week after infection ( B, D ). Infectious virus was measured by plaque assay in vaginal washes collected on the indicated days ( C, E ). Bars in A show mean with SD. Bars in B and D show median with interquartile range. Bars in C and E show mean. Data in A-C are pooled from two independent experiments; data in D-E were performed once. Statistical significance was analyzed by unpaired Student's t-test ( A ), repeated measures two-way ANOVA with ( B, D ) or without ( C, E ) Geisser-Greenhouse correction and Bonferroni's multiple comparisons test, *p<0.05. Comparisons in B-E were not significant. Raw values for each biological replicate, epsilon values, and specific p values are provided in .

Article Snippet: S100A8 was detected with a rat anti-mouse S100A8 primary antibody (clone 63N13G5, Novus Biologicals) and a goat anti-rat IgG conjugated to AlexaFluor 568 (Life Technologies) in a similar manner.

Techniques: Isolation, Incubation, Fluorescence, Flow Cytometry, Infection, Virus, Plaque Assay

Journal: eLife

Article Title: A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection

doi: 10.7554/eLife.65762

Figure Lengend Snippet: Female C57BL/6J mice were treated with DMPA and inoculated ivag with 10 4 PFU HSV-1 McKrae or HSV-2. ( A ) Inflammation scores were monitored for 7 d.p.i. (HSV-1: n = 14, HSV-2: n = 13). ( B ) Neutrophils were counted by flow cytometry in vaginal tissues at the indicated days after HSV-1 or HSV-2 infection (d0: n = 7, day 2: n = 8, day 4: n = 7, day 6: n = 8). ( C ) Vaginas were harvested from phosphate buffered saline (PBS)-inoculated (mock), HSV-1- or HSV-2-inoculated mice at 4 d.p.i., and tissue sections were probed with antibodies against HSV proteins (green) or S100A8 (red). 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to detect cell nuclei. Images are representative of six mice per group. ( D ) Mice were treated with isotype control or anti-Ly6G mAb as described in and then inoculated ivag with 10 4 PFU HSV-1 McKrae. Inflammation scores were monitored for 7 d.p.i. (isotype, anti-Ly6G mAb: n = 10). Data are pooled from three ( A ) or two ( B–D ) independent experiments. Data in C is representative of two independent experiments. Bars show median with interquartile range ( A, D ) or mean ( B ). Scale bars show 50 mm. Statistical significance was measured by repeated measures two-way ANOVA with Geisser-Greenhouse correction and Bonferroni's multiple comparisons test ( A, D ) or two-way ANOVA with Bonferroni's multiple comparisons test ( B ). *p<0.05, ****p<0.001, ns = not significant. Raw values for each biological replicate, epsilon values, and specific p values are provided in . Figure 2—source data 1. Excel file with individual inflammation scores, cell numbers, description of statistical tests, epsilon values and actual p values for .

Article Snippet: S100A8 was detected with a rat anti-mouse S100A8 primary antibody (clone 63N13G5, Novus Biologicals) and a goat anti-rat IgG conjugated to AlexaFluor 568 (Life Technologies) in a similar manner.

Techniques: Flow Cytometry, Infection, Saline, Control

Journal: eLife

Article Title: A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection

doi: 10.7554/eLife.65762

Figure Lengend Snippet: Mice were infected as described in . Vaginas were harvested at 5 d.p.i., and live cells were flow sorted and subjected to high-throughput single-cell RNA sequencing (scRNA-seq). ( A ) A t-Distributed Stochastic Neighbor Embedding (tSNE) visualization of 21,633 cells across all mice resolves 17 distinct clusters in the vaginal tissue. Clusters can be identified as myeloid cells (red border), epithelial cells (blue border), or lymphocytes (purple border). Neutrophils are encircled in black and contain three distinct clusters (0, 2, and 5). ( B ) Neutrophils are defined by high expression of S100a8 and Csf3r (G-SCFR). ( C ) tSNE plots of vaginal cell clusters from mock-inoculated or HSV-infected mice. Neutrophil populations are circled in black. ( D ) Distribution of expression for genes within the Hallmark IFNa Response gene set. ( E ) Production of IFNβ in vaginal washes collected at 4 and 5 d.p.i. as measured by ELISA (uninfected: n = 8, HSV-1: n = 9, HSV-2: n = 9). scRNA-seq in A-D was performed once. Data in E are pooled from two independent experiments. Statistical significance was measured by one-way ANOVA with Tukey's multiple comparisons test. *p<0.05, **p<0.01, ***p<0.005. Raw values for each biological replicate, specific p values, and complete lists of differentially expressed genes between clusters 0, 2, and 5 are provided in . Figure 3—source data 1. Excel file with individual ELISA measurements, lists of differentially expressed genes between different neutrophil clusters, description of statistical tests and actual p values for . Figure 3—source data 2. Excel file with lists of differentially expressed genes between neutrophils between HSV-1 and HSV-2 infected mice, individual delta Cq values, ELISA measurements, description of statistical tests and actual p values for – .

Article Snippet: S100A8 was detected with a rat anti-mouse S100A8 primary antibody (clone 63N13G5, Novus Biologicals) and a goat anti-rat IgG conjugated to AlexaFluor 568 (Life Technologies) in a similar manner.

Techniques: Infection, High Throughput Screening Assay, RNA Sequencing, Expressing, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection

doi: 10.7554/eLife.65762

Figure Lengend Snippet: ( A ) IFNAR1 expression on neutrophils and non-neutrophil hematopoietic cells from the bone marrow of naive Ifnar1 fl/fl x S100a8-Cre (IFNAR1 CKO) or Cre- littermate controls. Plot is gated on live CD45 + cells. CD11b + Ly6G + cells are neutrophils, Ly6G- cells are non-neutrophils. Gray histogram shows isotype staining, black open histogram is Cre- control, and red open histogram is IFNAR1 CKO. ( B ) Neutrophils were counted by flow cytometry in vaginal washes collected at the indicated days from IFNAR1 CKO (n = 4) or Cre- controls (n = 7) that were infected with HSV-2 as described in . ( C ) Inflammation scores for the first 7 d.p.i. of IFNAR1 CKO (n = 10–13) or Cre- controls (n = 8–11). ( D ) Histology of the vagina and genital skin at 6 d.p.i. Red arrows point to areas of epithelial denuding or damage, black arrows denote basement membrane ( E ) Infectious virus as measured by plaque assay from vaginal washes collected on the indicated days from IFNAR1 CKO (n = 5–8) or Cre- controls (n = 7–10). Data in C and E are pooled from three independent experiments; data in B are pooled from two independent experiments; and data in D are representative of two independent experiments. Bars in C show median with interquartile range, bars in B and E show mean. Scale bars show 50 mm. Statistical significance was measured by mixed-effects analysis with ( C ) or without ( B, E ) Geisser-Greenhouse correction and Bonferroni's multiple comparisons test. *p<0.05, **p<0.01, ns = not significant. Raw values for each biological replicate, epsilon values and specific p values are provided in . Figure 5—source data 1. Excel file with individual cell numbers, inflammation scores, viral titers, description of statistical tests, epsilon values and actual p values for .

Article Snippet: S100A8 was detected with a rat anti-mouse S100A8 primary antibody (clone 63N13G5, Novus Biologicals) and a goat anti-rat IgG conjugated to AlexaFluor 568 (Life Technologies) in a similar manner.

Techniques: Expressing, Staining, Control, Flow Cytometry, Infection, Membrane, Virus, Plaque Assay

Journal: eLife

Article Title: A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection

doi: 10.7554/eLife.65762

Figure Lengend Snippet: C57BL/6 mice were treated with anti-Ly6G (n = 11) or isotype control (n = 12) as described in ( A ), therapeutically treated with anti-IFNAR1 (n = 12) or isotype control (n = 14) as described in ( B ) and infected with HSV-2, or IFNAR1 CKO (n = 7) and Cre- controls (n = 7) were infected with HSV-2 as described in ( C ). ( A-C ) Vaginal IL-18 levels were measured by ELISA in washes collected at 5 d.p.i. 100 μg of anti-IL-18 neutralizing antibody (n = 18) or isotype control (n = 16) was administered ivag at 3, 4, and 5 d.p.i. ( D ) Inflammation scores of antibody-treated mice over the first 7 d.p.i. ( E ) Infectious virus as measured by plaque assay in vaginal washes collected on the indicated days (n = 9–14). ( F ) Immunofluorescent staining of vaginal tissues collected at 6 d.p.i. or from mock-inoculated mice. Green shows S100A8 (neutrophil), red shows IL-18 or isotype, blue is DAPI. Top row shows HSV-2-infected tissue probed with isotype control, and bottom two rows show representative images of tissues probed with anti-IL-18 antibody (middle = mock-infected, bottom = HSV-2-infected). White arrows point to IL-18 + neutrophils. Yellow square demarcates area shown in the inset. Data in A-C, D, and E are pooled from four independent experiments for each experimental setup. Data in F is representative of two independent experiments. Bars in A-C show mean and SD, bars in D show median with interquartile range, and bars in E show mean. Scale bars show 50 mm. Statistical significance was measured by unpaired t-test ( A–C ), repeated measures two-way ANOVA with ( D ) Geisser-Greenhouse correction and Bonferroni's multiple comparisons test, or mixed-effects analysis with Bonferroni's multiple comparisons test ( D ). *p<0.05, ***p<0.005, ns = not significant. Raw values for each biological replicate, epsilon values, and specific p values are provided in . Figure 6—source data 1. Excel file with individual ELISA measurements, inflammation scores, viral titers, description of statistical tests, epsilon values and actual p values for .

Article Snippet: S100A8 was detected with a rat anti-mouse S100A8 primary antibody (clone 63N13G5, Novus Biologicals) and a goat anti-rat IgG conjugated to AlexaFluor 568 (Life Technologies) in a similar manner.

Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Virus, Plaque Assay, Staining

Journal: eLife

Article Title: A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection

doi: 10.7554/eLife.65762

Figure Lengend Snippet:

Article Snippet: S100A8 was detected with a rat anti-mouse S100A8 primary antibody (clone 63N13G5, Novus Biologicals) and a goat anti-rat IgG conjugated to AlexaFluor 568 (Life Technologies) in a similar manner.

Techniques: Virus, Isolation, Injection, Blocking Assay, Staining, Control, Purification, Sequencing, ROS Assay, Amplification, Enzyme-linked Immunosorbent Assay, Modification, Saline, Sterility, Protease Inhibitor, Software, Imaging, Light Microscopy