Journal: Mucosal immunology

Article Title: A role for BATF3 in T H 9 differentiation and T-cell-driven mucosal pathologies.

doi: 10.1038/s41385-018-0122-4

Figure Lengend Snippet: Fig. 2 TL1A upregulates BATF and BATF3 expression during TH9 differentiation. a Transcriptional profiling of TH9 and TH9-TL1A cells by RNA sequencing. Heat map displaying RNA-sequencing data using the top 100 genes with the largest IQR (interquartile range). The dendrograms to the left and above the heat map represent clustering of genes (rows) and samples (columns). b Naive CD4+ T cells were differentiated into different TH subsets and Tregs for 3 days. Relative Batf mRNA expression was analyzed by qPCR. c Naive CD4+ T cells were differentiated under TH0- or TH9-polarizing conditions with or without TL1A for the indicated time periods. QPCR analysis of relative Batf mRNA expression. d Co- staining of BATF and IRF4 (top). Quantitative analysis of the percentages of BATF+IRF4+ cells (bottom). e Co-staining of BATF and IL-9. f–g Naive human CD4+ T cells were isolated from PBMCs of healthy volunteers and differentiated under TH9-polarizing conditions with or without TL1A. f Representative intracellular staining of IL-9 and BATF at 48 h. g Frequency of BATF+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual donor. N = 4. h Relative Batf3 mRNA expression in different murine TH subsets and Tregs was analyzed by qPCR. i Relative Batf3 mRNA expression at indicated time points. j Representative co-staining of BATF3 and IL-9. k Frequency of BATF3+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual experiment. N = 3. l–m Naive human CD4+ T cells were differentiated under TH9-polarizing conditions with or without TL1A. l Representative intracellular staining of IL-9 and BATF3 at 48 h. m Frequency of BATF3+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual donor. N = 4. Data represent means ± SD of one independent experiment out of two (a) or at least three (b–d, i, k) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005 as determined by Student’s t test

Article Snippet: Intracellular staining Cells were restimulated with 50 ng/ml PMA (phorbol 12-myristate 13-acetate), 500 ng/ml ionomycin, and monensin (eBioscience) for 4 h, stained with anti-CD4 (RM4-5, eBioscience), fixed and permeabilized using the FoxP3 staining buffer set (eBioscience), and stained with antibodies against murine IL-9 (RM9A4, BioLegend), IL-10 (JES5-16E3), IL-13 (13A), IL-17A (eBio17B7), IL-17F (eBio18F10), IFN-γ (XMG1.2), IL-4 (BVD6-24G2), IL-22 (1H8PWSR), Ki67 (SolA15), BATF (MBM7C7), IRF4 (3E4), human IL-9 (MH9A4, all eBioscience), and BATF3 (841792, R&D Systems).

Techniques: Expressing, RNA Sequencing, Staining, Isolation

Journal: The Journal of Infectious Diseases

Article Title: Use of Recombinant Chikungunya Virus expressing Nanoluciferase to Identify Chondrocytes as Target Cells in an Immunocompetent Mouse Model

doi: 10.1093/infdis/jiaf232

Figure Lengend Snippet: Generation of a chikungunya virus (CHIKV) expressing NanoLuc luciferase (NLuc). A , Genome organization of the recombinant CHIKV-NLuc. The NLuc gene was inserted in frame between the non-structural protein (nsP) 3 and nsp4 genes; arrows represent nsP2 cleavage sites. B , Stability of the recombinant CHIKV-NLuc. Vero E6 cells were serially infected for 10 passages with CHIKV-NLuc to assess the stability of the virus. At each passage, the viral titer of the supernatant was measured ( curve ), as well as the luciferase activity ( bars ), expressed as relative luminescence units (RLUs) in the infected cells. Curves and histograms show the median value and interquartile range of measurements from 4 replicates. Abbreviation: PFUs, plaque-forming units.

Article Snippet: The cells were washed and incubated with the 3E4 recombinant anti-CHIKV E2 murine antibody (Creative Biolabs).

Techniques: Virus, Expressing, Luciferase, Recombinant, Infection, Activity Assay

Journal: The Journal of Infectious Diseases

Article Title: Use of Recombinant Chikungunya Virus expressing Nanoluciferase to Identify Chondrocytes as Target Cells in an Immunocompetent Mouse Model

doi: 10.1093/infdis/jiaf232

Figure Lengend Snippet: Plantar pad infections with chikungunya virus–NanoLuc luciferase (CHIKV-NLuc) generate sufficient bioluminescent signal for monitoring. Mice were subcutaneously inoculated into the plantar pad of the right hind paw with the L15 medium alone (control, on the top) or CHIKV-NLuc (10 3 –10 6 plaque-forming units (PFUs) per mouse). Bioluminescent signal intensity is associated with a color code according to the scales provided to the left of the images; the dorsal view is presented here. A , Mice at 1, 3, 5, 7, and 9 days post inoculation (dpi; scale, 10 6 –2.10 7 photons per second per square centimeter per steradian [p/s/cm 2 /sr]). B , Mice at 12 and 15 dpi (scale, 10 4 –10 5 p/s/cm 2 /sr). C , Mice at 34 dpi; significant signal in the footpad of 1 mouse is indicated with a white arrow (scale, 10 3 –10 4 p/s/cm 2 /sr). Data shown are representative of 2 experiments. Abbreviations: Max, maximum; Min, minimum.

Article Snippet: The cells were washed and incubated with the 3E4 recombinant anti-CHIKV E2 murine antibody (Creative Biolabs).

Techniques: Virus, Luciferase, Control

Journal: The Journal of Infectious Diseases

Article Title: Use of Recombinant Chikungunya Virus expressing Nanoluciferase to Identify Chondrocytes as Target Cells in an Immunocompetent Mouse Model

doi: 10.1093/infdis/jiaf232

Figure Lengend Snippet: Assessment of bioluminescent signal in mice infected with chikungunya virus–NanoLuc luciferase virus (CHIKV-NLuc), in the whole body ( A ), inoculated paw ( B ), and contralateral paw ( C ) after subcutaneous inoculation (footpad) with 10 6 ( dots ), 10 5 ( squares ), 10 4 ( upward-facing triangle ), or 10 3 ( downward-facing triangle ) plaque-forming units (PFUs) of CHIKV-NLuc or noninfected control ( rhombuses ). Mice were imaged, regions of interest of the same area and location were defined, the signal was quantified at 1, 3, 5, 9, 12, 15, and 34 days post inoculation and expressed as total flux in photons per second (y-axis). Each point represents the value for a single mouse, and medians and interquartile ranges of measurements from 3 mice are also depicted. NS, not significant; * P < .05; ** P < .01; *** P < .001 (2-way analysis of variance).

Article Snippet: The cells were washed and incubated with the 3E4 recombinant anti-CHIKV E2 murine antibody (Creative Biolabs).

Techniques: Infection, Virus, Luciferase, Control

Journal: The Journal of Infectious Diseases

Article Title: Use of Recombinant Chikungunya Virus expressing Nanoluciferase to Identify Chondrocytes as Target Cells in an Immunocompetent Mouse Model

doi: 10.1093/infdis/jiaf232

Figure Lengend Snippet: Ex vivo visualization and measurement of luciferase activity and infection in the leg bone and the articular cartilage of the ankle/metatarsi, knees and hips of infected mice. Mice were inoculated into the footpad of the right hind leg with chikungunya virus–NanoLuc luciferase (CHIKV-NLuc; 10 5 plaque-forming units [PFUs]) or the diluent alone (control). A , Leg bones of control and infected animals were dissected at 12 days post inoculation (dpi) and analyzed with bioluminescence imaging. Left, Infected mouse. Right, Control. Abbreviation: p/s/cm 2 /sr, photons per second per square centimeter per steradian. B , With use of a binocular magnifying glass, articular cartilages (hip, knee, and ankle/metatarsus) were isolated at 6, 12 and 30 dpi, and luciferase activity was quantified. Each point represents the luciferase activity of a cartilage normalized to uninfected controls and measured in 2 independent experiments; medians and interquartile ranges are also shown. Abbreviations: NS, not significant; RLU, relative luminescence unit. * P < .05; ** P < .01; *** P < .001 (nonparametric Mann-Whitney test). C, D, Mice were euthanized at 6 dpi ( C ) and 30 dpi ( D ), and cells were isolated from metatarsal cartilage. Isolated cells were immediately fixed and stained. First line (blue) indicates DAPI; second line (green) collagen type II; and third line (red) CHIKV E2. Arrows indicate cells with double labeling of type II collagen and viral E2 protein, as shown in the merge image on the bottom right-hand corner. Scale bars represent 50 µm ( C ) and 20 µm ( D ).

Article Snippet: The cells were washed and incubated with the 3E4 recombinant anti-CHIKV E2 murine antibody (Creative Biolabs).

Techniques: Ex Vivo, Luciferase, Activity Assay, Infection, Virus, Control, Imaging, Isolation, MANN-WHITNEY, Staining, Labeling

Journal: The Journal of Infectious Diseases

Article Title: Use of Recombinant Chikungunya Virus expressing Nanoluciferase to Identify Chondrocytes as Target Cells in an Immunocompetent Mouse Model

doi: 10.1093/infdis/jiaf232

Figure Lengend Snippet: Infection of primary human chondrocytes (pHCs) with chikungunya virus (CHIKV). A , Assessment of viral RNA levels in wild-type CHIKV–infected pHCs. Viral RNA was quantified in the cells lysates using reverse-transcription quantitative polymerase chain reaction after infection at 3 multiplicities of infection (MOIs). B, Bioluminescence activity, expressed in relative luminescence units (RLUs), after infection with CHIKV–NanoLuc luciferase (CHIKV-NLuc), at 3 MOIs. C, Numbers of infectious virus particles in the supernatant of CHIKV-infected pHCs, assessed by plaque assay. Curves show the median value and interquartile range obtained from 3 independent experiments in triplicate. Abbreviation: PFUs, plaque-forming units. D , In vitro cultured pHCs were inoculated with CHIKV (infected cells [ right ]; MOI, 1) or vehicle (uninfected cells [ left ]) and observed with fluorescence microscopy (scale bars, 200 μm).

Article Snippet: The cells were washed and incubated with the 3E4 recombinant anti-CHIKV E2 murine antibody (Creative Biolabs).

Techniques: Infection, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Plaque Assay, In Vitro, Cell Culture, Fluorescence, Microscopy

Journal: The Journal of Infectious Diseases

Article Title: Use of Recombinant Chikungunya Virus expressing Nanoluciferase to Identify Chondrocytes as Target Cells in an Immunocompetent Mouse Model

doi: 10.1093/infdis/jiaf232

Figure Lengend Snippet: Virus-induced alterations in human chondrocyte (HC) biology. A , Increased matrix metalloprotease (MMP) 3 and MMP-9 messenger RNA (mRNA) synthesis after chikungunya virus (CHIKV) infection of HCs. HCs were infected with CHIKV (multiplicity of infection [MOI], 1). At 48 hours post inoculation, MMP-3 and MMP-9 mRNAs were assessed by means of reverse-transcription quantitative polymerase chain reaction. The amount of mRNA is expressed relative to uninfected controls, after normalization against a housekeeping gene ( GAPDH ). Histograms show the median value and interquartile range obtained in 3 independent experiments performed in triplicate. ** P < .01 (nonparametric Mann-Whitney test). B , TUNEL (terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) labeling was performed, and TUNEL-positive cells were visualized using a fluorescence microscope. Displayed are the average numbers of TUNEL-positive cells over 3 randomly observed fields (as percentages of the total). *** P < .001 (nonparametric Mann-Whitney test). C , Caspase 3 and 7 activity following CHIKV infection of primary HCs (pHCs) (multiplicity of infection, 1). At different times after infection, the cell monolayer was lysed, and caspase 3 and 7 activity was measured (Caspase Glo 3/7 Assay Kit; Promega); the bioluminescent signal is expressed in relative light units (RLUs). Curves show the median value and interquartile range obtained in 2 independent experiments performed in triplicate.

Article Snippet: The cells were washed and incubated with the 3E4 recombinant anti-CHIKV E2 murine antibody (Creative Biolabs).

Techniques: Virus, Infection, Reverse Transcription, Real-time Polymerase Chain Reaction, MANN-WHITNEY, TUNEL Assay, End Labeling, Labeling, Fluorescence, Microscopy, Activity Assay, Caspase-Glo Assay