Journal: Communications Biology

Article Title: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

doi: 10.1038/s42003-020-0882-8

Figure Lengend Snippet: a HOIPINs suppress the LPS-mediated NF-κB and IFN antiviral pathways. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 20 μg/ml LPS for the indicated period with HOIPINs. The cell lysates were immunoblotted with the indicated antibodies. b LPS-induced gene expression is suppressed by HOIPINs. BMDM cells were pre-treated with the indicated concentrations of HOIPINs for 30 min, and stimulated with 100 ng/ml LPS for 1 h. The mRNA levels were assessed by qPCR. c Suppression of IRF3 targets by HOIPIN-1. BMDM cells were stimulated with 100 ng/ml LPS for 8 h with HOIPIN-1, and interferon β ( n = 13) and Cxcl10 ( n = 10) were quantified by ELISA. d Suppression of antiviral signaling by HOIPIN-8. MEFs were stimulated with 10 μg/ml poly(I:C) for the indicated period with HOIPIN-8, and subjected to immunoblotting analysis. e Suppression of IRF3 targets by HOIPINs. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 10 μg/ml poly(I:C) for 2 h with HOIPINs. The mRNA levels were assessed by qPCR. f HOIPIN-1 inhibits ISRE-luciferase activity. MEF cells, transfected with the ISRE-luciferase reporter, were stimulated with 10 μg/ml poly(I:C) with or without HOIPIN-1 for 6 h, and the luciferase activities were analyzed. g LUBAC activity is indispensable for the IFN pathway. WT- and HOIP −/− -MEFs were treated with 10 μg/ml poly(I:C) and HOIPIN-1 for 2 h. Cell lysates were subjected to immunoblotting analysis. h HOIP is critical for the expression of IRF3-target genes. WT- and HOIP −/− -MEFs were treated as in g , and qPCR analyses were performed. i The Sendai virus (SeV)-induced antiviral response is suppressed by HOIPINs. MEFs were infected with SeV at a multiplicity of infection (MOI) of 10 for 8 h, and treated with the indicated concentrations of HOIPINs for 30 min. qPCR analyses were performed. In b , c , e , f , h , i , data are shown as mean ± SEM, n = 3 (sample numbers in c are indicated in the legend). NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Cell lysis, reverse transcription, and qPCR were performed with a SuperPrep Cell Lysis RT Kit for qPCR (TOYOBO) and Power SYBR Green PCR Master Mix (Life Technologies), according to the manufacturers’ instructions.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay, Transfection, Expressing, Virus, Infection

Journal: Communications Biology

Article Title: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

doi: 10.1038/s42003-020-0882-8

Figure Lengend Snippet: a HOIPIN-1 shows potent toxicity to ABC-DLBCL cell lines. GCB-DLBCL cells (BJAB, SU-DHL-4, and HT) and ABC-DLBCL cells (TK, DLBCL2, and OYB) were cultured in the presence of DMSO or 10 μM HOIPIN-1. Taking the cell viabilities in the presence of DMSO as 100%, the relative cell viabilities of respective cell line in the presence of HOIPIN-1 were assessed by a CellTiter-Glo luminescent cell viability assay. b HOIPIN-8 enhanced the cell death of ABC-DLBCL cells than HOIPIN-1. DLBCL2 cells or BJAB cells were treated with DMSO, 10 μM HOIPIN-1, or 10 μM HOIPIN-8 for the indicated period, and the relative cell viabilities were assessed as in a . c Caspase activation in ABC-DLBCL cells by HOIPIN-8. Cells were treated with or without 10 μM HOIPIN-8 for 24 h, and cell lysates were immunoblotted with the indicated antibodies. d Reduced NF-κB activation in ABC-DLBCL cells by HOIPIN-8. Cells were treated and analyzed as in c . e HOIPIN-1 suppresses the expression of NF-κB target genes in ABC-DLBCL cells. Cells were treated with 10 μM HOIPIN-1 for 24 h, and qPCR analyses were performed. f Intracellular linear ubiquitin and IκBα phosphorylation in ABC-DLBCL cells are diminished by HOIPIN-1. HBL1 and BJAB cells were treated with 10 μM HOIPIN-1 for the indicated period, and cell lysates were immunoblotted with the indicated antibodies. g HOIPIN-8 has potent inhibitory effects on NF-κB activation and linear ubiquitination in ABC-DLBCL cells than those in HOIPIN-1. HBL1 cells were treated with the indicated concentrations of HOIPIN-1 or -8 for 24 h, and cell lysates were immunoblotted with the indicated antibodies. In b , e , data are shown as mean ± SEM, NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Cell lysis, reverse transcription, and qPCR were performed with a SuperPrep Cell Lysis RT Kit for qPCR (TOYOBO) and Power SYBR Green PCR Master Mix (Life Technologies), according to the manufacturers’ instructions.

Techniques: Cell Culture, Cell Viability Assay, Activation Assay, Expressing, Ubiquitin Proteomics, Phospho-proteomics

Journal: PLoS ONE

Article Title: Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

doi: 10.1371/journal.pone.0071244

Figure Lengend Snippet: Systemic and topical OVA sensitization results in inflammation, disturbs epidermal barrier homeostasis, and induces PPARδ target gene expression in skin.

Article Snippet: FABP5 protein levels were determined in protein lysates prepared from whole mouse skin according to the protocol indicated in using the rabbit FABP5 polyclonal antibody purchased from ProteinTech (Chicago, IL).

Techniques: Targeted Gene Expression, Binding Assay

Journal: PLoS ONE

Article Title: Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

doi: 10.1371/journal.pone.0071244

Figure Lengend Snippet: ( a ) IL-4 serum levels after systemic with or without additional topical OVA sensitization (n = 8). ( b ) ATRA levels in mouse skin determined by HPLC MS-MS method upon systemic (i.p.) and systemic plus topical (i.p.+e.c.) OVA sensitization (n = 3/group). ( c ) Ratio of Fabp5 vs. Crabp2 expression in the skin of OVA-treated mice (n = 6/group) compared to control mice (PBS i.p.). Data are presented as mean values ± SEM. Statistical significance ( p ) is based on one-way ANOVA followed by Tukey’s multiple comparison test for gene expression results and ELISA data. For HPLC MS-MS results, significance was determined using Student’s t -test.

Article Snippet: FABP5 protein levels were determined in protein lysates prepared from whole mouse skin according to the protocol indicated in using the rabbit FABP5 polyclonal antibody purchased from ProteinTech (Chicago, IL).

Techniques: Tandem Mass Spectroscopy, Expressing, Control, Comparison, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

doi: 10.1371/journal.pone.0071244

Figure Lengend Snippet: ( a ) Fabp5 protein levels in the skin of mice with allergen-induced dermatitis. 150 µg proteins were loaded per lane and beta-actin was used as control for even protein loading. ( b ) Immunohistochemical analysis of Fabp5 protein expression in five-micrometer back skin sections of OVA-sensitized mice. ( c ) ATRA-induced nuclear receptor-mediated signaling pathways depending on the predominant cellular transport protein.

Article Snippet: FABP5 protein levels were determined in protein lysates prepared from whole mouse skin according to the protocol indicated in using the rabbit FABP5 polyclonal antibody purchased from ProteinTech (Chicago, IL).

Techniques: Control, Immunohistochemical staining, Expressing, Protein-Protein interactions

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: A microscopic and molecular assay to measure parasite load. (A) Confocal microscopy images of THP-1 cell-derived macrophage-like cells infected or not with L. donovani promastigotes at the MOI shown at 100× magnification. The infection was carried out for 24 h before fixing and staining. DAPI stain was used to stain both the nuclear and parasite DNA (the pseudocolor red is shown instead of the blue color of DAPI for better visualization; laser wavelength: 405.0, power: 4.3). The arrow indicates an amastigote inside a cell. Images were obtained under the DAPI channel using the NIS-Elements Imaging software (version 5.20.00). (B) The images obtained in A were manually scanned to count the number of cells infected and the number of amastigotes/cell and plotted. Then, 100–200 cells were counted per condition. (C) Parasite count in THP-1-derived cells infected with L. donovani (L. d) or not (C, control). The Ct values obtained from qPCR carried out on DNA isolated from infected cells were compared with the same represented as a standard curve obtained from qPCR carried out on promastigote DNA that was isolated from serially diluted parasite cultures in M199 medium; from this standard curve, the parasite numbers were deduced and shown as mean and SD from two experiments. (D) A new method to estimate parasite load after normalizing for host DNA concentration. The kDNA was measured from DNA isolated from infected cells by qPCR and normalized to copies of GAPDH DNA. The mean is shown from three separate experiments along with SD. NC, no infection control. (E) RAW264.7 cells infected with L. donovani were stained with Geimsa stain for LD body counting. Images were obtained in a bright field using Cell D software at 100× magnification. The arrow indicates an amastigote inside a cell. The number of amastigotes in 100 macrophage cells was counted using oil immersion lenses. (F) Data obtained from counting cells in E are plotted, showing the mean and SD. (G) Parasite load was estimated as kDNA normalized to gapdh from two experiments (shown as mean and SD) that were performed simultaneously with those shown in E. L. d, L. donovani; NC, no infection control.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Confocal Microscopy, Derivative Assay, Infection, Staining, Imaging, Software, Control, Isolation, Concentration Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 inhibits L. donovani growth in cells. (A) Human (left) or mouse(right) IFN-λ3 at the shown concentration was added along with parasites during infection, and kDNA was measured after 24 h. The data are from two experiments in THP-1-derived cells and four experiments from RAW264.7 cells, showing the mean and SD. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Human (left) or mouse (right) IFN-λ3 at 100 ng/mL was added at different time points after L. donovani infection, and kDNA was measured after 24 h of infection. The data are from two experiments in both cell lines, shown as the mean and SD. *P < 0.05. (C) IFN-λ4 at 6 µg/mL was added, and the experiment was as in A showing data from two experiments with mean and SD (**P < 0.01). A high concentration of IFN-λ4 was required as the specific activity of the recombinant protein preparation is low, and 6 µg/mL would give a comparable activity to 100 ng/mL of IFN-λ3, as detailed in our previous work (16). (D) Effect of IFN-λ3 and IFN-λ4 on cytokine secretion in L. donovani infected M2-macrophage-like cells. M2 macrophages were differentiated for 2 days from THP-1 cells as described in our previous work (16), and IFN-λ3 at 1 µg/mL or IFN-λ4 at 6 µg/mL were added during parasite infection. After 24 h, the supernatants and cells were collected, and qPCR and ELISA were carried out. The data are from two experiments, showing the mean and SD. *P < 0.05; **P < 0.01. L. d, L. donovani; NT, no treatment control.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Concentration Assay, Infection, Derivative Assay, Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 inhibits L. donovani by increasing ROS production in cells. (A) (Top) RAW264.7 cells infected with L. donovani for different time points as shown were subject to qPCR for kDNA (normalized to Gapdh) and Ifnl3 (normalized to Rps29). (Bottom) Immunoneutralization of secreted mIFN-λ3 increases parasite load. The experiment was similar to the one described in the top, except that a rat anti-mouse IFN-λ3 antibody (shown as α-mIFN-λ3) was added or not at 24 h pi, at increasing concentrations as shown (1.5, 3.0, and 4.5 µg/mL) in the medium, and kDNA copies in the cells were measured after an additional 12 h (i.e., 36 h pi) of incubation. The data from both the top and bottom are from two experiments, showing the mean and SD. *P < 0.05i, post-infection. (B) ROS levels are induced by IFN-λ3, as measured by the NBT assay. (Top) The indicated treatment (IFN-λ3, LPS, or L. d ± IFN-λ3) was for 2 h; IFN-λ3 was at 100 ng/mL and LPS was at 1 µg/mL. (Bottom) L. d infection was for indicated time points. The means from three experiments (top) and two experiments (bottom) are shown; error bars denote SD. NT, no treatment. *P < 0.05; **P < 0.001; L.D, L. donovani. (C) mIFN-λ3 increases ROS production in RAW264.7 cells in a dose-dependent manner, as measured by a fluorescence-based assay. RAW264.7 cells were incubated with the shown concentrations of mIFN-λ3 or LPS (1 µg/mL) or NAC (100 mM) + mIFN-λ3 (0.5 µg/mL) for 2 h before staining with DCFDA. The fluorescence intensity quantified is shown at the top (data from two experiments with mean and SD are depicted; *P < 0.05), and the bottom shows representative images taken at 20× magnification of the cells under the conditions shown. (D) IFN-λ3 can inhibit parasite load even when ROS production is inhibited. (top) L. donovani (L. d) infection was given in THP-1-derived cells as before for 24 h in the absence (NT, no treatment) or presence of IFN-λ3 and in the presence or absence of an increasing concentration of NAC added to the medium. The parasite load was estimated after 24 h pi. (Bottom) As in top, but the kDNA copies are shown after normalizing to NT and NAC at 50 mM; data are from two experiments showing the mean and SD. **P < 0.01. (E) (Top) THP-1 cells were pretreated with 50 ng/mL of IFN-λ3 for 48 h (incubated along with PMA), and then the media was removed and L. donovani (L. d) or mock infection (1:10 MOI) was given for 2 h. ROS levels were measured by the NBT assay. Data from two experiments with mean and SD are shown. *P < 0.05. NT, no treatment. (Bottom) kDNA levels shown in THP-1 cells that were pretreated with 100 ng/mL IFN-λ3 or not (incubated along with PMA for 48 h) were washed off the media and infected or not with L. donovani for 24 h, and kDNA was measured by qPCR as before. The data are from two experiments, showing the mean and SD. Similar experiments with IFN-λ4 are shown in Fig. S2B.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Incubation, Fluorescence, Staining, Derivative Assay, Concentration Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 targets an early event in parasite uptake. (A) IFN-λ3 at the indicated doses (the color key for the doses shown is the same for A, B, and C) were incubated along with the promastigotes, and slides were stained with DAPI, and the amastigotes presence and numbers were manually counted. The data are from three (for no treatment) and four (for IFN-λ3 treatment) independent experiments, each with 100–200 individual cells shown as the mean and SD. *P < 0.05. (B) (Left) Fluorescence microscope image of THP-1-derived cells incubated with CFSE-stained heat-killed L. donovani showing different stains. The arrow indicates a parasite taken up by the cell by phagocytosis. (Right) Manual counting of the parasite inside the cells and numbers was done as above and plotted. The data are from four independent experiments, each involving 100 to 200 individual cells, showing the mean and SD. For B and C, live parasites inside the cells were observed with the DAPI channel, while heat-killed parasites were observed under the GFP channel. Images were obtained under DAPI (imparts red pseudocolor), GFP (green), and Texas Red (imparts gray pseudocolor). (C) (Top) Fluorescence microscopic image of THP-1-derived macrophage-like cells that have taken up zymosan in a phagocytosis assay as described in the Materials and Methods; images were obtained under DAPI (imparts red pseudocolor), GFP (green), and Brightfield. The arrow indicates zymosan inside a cell. (Bottom) Manual counting of the cells with zymosan was performed as in B and plotted. The data are from two separate experiments with ~250 cells in each, showing the mean and SD. For B and C, image analysis was done using Leica Application Suite X software (version 3.7.2.22383) at 100× magnification. (D) Schematic representation of an experimental design with five (I-V) strategies. L donovani (L. d) infection (downward arrow) was given at 1:20 MOI for only 6 h, after which the uninfected promastigotes were removed after rigorous washing with PBS. IFN-λ3 treatment (inverted dark triangle) was given at the depicted time points, and after the indicated incubation periods, cells were collected and quantified for kDNA copies by qPCR (downward arrow with round head). (E and F). IFN-λ3 targets an early event during parasite uptake. Experiments as designed in the schematic representation shown in D were carried out, and results are presented in E (strategies I-IV) for 24 (E, top), 48 (E, middle), or 72 h pi (E, bottom) and F (strategies I for 48 h pi and V). 1:10 MOI and continuous infection conditions as described for previous experiments were also carried out, but the incubation periods included 48 and 72 h pi along with 24 h pi (shown in E middle, bottom, and top, respectively). qPCR was performed to estimate kDNA copies and ISG expression. The kDNA copies were normalized to NT (no IFN-λ3 treatment) and shown in E, while the actual fold changes are shown in F. The data in both E and F are from two experiments showing the mean and SD. *P < 0.05; **P < 0.01. The data shown in E are also shown with actual fold changes and without normalization to NT control samples in Fig. S2E and F.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Incubation, Staining, Fluorescence, Microscopy, Derivative Assay, Phagocytosis Assay, Software, Infection, Expressing, Control

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 before infection fails to inhibit parasite load in the mouse model of VL. (A) and (B) Gene expression changes measured by qPCR from mice spleen (A) or liver (B) pretreated with 4 µg/mouse of mIFN-λ3 or PBS 18 h before infection with L. donovani. The infection was allowed for 6 weeks, and gene expression was quantified using primers listed in Table S1 and SYBR green (Applied Biosystems) protocol. kDNA was also measured and normalized using the 2−∆∆Ct method. Six female mice were used in each group, and 18 data points are shown for the six samples carried out in technical triplicate. The statistical significance, however, was calculated with n = 6 by considering the average of the technical triplicates for each animal as the actual value for that animal. **P < 0.01. No significant differences were observed in the body weight and organ weights for the two groups.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Gene Expression, SYBR Green Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 treatment during infection fails to inhibit parasite load in the mouse model of VL. (A) Liver and spleens of the mice treated with PBS or 4 µg/mouse of mIFN-λ3 (in two doses of 2 µg/mouse in each dose given 24 h apart) at 12 weeks of infection were collected and weighed before subjecting them to qPCR for kDNA (liver and spleen) and other genes (spleen only). Each group had six animals (n = 6); 18 data points are shown for the six samples carried out in technical triplicates for qPCR data. The statistical significance, however, was calculated with n = 6 by considering the average of the technical triplicates for each animal as the actual value for that animal; for the organ weights, single measurements were taken for each organ belonging to each animal. *P < 0.01. (B) The sera from mice were subjected to ELISA for the three mouse cytokines shown.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Enzyme-linked Immunosorbent Assay