tlr4 md2 neutralizing antibody  (InvivoGen)

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages

doi: 10.3389/fimmu.2022.801182

Figure Lengend Snippet: Dual role for TLR4 and TLR2 in the induction of type I IFN by L. donovani in macrophages. Total RNA was extracted from L. donovani -infected macrophages from C57BL/6, tlr2 -/- and myd88 -/- mice. Thioglycolate-recruited peritoneal macrophages were infected with stationary-phase promastigotes of L. donovani wild-type or L. donovani expressing ISP2 ( L.d:ISP 2) at a 10:1 parasite:macrophage ratio. After 2 or 3 h of infection, cultures were washed and RNA was extracted or cultures were incubated in RPMI-BSA for 6 h before extraction. Total RNA was extracted using the RNeasy kit (Qiagen). cDNA was made and used as templates in qPCR for the determination of the relative mRNA levels for IFN-β and IFN-α. Graphs show IFN-β levels in (A) C57BL/6 and tlr2 -/- macrophages from the same experiment, and in (C) C57BL/6 and myd88 -/- macrophages from the same experiment. (D) Graphs of the IFN-α levels in C57BL/6 and myd88 -/- macrophages. Where indicated, cells were pre-treated with IgG or neutralizing anti-TLR4 antibodies for 30 min prior to infection. Uninfected cells were used as a control (Ctrl). The experiment was repeated 2 independent times. The graphs show representative experiments with the means ± SD of the triplicate technical replicates. Statistical significance was assessed using one-way ANOVA with Bonferroni posttest. * p < 0.05, ** p < 0.01, *** p < 0.001. Differences between the same time point or the same group are shown. (B) Peritoneal thioglycolate-recruited macrophages from C57BL/6 and tlr2 -/- mice were cultivated on glass coverslips overnight in RPMI-FCS and then washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI-BSA. After 3 h, the monolayers were washed with HBSS to remove extracellular parasites, fixed with methanol and Giemsa-stained, or further incubated for 72 h in RPMI-FCS at 37°C before fixation and staining. Where indicated, 200 ng/ml IFN-β was added to cultures that had been previously infected for 3 h and washed for removal of extracellular parasites and cultivated for 72 h in RPMI-FCS. The number of intracellular parasites was determined under a light microscope. The experiment was performed 3 independent times. The graphs show a representative experiment of the means ± SD of replicates in triplicate. Statistical significance was assessed using two-way ANOVA with Tukey’s and Sidak’s multiple comparison tests. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a; Thermo Fisher Scientific) were incubated for 30 min with the macrophages in RPMI-10% FCS and then washed twice before infection with the parasites in RPMI-0.1% BSA at 37°C and 5% CO 2 .

Techniques: Infection, Expressing, Incubation, Extraction, Control, Staining, Light Microscopy, Comparison

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages

doi: 10.3389/fimmu.2022.801182

Figure Lengend Snippet: Neutrophil elastase and PKR are required for the induction of anti-inflammatory genes in infected macrophages. (A) Nuclear extracts were obtained from C57BL6 macrophages infected for 2, 6, or 24 h with stationary-phase promastigotes of L. donovani at a 10:1 parasite:macrophage ratio. The nuclear extracts were processed for Western blot analysis with antibodies to IRF3, as well as nuclear lamin for the loading control. Macrophages incubated in medium alone were used as the baseline control (Ctrl). Poly I:C was used at 25 µg/ml. In (D) , total lysates were obtained from C57BL/6 macrophages infected for 1 h with stationary-phase promastigotes of L. donovani at a 10:1 parasite:macrophage ratio and processed for Western blot with antibodies to phospho-PKR, as well as actin for the loading control. Where indicated, monolayers were pre-treated with 100 nM of the TLR4 inhibitor, TAK-242 or with 10 µM of neutrophil elastase inhibitor (NEI) before infection. Total RNA was extracted from L. donovani -infected macrophages from (B, C, E) C57BL/6 and ela2 -/- mice or (F) 129Sv/Ev and pkr -/- mice. Thioglycolate-recruited peritoneal macrophages were infected with stationary-phase promastigotes of L. donovani wild-type or L. donovani expressing ISP2 ( L.d:ISP 2) at a 10:1 parasite:macrophage ratio for 2 h, cultures were washed, and RNA was extracted or cultures were incubated in RPMI-BSA until 6 h before extraction. cDNA was made and used as a template in qPCR for the determination of the relative mRNA levels for IL10, OASL2, SOD1, and PKR as indicated. The experiment was repeated 2 independent times. The graphs show representative experiments, with the means ± SD of the triplicate technical replicates. Statistical significance was assessed using one-way ANOVA with Bonferroni posttest. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a; Thermo Fisher Scientific) were incubated for 30 min with the macrophages in RPMI-10% FCS and then washed twice before infection with the parasites in RPMI-0.1% BSA at 37°C and 5% CO 2 .

Techniques: Infection, Western Blot, Control, Incubation, Expressing, Extraction

Journal: Frontiers in Immunology

Article Title: Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages

doi: 10.3389/fimmu.2022.801182

Figure Lengend Snippet: Schematic representation of the role of TLRs and PKR in L. donovani development in macrophages. L. donovani utilizes macrophage NE activity, susceptible to inhibition by the ecotin-like inhibitor of serine peptidase ISP2, via TLR4 and TLR2 during phagocytosis (1), to induce PKR phosphorylation. NE contributes to the modulation of Rab7 + endosomal fusion with the phagosome (2) and to IRF3 accumulation in the nucleus (3). IRF7 recruitment to parasite-containing phagosomes takes place during early stages of infection (2). TLR3 is recruited to the parasitophorous vacuole (4), and together with integrative signals conveyed through PKR (5) acts as a hub to induce the expression of IFN-I, OASL2, SOD1, IL10, and PKR genes (6). IFNβ ensures parasite survival and growth in macrophages (7).

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a; Thermo Fisher Scientific) were incubated for 30 min with the macrophages in RPMI-10% FCS and then washed twice before infection with the parasites in RPMI-0.1% BSA at 37°C and 5% CO 2 .

Techniques: Activity Assay, Inhibition, Phospho-proteomics, Infection, Expressing

Journal: The FASEB Journal

Article Title: Neutrophil elastase promotes Leishmania donovani infection via interferon-β

doi: 10.1096/fj.201900524R

Figure Lengend Snippet: TLR4 and TLR2 are required for development of L. donovani in macrophages, and NE acts through TLR4. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice, TLR2 knockout mice ( tlr 2 −/− ), or TLR4 knockout mice ( tlr 4 −/− ) ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further cultivated for 72 h in RPMI-FCS at 37°C ( A ). NE acts through TLR4. B ) Where indicated, macrophages were pretreated with 10 μg/ml of control IgG2b or with anti-TLR4 (MTS5) mAb for 30 min in RPMI-FCS and washed before infection. Where indicated (+), purified human NE was added at 200 ng/ml after removal of antibodies and before addition of the parasites. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment. The graphs show means ± sd . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a) were incubated for 30 min with macrophages in RPMI-FCS, and the monolayers were washed twice before infection with the parasites at 37°C in RPMI-BSA.

Techniques: Knock-Out, Infection, Staining, Control, Purification

Journal: The FASEB Journal

Article Title: Neutrophil elastase promotes Leishmania donovani infection via interferon-β

doi: 10.1096/fj.201900524R

Figure Lengend Snippet: Colocalization of NE and TLR4 with L. donovani in infected macrophages. Macrophages of C57BL/6 mice were infected with WT L. donovani or L. donovani : ISP 2 (clone 2) for 3 h in RPMI-BSA and processed for immunofluorescence or washed and cultured for 72 h in RPMI-FCS and processed for immunofluorescence. Coverslips were treated with anti-TLR4 antibodies washed and incubated with anti-rabbit–Alexa 488. Coverslips were subsequently incubated with antibodies to NE and washed and incubated with anti-rabbit–cyanine 3 antibodies. Coverslips were treated with DAPI for 5 min and washed and mounted for immunofluorescence. Samples were analyzed by confocal microscopy. White arrows point to the parasite. Scale bar, 2 μm.

Article Snippet: In the experiments of receptor neutralization, 10 μg/ml anti-mouse TLR4 neutralizing antibodies (CD284/MD2 complex clone MTS510; Thermo Fisher Scientific) or 10 μg/ml IgG (IgG2aK control clone eBR2a) were incubated for 30 min with macrophages in RPMI-FCS, and the monolayers were washed twice before infection with the parasites at 37°C in RPMI-BSA.

Techniques: Infection, Immunofluorescence, Cell Culture, Incubation, Confocal Microscopy