Journal: bioRxiv
Article Title: SLAMF7 and SLAMF8 receptors shape human plasmacytoid dendritic cell responses to intracellular bacteria
doi: 10.1101/2024.04.25.591064
Figure Lengend Snippet: A-D . CAL-1 and stably transduced (ShCTRL, shSLAMF7, ShSLAMF8) CAL-1 cells were infected with DsRed WT S. Typhimurium (M.O.I. of 25) for 3 h. Then, cells were fixed, permeabilized, and stained for Phospho-flow cytometry or confocal microscopy. A. Heatmap showing MFI levels for P-IRF3, P-NF- κB p65, P-IRF7, P-ERK-1/2, P-p38, P-STAT1 and P-AKT of Salmonella -infected cells at 3 h p.i. relative to Mock condition. Multiple comparison Kruskal-Wallis test, followed by post-hoc Dunn’s test. n=4. B. Column graphs showing relative MFI of phosphorylated proteins (left, P-NF-κB p65; middle, P-STAT-1; right, P-IRF7) in infected cells. Mean ± SD. n=5. C. Left, representative confocal images of P-NF-κB p65 in CAL-1 and stably transduced CAL-1 cells infected with Salmonella . Scale bars: 20 μm. Right, quantification of P-NF-κB p65 + cells. n=3. One-way ANOVA followed by Dunnett’s multiple comparison test. D. Left, representative flow cytometry histogram of EAT-2 adaptor expression (fluorescence intensity (FI) in CAL-1 cells with positivity indicated by a vertical grey bar. Right, percentages of EAT- 2 + CAL-1 and stably transduced cells infected (Green) or not (Grey) with S. Typhimurium. n=3. E. CAL- 1 and stably transduced CAL-1 cells were infected with DsRed WT Salmonella Typhimurium (M.O.I. of 25) for 1 h. After washes and resuspension in gentamicin-containing medium, cells were treated with MitoTEMPO (100µM) for 2 h. Phospho-flow cytometry was performed to analyze the phosphorylation levels of selected proteins. Column graphs show relative MFI of P-NF-κB p65 (left), P-STAT-1 (middle) and P-IRF7 (right). Mean ± SD. n=4. Two-way ANOVA followed by Sidak’s multiple comparison test. Only statistical differences are shown. *, p < 0.05; **, p < 0.01; *** p < 0.001; **** p < 0.0001. no p value, non-significant. F. Proposed model for SLAMF7 and SLAMF8 function in human pDC during intracellular bacterial infection. At the resting state, few SLAMF8 is expressed on the surface of human pDC in contrast to higher SLAMF7 levels. Infection with an intracellular bacterium, like Salmonella , engages SLAMF7 and SLAMF8 receptors, and elicits the phosphorylation and activation of IRF7, STAT- 1 and NF-κB in a SLAMF7/8-dependent manner . This induces the expression of SLAMF7, SLAMF8, pDC activation markers, as well as cytokine secretion (TNF-α, IL-6 and type I and III IFN) . Elevated levels of SLAMF7 or SLAMF8 restrain mitochondrial ROS production, and subsequently favor NF-κB activation as well as Salmonella persistence . This illustration was created using Biorender.
Article Snippet: Phospho-flow staining was performed by fixing the cells with 3.2% PFA for 10 min at 20°C, followed by permeabilization with cold methanol for 15 min. After washing, intracellular staining with antibodies for phospho-proteins (Phospho-NF-κB p65 (Ser536) clone 93H1, #3033; P-Stat1 (Tyr701) clone D4A7, #7649; P-IRF7 (ser477) clone D7E1W, #42016; Phospho-IRF-3 (Ser396) #29047; P-AKT (thr308) #9275; Phospho-p38 MAPK (Thr180/Tyr182), #9211; Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) clone D13.14.4E, #4370; all from Cell Signaling Technology) was done in 1xPBS 2%BSA.
Techniques: Stable Transfection, Infection, Staining, Flow Cytometry, Confocal Microscopy, Comparison, Expressing, Fluorescence, Phospho-proteomics, Activation Assay
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![The cGAS-STING pathway plays a pivotal role in regulating IRF7 activation and nuclear translocation. (A) Western blot analysis of the expression of key molecules (cGAS, p-STING, and TBK1) in the cGAS-STING-TBK1 pathway within the striatum of MPTP-treated mice ( n = 6 per group). (B, C) BV2 cells were pre-treated with either RU.521 (2 µM) or H151 (5 µM) for 12 hours before being exposed to MPP + for an additional 24 hours, and western blotting was performed to determine the relative protein levels of p-IRF7 and IRF7 in these cells ( n = 6 independent experiments). (D) Fluorescence staining for nuclei (blue: DAPI) and p-IRF7 (red: Cy3). p-IRF7 localization to the nuclei was assessed by fluorescence microscopy. The percentage of p-IRF7 nuclear translocation in the MPP + -induced group was significantly higher than that in the control group, and pre-treatment with H151 significantly reduced the percentage of MPP + -induced p-IRF7 nuclear translocation. Arrows indicate nuclear translocation; scale bars: 20 μm. The percentage of cells with significant IRF7 nuclear translocation was calculated from multiple randomly selected fields of view (control samples: three fields of view, at least 200 cells; MPP + stimulated samples: three fields of view, at least 200 cells) ( n = 3 independent experiments). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t -test [A] or one-way analysis of variance followed by Tukey’s post hoc test [B–D]). cGAS: Cyclic guanosine monophosphate adenosine monophosphate synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H-151: a STING inhibitor; IRF7: interferon regulatory factor 7; MPP + : 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin; ns: not significant; p-IRF7: phospho-IRF7; p-STING: phospho-STING; RU.521: a selective cGAS inhibitor; STING: stimulator of interferon genes; TBK1: TANK-binding kinase 1.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9022/pmc11759022/pmc11759022__NRR-20-2361-g005.jpg)
![Impact of IRF7 knockdown and H151 pre-treatment on microglial inflammatory responses and phenotypic markers in BV2 cells exposed to MPP + . (A) IRF7 mRNA expression in cells subjected to si-IRF7 knockdown was determined by RT-PCR, which showed an approximate 80% reduction ( n = 3 independent experiments). (B) BV2 cells were pre-treated with si-IRF7 for 60 hours and then exposed to MPP + for an additional 24 hours. Western blot analysis was used to detect the relative protein levels of IRF7 and IFN-β in BV2 cells stimulated with MPP + after si-IRF7 pre-treatment ( n = 5 independent experiments). (C) Western blotting was used to detect the relative protein levels of iNOS and TNF-α in BV2 cells stimulated with MPP + after si-IRF7 pre-treatment ( n = 5 independent experiments). (D) BV2 cells were pre-treated with H151 (5 µM) for 12 hours and then exposed to MPP + for another 24 hours. RT-PCR was used to detect markers of the M1 ( CD16 , CD32 , and CD86 ) and M2 ( ARG1 , YM1 ) phenotypes ( n = 6 independent experiments). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t -test [A] or one-way analysis of variance followed by Tukey’s post hoc test [B–D]). ARG1: Arginase 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H-151: a STING inhibitor; IFN-β: interferon-beta; IRF7: interferon regulatory factor 7; iNOS: inducible nitric oxide synthase; MPP+: 1-methyl-4-phenylpyridinium; ns: not significant; RT-PCR: real-time polymerase chain reaction; si-IRF7: small interfering RNA targeting IRF7; TNF-α: tumor necrosis factor-alpha.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9022/pmc11759022/pmc11759022__NRR-20-2361-g006.jpg)