We decided to study MyD88 levels in a time-course infection with by western blot, demonstrating that the quantity of this molecule was the same between and DCs and remaining stable during infection (S6 Fig part C). of the spleen. A) Dot-plots showing the gating of myeloid populations of spleen. Dot-plots showing SSC-A versus FSC-A indicates p1, FSC-H versus FSC-W and SSC-H versus SSC-W were used to avoid doublets and FSC-H versus viability shows live and dead cells. Singlets and live cells were used to choose CD3-CD19-DX5-Ly6G+ cell population. From this population, neutrophils were gated as Ly6G+Ly6C+ cells, monocytes as CD11b+CD11clo, Tips DCs as intermedium levels of CD11b and CD11c, conventional dendritic cells (cDCs) as CD11chi; inside this population cDCs CD8- were distinguish as CD11chiCD11b+ and cDCs CD8- as CD11chiCD11blo. B) Representative CGRP 8-37 (human) histograms of different splenic populations (monocytes, neutrophils, Tips DCs, total cDCs, cDCs CD8- and cDCs CD8+) show signal of and mice injected with a lethal dose of at 6 hpi. A pool of CGRP 8-37 (human) and spleens non-infected was used as a control sample without infection (NI). **p0.01, * p0.05; n = 6.(TIF) ppat.1006799.s002.tif (2.0M) GUID:?63F7F8AC-4519-43FE-BA27-B554A3852E01 S3 Fig: Control vehicles and autophagy markers. A) Total CFUs at 0 and 6 hpi in and BMDCs at 6, 12 and 24 hpi. ***p0.001, ** p0.01, * p0.05, ns>0.05 non-significant; n = 5.(TIF) ppat.1006799.s004.tif (456K) GUID:?86A7DADB-7FAD-4D0D-8064-8113B3EE215B S5 Fig: TLR expression and TLR-signalling pathway activation by LPS and HKLM. A) Western-blot analysis in and BMDCs over the time-course of LPS or HKLM treatment. Total and phosphorylated AKT were detected for both treatments. Accompanying charts on the right show quantification, indicating the percentage of phAKT/total AKT ratio. ** p0.01, * p0.05; n = 4. B) PCR analysis of TLR-1, 2 and 6 (arbitrary units) in and BMDCs non-infected (NI) and after and FLT3L-DCs activated with LPS, Imiquimod, Pam3GSK4, HKLM, HKST, and BMDCs. Western-blot analysis of MyD88 over the time-course of infection in and BMDCs (left). Accompanying charts on the right show quantification of the percentage of MyD88; ns non-significant; n = 5. D) Immunoprecipitation of HA (MyD88) followed by western-blot for HDAC6 and MyD88. Immunoprecipitations were carried out using different HDAC6-eGFP plasmids co-transfected with MyD88-HA in HEK cell line. Over-expressed (HDAC6-eGFP, 160 kDa) is indicated CGRP 8-37 (human) at right of western-blot. E) Immunoprecipitation of HA (MyD88) followed by mass spectrometry analysis. Immunoprecipitations were carried out using different HDAC6-eGFP plasmids co-transfected with MyD88-HA in HEK cell line. The number of unique MyD88 and HDAC6 peptides identified is indicated. (*) indicates the presence of acetylated MyD88 peptides. Similar results were obtained in three independent experiments. F) MS2 fragmentation spectra from the peptides showing at 1217.0699 (Top), and 599.3803 (Bottom). Ion adscription to carboxy- (ions, blue) and amino-terminal (ions, red) fragmentation series is indicated. denotes acetylated lysine and indicates carbamidomethylated cysteine. CGRP 8-37 (human) Fragment ion sequence coverage is schematically indicated. Similar results were obtained in three independent experiments.(TIF) ppat.1006799.s006.tif (1.8M) GUID:?2A6F930C-6F52-455B-AED4-B8A6267E20AC S1 Table: Antibody table. Table of antibodies used in experimental procedures disclosed by reference, brand, host, application and dilution.(PDF) ppat.1006799.s007.pdf (478K) GUID:?7BCFD370-7D51-46D3-B463-E8E9A75AEEBB S2 Table: qPCR primers. Table of qPCR primers used in experimental procedures disclosed by gene name and sequence 5-3.(PDF) ppat.1006799.s008.pdf (190K) GUID:?FB6E91A7-92B1-42A2-BA41-03244368DF14 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the (bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. BMDCs have a weakened phosphorylation of MAPK signalling in response to illness, suggesting modified Toll-like receptor signalling (TLR). Compared with counterparts, GM-CSF-derived and FLT3L-derived dendritic cells display weaker pro-inflammatory cytokine secretion in response to numerous TLR CGRP 8-37 (human) agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation main response gene 88 (mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic illness with BMDCs accumulate higher levels of the autophagy marker GPIIIa p62 and display defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results therefore demonstrate an important regulatory part for HDAC6 in the innate immune response to intracellular bacterial infection. Author summary is definitely a food-borne intracellular bacterium that causes listeriosis to 1 1.600 people each.