Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: In silico compound screen and validation of transcriptomic modulators for the DAM1/DAM2 signature. A. Graph depicting the in silico approach to identify compounds mimicking the DAM cluster signatures using the CMAP resource (Connectivity Map resource; ) followed by a validation approach. B. CMAP predictions from microglial single-cell RNA- Seq Cluster 11 , single-nucleus RNA-Seq Microglia 13 cluster , and the overlapping upregulated gene set. The Connectivity Map (CMAP) was used to identify compounds that are predicted to upregulate or downregulate gene sets associated with either of the DAM-like clusters from single-cell or single-nucleus data. Heatmaps depict the Z-scored tau score for each compound following query analysis. Clustering of tau scores across microglial clusters 11, 13, or the merged signature was performed with absolute linkage. C. Screening of identified candidate drugs via RT-qPCR in HMC3 microglia-like cells. HMC3 cells were exposed to selected compounds predicted to upregulate (upper panel; DMSO control: n=6; Vorinostat: n=6; Entinostat: n=6; Wiskostatin: n=6; Flavokavain B: n=6; Trimipramine: n=6;) or downregulate (lower panel; DMSO control: n= 4; Geranylgeraniol: n=4; Valproic acid: n=4 ; Cholic acid: n=4; Temozolomide: n=4; Ramipril: 0.01mM n=8, 0.1mM n=4; Naftopidil: n=6) the DAM signature at 6 hrs. or 24 hrs. Selected DAM marker gene expression ( CD9, SPP1, CTSD ) was assessed via RT-qPCR. CT values were normalized to HPRT1 . Bars represent fold- change gene expression in relation to DMSO control. For statistical analysis Kruskal Wallis test followed by Dunn’s multiple comparisons test was performed.*p.adj ≤ 0.05; **p.adj ≤ 0.01; ***p.adj ≤ 0.001; ****p.adj ≤ 0.0001.

Article Snippet: Each concentration of drug was plated in triplicate with early-passage HMC3 cells (ATCC; Cat #: CRL-3304), and cell viability was read out using using MTT assay, by incubating the cells in 0.25mg/ml MTT (invitrogen; Catalog #: M6494) in PBS (Corning, Cat #:21-040-CV) for 1hr at 37°C, following removal of MTT solution and addition of 200μl DMSO (Sigma-Aldrich, Cat #:472301) and further incubation of the cells for 15 min at 37°C before measuring the absorbance at 570 nm with a Tecan Infinite® 200 PRO plate reader (Tecan; Cat#: 30050303).

Techniques: In Silico, Biomarker Discovery, RNA Sequencing, Quantitative RT-PCR, Control, Marker, Gene Expression

Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: Bulk RNAseq data from the HMC3 DAM model. A. Heatmaps showing the expression of Cluster 11 (left), Microglia 13 (middle) and DAM1/DAM2 (right) marker gene sets in bulk RNAseq data generated 24hrs following exposure to DMSO (control), Entinostat (red; 10µM) or Vorinostat (green; 1µM). Each column represents a single sample, each row a single gene represented in the respective marker set. Pairwise differential testing between DMSO control and each of the treatment conditions (Entinostat, 10µM; Vorinostat, 1µM) was conducted using a Wald test with the Benjamini-Hochberg correction (FDR alpha < 0.05). The legend represents Z scores, with lower scores indicated in red and higher scores indicated in blue. Data represents n=3 independent experiments for each treatment group with each n for all compounds being performed at the same time. B. Volcano Plots depicting the distribution of differentially expressed genes from different signatures (DAM1, DAM2 , Cluster 11 , Microglia 13 ) for each treatment condition (Entinostat or Vorinostat) in comparison to DMSO control. HMC3 microglia were treated for 24hrs with DMSO as control, Entinostat (10µM) or Vorinostat (1µM) followed by bulk RNA-Seq. Volcano plots depict all genes present in each marker set (DAM1: 10 genes; DAM2: 20 genes; Cluster 11: 89 genes, Microglia 13: 127 genes) plotted based on log2FC (fold change expression) and -log10(p value) with the ones significantly upregulated marked in red and labelled with the gene name. Plots are organized from Cluster 11 (top left), to DAM1 (bottom left), to DAM2 (top right) to Microglia 13 (bottom right). C. PCA plot of bulk RNAseq results from HMC3 microglia treated with DMSO, Vorinostat or Entinostat . Principal component analysis (PCA) was calculated on log-normalized bulk RNA-Seq data derived from compound-treated HMC3 microglia following 24hrs of exposure to DMSO (control; blue), Entinostat (10µM; red) or Vorinostat (1µM; green). Data represents n=3 independent experiments for each of the treatment group with each n for all compounds being performed at the same time. D. Pie chart depicting the number of significantly upregulated genes by Entinostat or Vorinostat for each of the queried marker signatures DAM1, DAM2, Cluster 11 , Microglia 13 . The number of significantly upregulated genes across all three replicates for each treatment group (Entinostat or Vorinostat) in comparison to DMSO control was identified and converted to a percentage of marker genes upregulated/ marker set. Data for DAM1 are depicted in purple, for DAM2 depicted in red, for Cluster 11 depicted in violet and for Microglia 13 depicted in teal. E. Signature-specific markers induced by Vorinostat and Entinostat. Markers significantly induced by Vorinostat and Entinostat for each signatured are depicted for DAM1 (purple), DAM2 (red), Cluster 11 (violet), Microglia 13 (teal).

Article Snippet: Each concentration of drug was plated in triplicate with early-passage HMC3 cells (ATCC; Cat #: CRL-3304), and cell viability was read out using using MTT assay, by incubating the cells in 0.25mg/ml MTT (invitrogen; Catalog #: M6494) in PBS (Corning, Cat #:21-040-CV) for 1hr at 37°C, following removal of MTT solution and addition of 200μl DMSO (Sigma-Aldrich, Cat #:472301) and further incubation of the cells for 15 min at 37°C before measuring the absorbance at 570 nm with a Tecan Infinite® 200 PRO plate reader (Tecan; Cat#: 30050303).

Techniques: Expressing, Marker, Generated, Control, Comparison, RNA Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: Bulk RNA-Seq of the human iPSC-derived microglia (iMG) DAM model. A. Volcano Plots depicting the distribution of differentially expressed genes from different signatures (Cluster 11 , Microglia 13 , iMG Cluster 2+8 ) for Vorinostat treatment in comparison to DMSO control. iPSC-derived microglia at Day 28-29 of differentiation were treated for 24hrs with DMSO as control or Vorinostat (0.1µM) followed by bulk RNAseq. Volcano plots depict all genes present in each marker set (Cluster 11: 89 genes, Microglia 13: 127 genes, iMG Cluster 2+8: 134 genes) plotted based on log2FC (fold change expression) and -log10(p value) with the ones significantly upregulated marked in red and of the most significantly changed genes, a selection of nine genes was labeled with the gene name. Plots are organized from Cluster 11 (left), to Microglia 13 (middle), to iMG Cluster 2+8 (right). B. Heatmaps showing the expression of Cluster 11 (left), Microglia 13 (middle) and iMG Cluster 2+8 (right) marker sets in bulk RNAseq data generated 24hrs following compound treatment with DMSO (control) or Vorinostat (green; 0.1µM). Each column represents a single sample, each row a single gene represented in the respective marker set. Pairwise differential testing between DMSO control and each of the treatment conditions (Entinostat, 10µM; Vorinostat, 1µM) was conducted using a Wald test with the Benjamini-Hochberg correction (FDR alpha < 0.05). The legend represents Z scores, with lower scores indicated in red and higher scores indicated in blue. Data represents n=5 independent experiments per treatment group from one batch of iPSC-derived human microglia. For data replication in a second batch see Supple. . C. Venn diagram depicting significantly induced markers across the signatures for Cluster 11 , Microglia 13 and iMG Cluster 2+8 in Vorinostat-treated iMGs. Each circle shows significantly induced markers from each marker set - Cluster 11 (violet), Microglia 13 (green), Dolan et al. (red). Overlays of circles depict induced marker genes shared across different combinations of marker sets. Percentage indicates ratio of each marker set in relation to the total number of significantly induced markers across all three signatures. D. MITF expression in HMC3 and iMG DAM models. Violin plots depict the expression of the transcription factor MITF in transcripts per million (TPM) across treatment conditions in HMC3 microglia (top; DMSO (blue), Vorinostat (1µM; green), Entinostat (10µM; red); n=3/group) and iMG (bottom; DMSO (blue), Vorinostat (0.1µM; green); n=6 per group, one iMG batch; for data replication see Suppl. ). For statistical analysis of HMC3 data, one-ay ANOVA followed by Dunnett’s multiple comparisons test was performed. For iMG data, unpaired t-test was performed. Each dot represents a replicate, central interrupted line represents the median and fine dotted lines represent the interquartile range. *p.adj ≤ 0.05, **p.adj ≤ 0.01, ***p.adj ≤ 0.001 test

Article Snippet: Each concentration of drug was plated in triplicate with early-passage HMC3 cells (ATCC; Cat #: CRL-3304), and cell viability was read out using using MTT assay, by incubating the cells in 0.25mg/ml MTT (invitrogen; Catalog #: M6494) in PBS (Corning, Cat #:21-040-CV) for 1hr at 37°C, following removal of MTT solution and addition of 200μl DMSO (Sigma-Aldrich, Cat #:472301) and further incubation of the cells for 15 min at 37°C before measuring the absorbance at 570 nm with a Tecan Infinite® 200 PRO plate reader (Tecan; Cat#: 30050303).

Techniques: RNA Sequencing, Derivative Assay, Comparison, Control, Marker, Expressing, Selection, Labeling, Generated

Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: HCM3 microglia-like cells were pretreated each compound or DMSO as control for 24hrs. Subsequently, they were exposed to pHrodo-labeled Dextran for 1hr, and subsequently the uptake of pHrodo-labeled Dextran was assessed using flow cytometry. Individual experiments are depicted as individual dots in the bar graphs depicting mean ± SEM (Vorinostat – green; Entinostat– purple). Phagocytosis was normalized to percent DMSO (% DMSO) control and for statistical analysis, log-fold change values in comparison to DMSO-treated control samples were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. *p.adj ≤ 0.05; **p.adj ≤ 0.01. C. Vorinostat and Entinostat induce Aß1-42 phagocytosis in HMC3 microglia-like cells. HCM3 microglia were pretreated with each compound or DMSO as control for 24hrs, subsequently exposed to AlexaFluor 647-labeled Aß monomers for 1hr and subsequently the uptake of AlexaFluor 647-labeled Aß monomers was assessed using flow cytometry Individual experiments are depicted as individual dots in the bar graphs depicting mean ± SEM (Vorinostat - green; Entinostat - purple). Phagocytosis was normalized to % DMSO control and for statistical analysis, log-fold change values in comparison to DMSO-treated control samples were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. *p.adj ≤ 0.05; ** p.adj ≤ 0.01. D. Vorinostat reduces phagocytosis of pHrodo-labeled E.coli. HCM3 microglia-like cells were pretreated with each compound or DMSO as control for 24hrs, subsequently exposed to pHrodo-labeled E.coli particles for 1hr and subsequently the uptake of pHrodo-labeled E.coli particles was assessed using flow cytometry. Individual experiments are depicted as individual dots in the bar graphs depicting mean ± SEM (Vorinostat - green; Entinostat - purple). Phagocytosis was normalized to % DMSO control and for statistical analysis, log-fold change values in comparison to DMSO-treated control samples were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. *p.adj ≤ 0.05; **p.adj ≤ 0.01. E. Vorinostat and Entinostat, decrease the secretion of pro-inflammatory cytokine MCP-1. HMC3 microglia-like cells were pre-treated with Vorinostat or Entinostat for 24hrs, and subsequently stimulated with either TNF-α (0.3 µg/mL), IFN-y (0.3 µg/mL) or H2O as control for 12 or 24 hrs. Supernatant was collected and pro-inflammatory cytokine secretion assessed using a human pro-inflammatory cytokine discovery assay. Heatmap depicts measured amount of cytokines IL-6, IL-8, MCP-1 (mean ± SEM) in pg/ml for DMSO control- treated samples (white, light gray, gray) or compound-treated samples (light green, green dark green/light purple, purple, dark purple). For statistical analysis, one-way ANOVA followed by Tukey’s multiple comparisons test with a single pooled variance was performed. *p.adj ≤ 0.05; **p.adj ≤ 0.01; ***p.adj ≤ 0.001; ****p.adj ≤ 0.0001. F. Summary graph depicting overall results from this study.

Article Snippet: Each concentration of drug was plated in triplicate with early-passage HMC3 cells (ATCC; Cat #: CRL-3304), and cell viability was read out using using MTT assay, by incubating the cells in 0.25mg/ml MTT (invitrogen; Catalog #: M6494) in PBS (Corning, Cat #:21-040-CV) for 1hr at 37°C, following removal of MTT solution and addition of 200μl DMSO (Sigma-Aldrich, Cat #:472301) and further incubation of the cells for 15 min at 37°C before measuring the absorbance at 570 nm with a Tecan Infinite® 200 PRO plate reader (Tecan; Cat#: 30050303).

Techniques: Control, Labeling, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Virus and Cell Specific HMGB1 Secretion and Subepithelial Infiltrate Formation in Adenovirus Keratitis

doi: 10.1101/2025.01.07.631509

Figure Lengend Snippet: (A) Cytoplasmic and nuclear extracts prepared from uninfected or HAdV-D37-infected for 2, 12, 24, and 48 hpi. were resolved on 4-20% SDS-PAGE. Western blots for HMGB1 in the nuclear (Nuc), cytoplasmic (Cyt), and supernatant (Sup) extracts with TBP (nuclear) β-actin (cytoplasmic) as loading controls in cell types THE, PHCE, HCF, A549, and HEK293 (left to right). (B) Densitometric analysis of HMGB1 band intensity of cytoplasmic and nuclear extracts in THE, PHCE, HCF, A549, and HEK293 (left to right). p values were determined by t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ( n=3 ). (C) Quantification of extracellular HMGB1 measured from supernatants collected at indicated time points pi in THE and PHCE cells ( n=3 ). (D) Schematic representation (created using Biorender.com ) for HAdV-D37 induced translocation of HMGB1 from the cell nucleus to the cytoplasm and then into the extracellular space.

Article Snippet: A549 (CCL-185), a human lung carcinoma cell line, and HEK293 (CRL-1573), a human embryonic kidney cell line, were purchased from American Type Culture Collection (ATCC).

Techniques: Infection, SDS Page, Western Blot, Translocation Assay

Journal: bioRxiv

Article Title: Virus and Cell Specific HMGB1 Secretion and Subepithelial Infiltrate Formation in Adenovirus Keratitis

doi: 10.1101/2025.01.07.631509

Figure Lengend Snippet: (A) Western blot analysis for HMGB1 expression along with β-actin for load control from whole cell lysates of uninfected (M) or HAdV-D37-infected (V) THE cells for 2, 12, 24, and 48 hpi. qRT-PCR analysis of HMGB1 gene expression for mock and virus infected cells at the same times pi is shown as bar graphs below the Western blot. (B) Bar graph for qRT-PCR of the viral early gene E1A expression, a surrogate marker for viral entry, and normalized to human ACTG gene for quantification. (C) Viral late protein pIIIa expression in cytoplasmic and nuclear extracts prepared from uninfected or HAdV-D37-infected THE, PHCE, HCF, A549 and HEK293 for 2, 12, 24, and 48 hpi show successful infection of all cell types.

Article Snippet: A549 (CCL-185), a human lung carcinoma cell line, and HEK293 (CRL-1573), a human embryonic kidney cell line, were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Control, Infection, Quantitative RT-PCR, Gene Expression, Virus, Marker