Journal: Cell Reports

Article Title: Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19

doi: 10.1016/j.celrep.2022.111020

Figure Lengend Snippet:

Article Snippet: CD64 , Acrobiosystems , Cat #FCA-H82E8-25ug.

Techniques: Recombinant, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: TLR7-agonist and antineoplastic MEK1/2-inhibitor combination unlocks interferon responses from macrophages

doi: 10.1101/615609

Figure Lengend Snippet: ( a , b ) mRNA expression of three M1 phenotype genes ( Nos2 , Il-12b and Tnf-α ) ( a ) and two M2 phenotype genes ( Mrc1 and Arg1 ) ( b ). BMDM were activated via TLR7 agonist R848 for six hours in the presence or absence of three MEK1/2 inhibitors. ( c , d , e ) Surface expression of M1 phenotype marker MHC-II ( c ), M2 phenotype markers CD80 and CD163 ( d ) and phagocytosis marker CD64 ( e ) within the indicated cell populations. BMDM were stimulated with R848 for twenty-four hours in the presence or absence of MEKi-U. Cells were then harvested and assayed for flow cytometry analysis. ( f, g ), mRNA expression of four M1 phenotype genes ( Il-12b, Il-6, Tnf-α and Nos2 ) in BMDM generated from WT and Stat1 -/- mice ( f ) or from WT and Irf1 -/- mice ( g ). Cells were stimulated with R848 for six hours in the presence or absence of MEKi-U. ( h ) Percentages of MHC II + CD163 - M1 and MHC II - CD163 + M2 phenotype markers shown by representative flow cytometry analysis (left) and corresponding quantifications (right) in BMDM generated from WT and Irf1 -/- mice. Cells were similarly treated and processed as that in ( c-e ). *0.01< P <0.05, **0.001< P <0.01, *** P <0.001 (one-way ANOVA with Bonferroni’s correction or two-way ANOVA with Tukey’s correction). Data are presented as mean ± S.D. or one representative of four ( a - g ) or two ( h - k , n=4 per group) independent experiments. See also .

Article Snippet: Cells were stained with fixable viability dye eFluorTM 506 (ebioscience, Thermo Fisher Scientific), blocked with 2.5 μg/ml anti-CD16/32 (clone 93; ebioscience, Thermo Fisher Scientific) and then stained for flow cytometry with the following antibodies: CD45-eF450 (104), CD11b-FITC (M1/70), MHC II-PE-Cy7 (M5/114.15.2), F4/80-PE-Cy5 (BM8), F4/80-biotin (BM8), Ki67-PerCP-eF710 (SolA15), CD3ε-biotin (145-2C11), CD4-APC-Cy7 (GK1.5), CD4-PE (GK1.5), CD8α-PerCP-eF710 (53-6.7), CD19-biotin (1D3), NK1.1-APC (PK136), CD80/B7.1-PE (16-10A1), granzyme B-PerCP-eF710 (NGZB) and Foxp3-PE-Cy7 (fjk-16s) from ebioscience, Thermo Fisher Scientific; Ly6G-BV605 (1A8), streptavidin-BV605, CD8α-PE (53-6.7), CD68-PE (FA-11), CD206-PE (MR6F3) and CD163-PE (TNKUPJ) from Biolegend and CD64-APC (REA286) from Miltenyi Biotec.

Techniques: Expressing, Marker, Flow Cytometry, Generated

Journal: PLoS Pathogens

Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing

doi: 10.1371/journal.ppat.1006582

Figure Lengend Snippet: (A) Static biofilm-forming ability of isogenic RD2 (T4ΩRD2) and RD12 (T4ΩRD12) deficient mutants relative to TIGR4 (T4) was assessed in a 48-hour 6-well polystyrene plate model (n = 7 experiments). Biofilm biomass was measured using crystal violet staining. Statistical analysis was performed using Student’s t -test. Shown is a representative image of crystal violet stained biofilms. (B) Representative Z-stacked images of TIGR4, T4ΩRD2, and T4ΩRD12 biofilms grown on coverslips for 24 hours. Biofilms were examined for viability using Live/Dead staining. Whole (live and dead) bacterial biomass stain green whereas non-viable bacteria stain red (n = 3 experiments). (C) Pneumococcal titers in the blood of mice (n = 5 per group) infected with TIGR4 (T4), T4ΩRD2 (ΩRD2), or T4ΩRD12 (ΩRD12) 30 hours post-infection. Mann-Whitney test comparing the mutant strain titers to the wildtype TIGR4 titers was performed. (D) Representative immunofluorescent stained images of cardiac sections from mice infected with TIGR4 (T4), T4ΩRD2 (ΩRD2), or T4ΩRD12 (ΩRD12) 30 hours post infection (n = 5 mice per group). Cardiac sections were stained using serotype 4 capsule polysaccharide antisera ( green ) and DAPI ( blue ). (E) Absolute numbers of infiltrated neutrophils in hearts of mice (n = 5 per group) infected with TIGR4, and T4ΩRD12 (ΩRD12) 30-hours post-infection. Neutrophils were identified as Gr-1 + CD11b + Ly-6G + F4/80 - cells. Statistical analysis was performed using student’s t- test. (F) In vitro antimicrobial tolerance assays for survival of paired blood-isolated pneumococci (BIP) and heart-isolated pneumococci (HIP) from T4ΩRD12 infected mice (n = 5). Tolerance to penicillin and erythromycin killing at the designated concentration was tested. Statistical analysis was performed using Mann-Whitney test. No statistically significant differences were observed. (G) LDH release cytotoxicity assay of J774A.1 macrophages challenged with equal biomass of TIGR4 (T4) and T4ΩRD12 (ΩRD12) as determined at 0, 1, 2, 4 hours post-infection (n = 3 biological replicates, each with 3 technical replicates). Statistical analysis for comparisons of cytotoxicity at each time point was performed using Mann-Whitney test. (H) TNFα and CXCL2 production by J774A.1 macrophages and HL-1 cardiomyocytes following 4-hour exposure to an equal biomass of planktonic TIGR4 (T4), biofilm TIGR4 (T4), or planktonic T4ΩRD12 ( ΩRD12) (n = 3 biological replicates, each with 3 technical replicates). Statistical analysis was performed using non-parametric One-way ANOVA (Kruskal-Wallis Test). P value: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001; data are represented as mean ± SEM.

Article Snippet: These cell suspensions were then neutralized with IDMEM containing 10% FBS and filtered through 0.45μm strainer before blocking with 2.4G2 (BD Pharmingen, Cat #553141) for 30 minutes on ice, and staining with Gr-1-APC (Clone RB6-8C5, eBioscience), CD11b-APC-Cy7 (M1/70, BD Pharmingen), Ly 6C-PerCP-Cy5.5 (HK1.4, eBioscience), Ly6G-FITC (1A8, eBioscience), MHC-II PE-Cy5 (M5/114.15.2, eBioscience), MerTK-PE (DS5MMER, eBIoscience), F4/80-PE-Cy5 (BM8, eBioscience) and CD64-Biotin (X54-5/7.1, Biolegend and used in conjunction with Streptavidin PE-Cy7, eBioscience) antibodies for 30 minutes on ice protected from light.

Techniques: Staining, Infection, MANN-WHITNEY, Mutagenesis, In Vitro, Isolation, Concentration Assay, Cytotoxicity Assay

Journal: PLoS Pathogens

Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing

doi: 10.1371/journal.ppat.1006582

Figure Lengend Snippet: (A) Western blots for pneumolysin levels in equal biomass of whole cell lysates (pellets) and supernatants of planktonic- wildtype TIGR4 (n = 3), biofilm- wildtype TIGR4 (n = 3), and planktonic T4ΩRD12 (ΩRD12) (n = 2). An isogenic pneumolysin deficient TIGR4 strain (T4 Δ ply ) was tested as the negative control. Normalized densitometric quantification of pneumolysin levels in the supernatant is provided. Statistical analysis was performed by comparison supernatant pneumolysin levels from planktonic (PK)- wildtype TIGR4 (n = 3), and planktonic T4ΩRD12 (ΩRD12) (n = 2) to biofilm (BF)- wildtype TIGR4 (n = 3) using Welch’s t -test. (B) LDH release cytotoxicity assay of J774A.1 macrophages challenged with equal biomass of planktonic-, biofilm- TIGR4 (T4), planktonic-, biofilm- T4 Δ ply and planktonic-, biofilm- T4 Δ ply complemented with exogenous recombinant pneumolysin (rPLY, 0.3μg/mL) as determined at 0, 1, 2, 4 hours post-infection (n = 3 biological replicates, each with 3 technical replicates). Statistical analysis was performed using ordinary one-way ANOVA. (C) TNFα production by J774A.1 macrophages at designated time points following exposure to an equal biomass of planktonic-, biofilm- TIGR4 (T4), planktonic-, biofilm- T4 Δ ply and planktonic-, biofilm- T4 Δ ply complemented with exogenous recombinant pneumolysin (rPLY, 0.3μg/mL) as determined at 0, 1, 2, 4 hours post-infection (n = 3 biological replicates, each with 3 technical replicates). Statistical analysis was performed using ordinary one-way ANOVA. (D) Representative transmission electron microscopy (TEM) image of cardiac sections (magnification: 2,500X) from BALB/cJ mice infected with T4 Δ ply 30 hours post-infection (n = 3). (E) Representative high magnification immunofluorescent microscopy images of cardiac microlesions from uninfected-, passively immunized (αPly)- and naïve- mice infected with HIP or BIP 30 hours post infection, showing presence of: capsule (stained with anti-serotype 4 capsule antibody [CPS], red ), cardiac macrophages (stained using anti-Mac-3 antibody [Mac-3], green ), and infiltrated neutrophils (stained with anti-Ly-6G antibody [Ly-6G], green ). A minimum of 4 stained heart sections were examined. (F) Absolute numbers of infiltrated neutrophils in hearts of uninfected-, passively immunized (αPly)- and naïve- mice infected with HIP or BIP 30 hours post infection. Neutrophils were identified as Gr-1 + CD11b + Ly-6G + F4/80 - MHC-II - cells. Statistical analysis was performed using student’s t- test. (G) Absolute numbers of cardiac macrophages in hearts of uninfected-, passively immunized (αPly)- and naïve- mice infected with HIP or BIP 30 hours post infection. Macrophages were identified as CD64 + MerTK + F4/80 + CD11b + cells. Statistical analysis was performed using student’s t- test. P value: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001; data are represented as mean ± SEM.

Article Snippet: These cell suspensions were then neutralized with IDMEM containing 10% FBS and filtered through 0.45μm strainer before blocking with 2.4G2 (BD Pharmingen, Cat #553141) for 30 minutes on ice, and staining with Gr-1-APC (Clone RB6-8C5, eBioscience), CD11b-APC-Cy7 (M1/70, BD Pharmingen), Ly 6C-PerCP-Cy5.5 (HK1.4, eBioscience), Ly6G-FITC (1A8, eBioscience), MHC-II PE-Cy5 (M5/114.15.2, eBioscience), MerTK-PE (DS5MMER, eBIoscience), F4/80-PE-Cy5 (BM8, eBioscience) and CD64-Biotin (X54-5/7.1, Biolegend and used in conjunction with Streptavidin PE-Cy7, eBioscience) antibodies for 30 minutes on ice protected from light.

Techniques: Western Blot, Negative Control, Cytotoxicity Assay, Recombinant, Infection, Transmission Assay, Electron Microscopy, Microscopy, Staining