Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Frequency of classical (CD14 + CD16 neg ) and intermediate (CD14 + CD16 + ) peripheral CD14 + monocyte subsets with and without OA synovial fluid (SF). (B) Percent intermediate (CD14 + CD16 + ) monocytes in CD45 + cells with indicated treatments. (C, D) Mean fluorescence intensity (MFI) of CD163, CD206, HLA-DR, and CD86 surface markers, and HLA-DR/CD163 MFI ratio on peripheral CD14⁺ monocytes from late-OA patients and healthy donors, with or without late-stage OA SF, following lipopolysaccharides (LPS, 2.5 ng/mL) stimulation. (E-I) MFI expression of CD163, CD206, HLA-DR, and CD86 cell surface markers and HLA-DR/CD163 MFI expression ratio on healthy peripheral CD14 + monocytes at the indicated conditions. (J) Tumor necrosis factor (TNF) soluble factor production by peripheral CD14 + monocytes in indicated treatments, with LPS (2.5 ng/mL) stimulation. Peripheral CD14 + monocytes were cultured for 72h with indicated treatments in (A-I) for flow cytometry assay and 48 hours in (J) for TNF assay; IL-6 recombinant (5 ng/mL); IL-6 neutralizing antibody (Ab; 2.5 µg/mL); 30% pooled mid- or late-OA SF. Values are shown as mean ± SD; N=3 biological replicates; n ≥ 3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Fluorescence, Expressing, Cell Culture, Flow Cytometry, Recombinant

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Schematic representation of dual IL-6 signaling in cells such as monocytes/macrophages (MΦs) expressing IL-6 receptor (IL-6R): i) trans-signaling through glycoprotein (gp)130 and soluble IL-6R (sIL-6R shed by a disintegrin and metalloproteinase 17 (ADAM 17)); ii) classical signaling through membrane-bound (m) IL-6R and gp130. (B) Median and interquartile range of sIL-6R concentration in synovial fluid (SF) samples from not-OA (N=7); mid-OA (N=24); and late-OA (N=43). (C-F) Mean fluorescence intensity (MFI) expression of CD163, CD206, HLA-DR and CD86 cell surface markers in the presence of late-OA SF and physiological levels of IL-6 (0.5 ng/mL) or/and sIL-6R (10 ng/mL), soluble(s) gp130 (10 ng/mL). (G) mIL-6R expression on CD14 + monocytes at the indicated treatments. (H) IL-6: sIL-6R complex levels in not-OA SF (N=7); mid-OA SF (N=24); late-OA SF (N=72), psoriatic arthritis (PsA) SF (N=2) and rheumatoid arthritis (RA) SF (N=1). Peripheral CD14 + monocytes were cultured for 72h with 30% pooled mid- or late-OA SFs, IL-6 neutralizing antibody (Ab; 2.5 µg/mL), and indicated treatments. MFI expression of monocyte cell surface markers is shown as mean ± SD; N=3 biological replicates; n=3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Expressing, Membrane, Concentration Assay, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) An experimental timeline for CD14⁺ monocytes indicating treatment conditions at each time point (arrows) for both short- and long-term exposure periods. (B) Representative western blot images from CD14 + monocytes at short treatment exposures. (C) Representative western blot images from CD14 + monocytes at long treatment exposures. (D-F) Bar graphs illustrating phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), phosphorylated-c-Jun terminal kinase (p-JNK), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) levels (normalized to total glyceraldehyde-3-phosphate dehydrogenase; GAPDH) in short and long treatment exposures. (G) Suppressor of cytokine signaling 3 ( SOCS3 ) expression in CD14 + monocytes with indicated treatments. (H) Chemokine (C-C motif) ligand 2 ( CCL2 ) expression in CD14 + monocytes with indicated treatments. (I) Tumor necrosis factor ( TNF ) expression by CD14 + monocytes in indicated treatments and lipopolysaccharides (2.5 ng/mL) stimulation. Peripheral CD14 + monocytes were cultured for 48h (G-I); IL-6 recombinant (5 ng/mL); S100A8/9 recombinant (1 µg/mL); IL-6 neutralizing antibody (Ab; 2.5 µg/mL); 30% pooled late-OA synovial fluid (SF); TAK242 (1 µM/mL). Values are shown as mean ± SD; N=3 biological replicates; n≤3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Experimental timeline and schematic workflow for CD14 + monocytes directly co-cultured with MSC(M), 30% pooled mid- or late-OA synovial fluid (SF) or IL-6 neutralizing antibody (Ab; 2.5 µg/mL) for 72h. (B-F) Mean fluorescence intensity (MFI) expression of CD163, CD206, HLA-DR, CD86 cell surface markers and the ratio of HLA-DR/CD163 MFI expression on CD14 + monocytes directly co-cultured with MSC(M) and indicated treatments. Values are shown as mean ± SD; N=3 biological replicates; n≤3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p<0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Cell Culture, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Experimental timeline and schematic workflow for CD14 + monocytes treated with soluble factors from licensed MSC(M) CM in OA environment for the indicated times. (B) Representative western blot images. (C-E) Bar graphs illustrating phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), phosphorylated-c-Jun terminal kinase (p-JNK), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) levels (normalized to total glyceraldehyde-3-phosphate dehydrogenase; GAPDH) after 24h of treatments. (F) Expression of the suppressor of cytokine signaling 3 ( SOCS3) gene in treated CD14 + monocytes. (G and H) JNK downstream chemokine (C-C motif) ligand 2 ( CCL2) and tumor necrosis factor ( TNF) genes expression in treated CD14 + monocytes. (I) TNF soluble factor production by peripheral CD14 + monocytes in indicated treatments and lipopolysaccharides (2.5 ng/mL) stimulation. (J) Phagocytosis of pHrodo Deep Red E. coli BioParticles by macrophages in indicated treatments for 72h. Peripheral CD14 + monocytes were treated with CMs of directly co-cultured monocytes with MSC(M), and mid- or late OA SF ± IL-6 neutralizing antibody (Ab, 2.5 µg/mL); 30% pooled mid- or late synovial fluid (SF). Gene expression in (F-H) and TNF soluble in (I) were assessed after 48h. Values are shown as mean ± SD; N=3 biological replicates; n ≥ 3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Different letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Cell Culture, Gene Expression

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: The percentage of the neutrophil extracellular trap (NET) area in the SS and FS plots of flow cytometry was compared among untreated neutrophils and samples following NET induction with E. coli DH5α or PMA. (a) Expression intensities of Cit-H3, flavocytochrome b558, SYTOX™ Green, CD14, TLR2, TLR4, and LL-37 were compared between untreated neutrophil and NET areas after E. coli DH5α stimulation. (b) NET ratios were compared among untreated neutrophils and samples treated with E. coli DH5α or PMA using enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. ** p < 0.01. (c) Western blotting was performed to examine CD14 and Cit-H3 protein levels in untreated neutrophils and those stimulated with E. coli DH5α or PMA. Band intensities were quantified using ImageJ and normalized to β-actin. Data are presented as mean ± standard deviation (SD) (n = 3). Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s HSD test. ** p < 0.01

Article Snippet: Details of the reagents used in this study and their sources are as follows: Polymorphprep ™ (Cat. No.1114683; Axis-Shield, Dundee, Scotland), RPMI-1640 Medium (Cat. No. R8758; Sigma-Aldrich, MO, USA), phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, MO, USA), Escherichia coli DH5α competent cells ( E. coli DH5α; Cat. No. 9057; Takara Bio, Shiga, Japan), EasySep ™ Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada), CellXVivo TM Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep Permeabilizaton Reagent (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), cytochalasin D (Cat. No. 11330; Cayman Chemical, Ann Arbor, MI, USA), wortmannin (Cat. No. AG-CN2-0023-M001; Adipogen Life Sciences, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF; Cat. No. 195381; MP Biomedicals, Irvine, CA, USA), elafin (Cat. No. 4243-v; Peptide Institute, Osaka, Japan), May–Grünwald’s stain solution (Cat. No. 15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No. 15002; Mutokagaku, Tokyo, Japan), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No. 15612; Mutokagaku, Tokyo, Japan), 4% paraformaldehyde phosphate buffer solution (Cat. No. 09154-85; Nacalai Tesque, Kyoto, Japan), Triton ™ X-100 (Cat. No. 194854; MP Biomedicais, Irvine, CA, USA), sample buffer solution with 2-mercaptoethanol (Cat. No. 30566-22; Nacalai Tesque, Kyoto, Japan), skim milk powder (Cat. No. 190-12865; Fujifilm Wako Pure Chemical, Tokyo, Japan), Chemi-Luna One Super (Cat. No. 02230; Nacalai Tesque, Kyoto, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Tokyo, Japan), SYTOX ™ Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechest ® 33342 (Cat. No. 346-07951; Fujifilm, Tokyo, Japan), Recombinant human CD14 protein (rCD14; Cat. No. 383-CD; R&D systems, Minneapolis, MN, USA), and recombinant human PR3 protein (Cat. No. 13699-H08H1; Sino Biological, Kanagawa, Japan).

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: (a) Presepsin (P-SEP) levels in the supernatant were measured after addition of PR3 to rCD14. Conditions with PR3 inhibition using the inhibitors phenylmethylsulfonyl fluoride or elafin were also compared. (b) Western blot analysis of CD14 and P-SEP proteins, with band intensities quantified using ImageJ. Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey–Kramer’s HSD test. ** p < 0.01.

Article Snippet: Details of the reagents used in this study and their sources are as follows: Polymorphprep ™ (Cat. No.1114683; Axis-Shield, Dundee, Scotland), RPMI-1640 Medium (Cat. No. R8758; Sigma-Aldrich, MO, USA), phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, MO, USA), Escherichia coli DH5α competent cells ( E. coli DH5α; Cat. No. 9057; Takara Bio, Shiga, Japan), EasySep ™ Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada), CellXVivo TM Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep Permeabilizaton Reagent (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), cytochalasin D (Cat. No. 11330; Cayman Chemical, Ann Arbor, MI, USA), wortmannin (Cat. No. AG-CN2-0023-M001; Adipogen Life Sciences, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF; Cat. No. 195381; MP Biomedicals, Irvine, CA, USA), elafin (Cat. No. 4243-v; Peptide Institute, Osaka, Japan), May–Grünwald’s stain solution (Cat. No. 15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No. 15002; Mutokagaku, Tokyo, Japan), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No. 15612; Mutokagaku, Tokyo, Japan), 4% paraformaldehyde phosphate buffer solution (Cat. No. 09154-85; Nacalai Tesque, Kyoto, Japan), Triton ™ X-100 (Cat. No. 194854; MP Biomedicais, Irvine, CA, USA), sample buffer solution with 2-mercaptoethanol (Cat. No. 30566-22; Nacalai Tesque, Kyoto, Japan), skim milk powder (Cat. No. 190-12865; Fujifilm Wako Pure Chemical, Tokyo, Japan), Chemi-Luna One Super (Cat. No. 02230; Nacalai Tesque, Kyoto, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Tokyo, Japan), SYTOX ™ Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechest ® 33342 (Cat. No. 346-07951; Fujifilm, Tokyo, Japan), Recombinant human CD14 protein (rCD14; Cat. No. 383-CD; R&D systems, Minneapolis, MN, USA), and recombinant human PR3 protein (Cat. No. 13699-H08H1; Sino Biological, Kanagawa, Japan).

Techniques: Inhibition, Western Blot

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: Upon bacterial infection of blood vessels, neutrophils release neutrophil extracellular traps (NETs) as a host defense mechanism. M1 MΦs phagocytose CD14-rich NETs, and the engulfed CD14 is degraded by the proteolytic enzyme PR3 within the macrophages, resulting in the generation of presepsin (P-SEP). Subsequently, P-SEP is released extracellularly from M1 MΦs, leading to elevated blood levels of P-SEP.

Article Snippet: Details of the reagents used in this study and their sources are as follows: Polymorphprep ™ (Cat. No.1114683; Axis-Shield, Dundee, Scotland), RPMI-1640 Medium (Cat. No. R8758; Sigma-Aldrich, MO, USA), phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, MO, USA), Escherichia coli DH5α competent cells ( E. coli DH5α; Cat. No. 9057; Takara Bio, Shiga, Japan), EasySep ™ Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada), CellXVivo TM Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep Permeabilizaton Reagent (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), cytochalasin D (Cat. No. 11330; Cayman Chemical, Ann Arbor, MI, USA), wortmannin (Cat. No. AG-CN2-0023-M001; Adipogen Life Sciences, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF; Cat. No. 195381; MP Biomedicals, Irvine, CA, USA), elafin (Cat. No. 4243-v; Peptide Institute, Osaka, Japan), May–Grünwald’s stain solution (Cat. No. 15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No. 15002; Mutokagaku, Tokyo, Japan), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No. 15612; Mutokagaku, Tokyo, Japan), 4% paraformaldehyde phosphate buffer solution (Cat. No. 09154-85; Nacalai Tesque, Kyoto, Japan), Triton ™ X-100 (Cat. No. 194854; MP Biomedicais, Irvine, CA, USA), sample buffer solution with 2-mercaptoethanol (Cat. No. 30566-22; Nacalai Tesque, Kyoto, Japan), skim milk powder (Cat. No. 190-12865; Fujifilm Wako Pure Chemical, Tokyo, Japan), Chemi-Luna One Super (Cat. No. 02230; Nacalai Tesque, Kyoto, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Tokyo, Japan), SYTOX ™ Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechest ® 33342 (Cat. No. 346-07951; Fujifilm, Tokyo, Japan), Recombinant human CD14 protein (rCD14; Cat. No. 383-CD; R&D systems, Minneapolis, MN, USA), and recombinant human PR3 protein (Cat. No. 13699-H08H1; Sino Biological, Kanagawa, Japan).

Techniques: Infection

Journal: The Journal of Headache and Pain

Article Title: Expression of miR-155 in monocytes of people with migraine: association with phenotype, disease severity and inflammatory profile

doi: 10.1186/s10194-024-01842-y

Figure Lengend Snippet: Percentage of events recorded for the different monocytic sub-populations among the three study groups. Legend: Panel A : percentage of M1 monocytes (CD80 +) sub-populations. Panel B : percentage of M2 monocytes (CD163 +) sub-populations. CM-MO: chronic migraine with medication overuse, EM: episodic migraine, HCs: healthy controls, classical: “classical” monocytes (CD14 + /CD16– expression), non-classical: “non classical-intermediate” monocytes (CD14 + /CD16 + expression), M1: pro-inflammatory “M1” monocytes (CD80 + expression), M2 : anti-inflammatory “M2” monocytes (CD163 + expression). Box-plot: the range between the upper and lower border of the box indicates the interquartile range (IQR), spanning from the 25 th to the 75 th percentile. Within the box, the line indicates the median, and the cross denotes the mean. The upper and lower whiskers extend to the maximum and minimum values, excluding outliers. Symbols positioned above the upper whisker represent outliers, defined statistically as values beyond the 75 th percentile plus 1.5 times the IQR. Kruskal–Wallis Test was used for intergroup comparisons

Article Snippet: Subsequently, they were incubated for 30 min at 4 °C in the dark with all of the following monoclonal antibodies: Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti-human CD14 (BD Biosciences, 20µL per 1 × 10 6 cells), R-phyco-erythrin-cyanine7 (PE-Cy7)-conjugated anti-human CD16 (BD Biosciences, 5µL per 1 × 106 cells), and R-phycoerythrin (PE)-conjugated CD163 (BD Biosciences, 5µL per 1 × 106 cells) or R-phycoerythrin (PE)-conjugated CD80 (BD Biosciences, 5µL per 1 × 106 cells).

Techniques: Expressing, Whisker Assay

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: Screening for CD14 incorporation by lab-adapted HIV-1 isolates. ( A ) Schematic describing the virion capture assay used throughout. Briefly, antibodies or LPS (not depicted) were adsorbed onto 96-well plates and blocked before adding virus samples to allow precipitation of virus particles bearing the antigen of interest. Unbound virions were washed away and captured virions were lysed for quantitation of HIV-1 by Gag p24 ELISA. ( B ) Virion capture assay was performed on four HIV-1 isolates (IIIB, NL4-3, BaL, and SF162) produced by infected PBMCs using anti-CD14 (orange) and anti-gp120 (blue). Bars are isotype control-subtracted mean ± SD Gag p24 of duplicate assays and are representative of two independent experiments. ( C ) Western blot corroborating the incorporation of CD14. HIV-1 grown in monocyte-derived macrophages (MDMs) (BaL and SF162, two donors each) and PBMCs (IIIB, two donors; NL4-3, four donors) were concentrated, lysed, and resolved by SDS-PAGE before blotting for p24 (loading control), gp120 (positive control for viral surface protein), and CD14. Data are representative of three independent blots.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Virus, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Produced, Infection, Control, Western Blot, Derivative Assay, SDS Page, Positive Control

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: HIV-1 pseudovirus reliably models virion incorporation of CD14. ( A ) Schematic depicting the process of transfecting 293T cells with plasmids encoding the HIV-1 backbone (pSG3ΔEnv) and CD14 to produce PV bearing CD14 on the surface. ( B ) Confirming the transfection efficiency of 293T cells ectopically expressing CD14. Cells were transfected with backbone only (black histogram) or backbone plus CD14 (orange histogram) to produce WT or CD14 + PVs, respectively. Cells were collected and stained with PE-conjugated anti-CD14 to confirm cell-surface expression of CD14 available for incorporation. ( C ) Gating strategy to identify PV populations by FVM. Phosphate-buffered saline (PBS) or WT virions were acquired, and data are displayed on pseudocolored dot plots. Fluorescence data (y-axis) were calibrated to produce quantitative units of MESF (molecules of equivalent soluble fluorophore), which describe the number of fluorophores and, indirectly, the number of antigens detected. Side scatter data (x-axis) were calibrated to produce arbitrary units of scattering cross section (nm 2 ), which relates to the size and refractive index of particles. Gates to the far left (PBS and WT PV) represent the instrument’s optical noise and are present of every FVM plot. Virions are identified as the dense, monodispersed population (right gate) adjacent to the optical noise. ( D ) WT (top row) and CD14 + (bottom row) virions were stained with PE-conjugated isotype control (left column) or anti-CD14 (right column) (0.4 µg/mL) and analyzed by FVM. Gates were positioned above the unstained PV populations, identifying PE + events, and were set respective to each isotype control stain. PE MESF values are enumerated where PE + events are present (mean ± SD, n = 3 independent stains), describing the number of fluorophores detected in each PE + gate. Virus stocks used in C and D had a titer of 187 ng/mL (WT) and 114 ng/mL (CD14 + ) of p24 as measured by ELISA. ( E ) Virion capture assay was performed on WT (teal) and CD14 + (orange) PVs to corroborate CD14 incorporation. “Nil” represents PBS buffer, “mIgG2a” is an isotype-matched control, and “αCD14” is anti-CD14 mAb M5E2. Bars are mean ± SD p24 of four independently produced virus stocks and are representative of at least two independent assays.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Transfection, Expressing, Staining, Saline, Fluorescence, Refractive Index, Control, Virus, Enzyme-linked Immunosorbent Assay, Produced

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: CD14 remains functional in the viral envelope and binds to LPS. ( A ) FVM was used to read out a checkboard titration of LPS-bio and PE-SA on CD14 + virions to identify optimal staining concentrations. LPS-bio was incubated overnight at 4°C with virus samples and PE-SA was added for 4 h at 4°C the next day before acquisition. Gates to the left identify optical noise used for ( C ), and gates to the right identify PE + events that were set respective to each negative control PE-SA stain (top row). CD14 + virus stock shown in FVM plots had a titer of 57 ng/mL p24 as measured by ELISA. ( B ) The number of particles in the PE + gates of ( A ) were tabulated and heat mapped with blue, yellow, and red identifying low, intermediate, and high values, respectively. Higher particle counts identify LPS-bio and PE-SA stains that support CD14 + virions binding. ( C ) SI was calculated and plotted for each PE-SA concentration using the PE MESF measured in the PE + gates of ( A ). Peak SI identifies optimal staining pairs (orange stars and blue inverted triangles). ( D ) Incubation times for optimal PE-SA pairs from ( C ) were evaluated over a 16-h time course. Particle counts in PE + gates (right gates) are enumerated on each plot. CD14 + virus stock shown in FVM plots had a titer of 73 ng/mL p24 as measured by ELISA. ( E ) SI of PE MESF measured in ( D ) were plotted over time. All titration data were reproducible across two independent experiments using two independently produced virus stocks. All LPS-bio and PE-SA concentrations in A–E are in units of ng/mL and µg/mL, respectively.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Functional Assay, Titration, Staining, Incubation, Virus, Negative Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Produced

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: LPS binds to HIV-1 in a CD14-specific manner and does not passively associate with the lipid envelope. ( A ) Optimized LPS-bio (300 ng/mL, overnight, 4°C) and PE-SA (0.2 µg/mL, 4 h, 4°C) staining on CD14 + virions. Particle counts in PE + gates are enumerated on each plot. ( B ) Virion capture assay using immobilized untagged LPS. Ninety-six-well plates were coated with buffer (PBS) or LPS and virions with (CD14 + , orange bars) or without CD14 (WT, teal bars) were added. Captured virions were quantified by Gag p24 ELISA. Bars are mean ± SD of duplicate capture assays and are representative of three independent experiments. ( C ) FVM evaluating the contribution of recombinant LPS-binding protein (LBP) on LPS-bio binding to CD14 + virions. Varying amounts of recombinant LBP (rLBP) were mixed with LPS-bio prior to incubating with WT (top row) and CD14 + (bottom row) virus samples and staining with PE-SA. Gates define PE + events based on negative control (PE-SA only). WT and CD14 + virus stocks shown in FVM plots for ( A ) and ( C ) had a respective titer of 76 and 57 ng/mL p24 as measured by ELISA. ( D ) Particle counts in PE + gates from ( C ) were plotted as a function of PE-SA concentrations for WT (teal triangles) and CD14 + virions (orange circles). Data are mean ± SD of two independent virus stocks. ( E ) FVM measuring neutralization of LPS-bio binding to virions. Anti-CD14 (M5E2) was added to WT (top row) and CD14 + (bottom row) virions before incubation with LPS-bio and PE-SA. Gates are as described for ( C ). WT and CD14 + virus stocks shown in FVM plots had a respective titer of 368 and 141 ng/mL p24 as measured by ELISA. ( F ) Particle counts in PE + gates from ( E ) were plotted over the range of anti-CD14 concentrations tested for WT (teal triangles) and CD14 + (orange circles) virions. Data are representative of two independent virus stocks.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Virus, Negative Control, Neutralization, Incubation

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: CD14 + HIV-1 can trigger TLR4 signaling and NF-κB activation by delivering LPS to human monocytes. ( A ) Schematic describing how virions were “loaded” with LPS before overlaying onto TLR4-expressing cells. ( B ) NF-κB was proxied by measuring SEAP activity from TLR4-expressing (Tlr4 +/+ ) THP1-Dual cells induced after 24-h stimulation with samples. Bars are mean ± SD across four to five independent experiments, and where indicated, at least four different virus stocks were tested each time. Statistical significance was evaluated with one-way analysis of variance (ANOVA) (α = 0.05) and Tukey’s correction for multiple comparisons. From left to right: n = 21, n = 11, n = 12, n = 21, n = 16, n = 22, and n = 22. ns, non-significant; **** P < 0.0001. ( C ) Experiment as in ( B ) but done on TLR4-KO (Tlr4 -/- ) THP1-Dual cells. Data are mean ± SD of two independent experiments using four different virus stocks. Statistical significance was evaluated as in ( B ). From left to right: n = 7, n = 6, n = 7, n = 4, n = 4, n = 6, and n = 8. ns, non-significant; ** P = 0.0014; **** P < 0.0001. ( D ) Secreted TNF-α was measured by ELISA from stimulated (24 h) THP-1 cells. Data are mean ± SD across two independent experiments with four different virus stocks. Statistical significance was evaluated with one-way ANOVA (α = 0.05) and Tukey’s correction for multiple comparisons. From left to right: n = 8, n = 8, n = 8, n = 4, n = 4, n = 8, and n = 8. ns, non-significant; ** P = 0.0052; **** P < 0.0001.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Activation Assay, Expressing, Activity Assay, Virus, Enzyme-linked Immunosorbent Assay