Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Exosomes shed from late sepsis MDSCs inhibit LPS-induced secretion of S100A9 protein from early sepsis MDSCs. Gr1 + CD11b + cells were isolated from bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were cultured for 24 hr in serum-free media. Culture supernatants were harvested, and exosomes were purified using exoEasy Maxi kit. Early sepsis Gr1 + CD11b + cells were cultured in 12-well plates with exosomes (50 μg/well) for 24 hr with or without 0.1 μg/ml of E. coli lipopolysaccharide (serotype 0111:B4; Sigma-Aldrich, St. Louis, MO). Levels of S100A9 protein in the culture super natants were measured by ELISA. Data are expressed as means ± SD of 6-8 mice (6-8 cultures/group) from three experiments. * p /** p < 0.05, versus exosomes from sham or early sepsis.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Isolation, Selection, Magnetic Beads, Cell Culture, Purification, Enzyme-linked Immunosorbent Assay

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Late sepsis MDSC-derived exosomes switch naïve Gr1 + CD11b + cells into the immnosuppressive phenotype. Gr1 + CD11b + cells were isolated from the bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were cultured for 24 hr in serum-free media, and exosomes were purified from the culture supernatants using exoEasy Maxi kit. CD4 + T cells were isolated from splenocytes of naive mice with anti-CD4 antibody and labeled with the fluorescent dye CFSE. The CD4 + T cells were cultured with naive Gr1 + CD11b + cells (1:1 ratio) in the presence of exosomes (50 μg/well) for 3 days, and anti-CD3 plus anti-CD28 antibodies (1 μg/ml each) were added to the culture to activate T cells. The cells were harvested, and T cell proliferation was determined by the step- wise dilution of CFSE dye in dividing CD4 + T cells using flow cytometry. (A) Representative dot plots of CFSE positive T cells gated on CD4 are shown. (B) Summary data of flow cytometry. (C) The culture supernatants were used to determine IFNϒ levels by ELISA. Data are expressed as means ± SD of 6-9 mice (6-9 cultures/group) from three experiments. * p < 0.05.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Derivative Assay, Isolation, Selection, Magnetic Beads, Cell Culture, Purification, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Hotairm1 expression is increased in late sepsis MDSCs. Gr1 + CD11b + cells were isolated from the bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. (A) The cells were cultured for 24 hr in serum-free media. Exosomes were purified from the culture supernatants, and exosomal RNA was extracted with exoRNeasy Starter kit. Levels of Hotairm1 were determined by RT-qPCR using RT lncRNAqPCR Assay Primers (Qiagen). Values were normalized to 18S RNA. (B) Total RNA was isolated from Gr1 + CD11b + cells using TRIzol reagent, and levels of Hotaim1 were determined as in A. Values were normalized to GAPDH RNA. (C) Gr1 + CD11b + cells isolated from naïve mice were cultured for 24 hr without or with exosomes (50 μg/well), purified from late sepsis MDSC culture. The cells were harvested, total RNA was extracted and levels of Hotairm1 were determined as in B. PCR was performed in duplicate. Data are presented relative to sham or media control (1-fold). Data in A and B are expressed as means ± SD of 6-9 mice/group from three experiments. Data in C are expressed as means ± SD of 7 cultures from two experiments. * p < 0.05.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Expressing, Isolation, Selection, Magnetic Beads, Cell Culture, Purification, Quantitative RT-PCR, Control

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Knockdown of Hotairm1 in late sepsis MDSCs attenuates their immunosuppressive functions. Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were transfected with Hotairm1-specific or scramble siRNAs for 36 hr. (A) Effects of MDSCs on T cell proliferation and activation. CD4 + T cells were isolated from splenocytes of naive mice with anti-CD4 antibody and labeled with the fluorescent dye CFSE. The late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were then co-cultured (1:1 ratio). T cells were stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each). After 3 days, the cells were harvested, and T cell proliferation and IFNϒ production were determined as in . (B and C) Effect of exosomes lacking Hotairm1 on T cells. Late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were cultured for 24 hr in serum-free media. Culture supernatants were harvested, and exosomes were purified using exoEasy Maxi kit. (B) Levels of Hotairm1 in exosomal RNA was determined by RT-qPCR. Values were normalized to 18S RNA. (C) Spleen CD4 + T cells were labeled and cultured with naive Gr1 + CD11b + cells (as described in ) in the presence of Hotairm1-lacking exosomes. T cell proliferation and IFNϒ production were measured as in A. Data are expressed as means ± SD of 5-6 mice/group from three experiments. * p < 0.05.KD, knockdown.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Knockdown, Isolation, Selection, Magnetic Beads, Transfection, Activation Assay, Labeling, Cell Culture, Purification, Quantitative RT-PCR

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Hotairm1 binds to S100A9 in late sepsis MDSCs. Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice using anti-Gr1 antibody and magnetic beads. (A) The cells (pooled from 2 mice) were treated with formaldehyde for reversible cross-linking of RNA-protein complexes. Cell lysates were prepared and immunoprecipitated with S100A9 or IgG antibody, and S100A9 levels were determined by western blot. The cross-linked RNA was extracted from the immunoprecipitated complexes using TRIzol reagent, and Hotairm1 levels were determined by standard PCR. (B) Hotairm1 knockdown relocalizes S100A9 in the cytosol. Late sepsis Gr1 + CD11b + cells were transfected with Hotairm1-specific or scramble siRNAs for 36 hr. Cytoplasmic and nuclear proteins were extracted, and levels of S100A9 were determined by Western blot. (C) Ectopic expression of Hotairm1 in early sepsis Gr1 + CD11b + cells moves S100A9 to the nucleus. The cells were transfected with Hotairm1 plasmid or a control vector for 24 hr. Cell lysates were prepared and immunoprecipitated with S100A9 or IgG antibody, and S100A9 levels were determined by Western blot. RNA was extracted from S100A9 IP and Hotairm1 levels were determined by RT-qPCR. Values were normalized to IgG IP samples and presented relative to vector. (D) Protein extracts were prepared, and levels of S100A9 were determined by Western blot. The results are representative of three experiments.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Isolation, Magnetic Beads, Immunoprecipitation, Western Blot, Knockdown, Transfection, Expressing, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Overexpression of Hotairm1 in early sepsis Gr1 + CD11b + cells switches them to the immunosuppressive phenotype. Gr1 + CD11b + cells were isolated from the bone marrow of early septic mice using anti-Gr1 antibody and magnetic beads. The cells were transfected with Hotairm1 expression plasmid or an empty vector and cultured with 10 ng/ml of recombinant mouse IL-10. (A) After 24 hr, a portion of the cells was used for Hotairm1 expression measurement by RT-qPCR. The remainder of the cells were washed and stimulated with 1 μg/ml of of E. coli lipopolysaccharide (serotype 0111:B4; Sigma) for 24 hr. (B) The cells were harvested, lysed and analyzed for arginase activity. (C and D) Levels of IL-10 and TNFα in the culture supernatants were measured by ELISA. Data are expressed as means ± SD of 5-6 mice (5-6 cultures/group) from two experiments, * p < 0.05.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Over Expression, Isolation, Magnetic Beads, Transfection, Expressing, Plasmid Preparation, Cell Culture, Recombinant, Quantitative RT-PCR, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: High levels of Hotairm1 in plasma exosomes and MDSCs from late septic patients. (A) Exosomes were purified from plasma and RNA was extracted using exoRNeasy Starter kit. (B) Peripheral blood CD33 + CD11b+HLA-DR − cells were isolated by magnetic cell separation. PBMCs were first purified and depleted of the HLA-DR + cells. The HLA-DR − cell population was then subjected to positive selection with biotin-coupled anti-CD33 antibody, followed by anti-CD11b antibody. Total RNA was extracted using TRIzol reagent. Levels of Hotairm1 were determined by RT-qPCR as in . Values in A were normalized to 18S RNA, and values in B were normalized to GAPDH RNA. Data are expressed as means ± SD of 7-9 subjects/group and are presented relative to HC (1-fold), * p < 0.05, versus HC or early septic; ** p < 0.05, versus late septic. HC, helthy control. (C) S100A9 accumulates in cytosol in CD33+CD11b+HLA-DR − cells during late sepsis. Cytoplasmic and nuclear proteins were extracted from CD33 + CD11b + HLA-DR − cells, and levels of S100A9 were determined by Western blot. The results are representative of two Western blots. (D) Hotairm1 binds S100A9 protein during late sepsis. Cell lysates were prepared from CD33 + CD11b + HLA-DR − cells isolated from late septic patients (n=4) and immunoprecipitated with S100A9 or IgG antibody. S100A9 levels were determined by Western blot. RNA was extracted from S100A9 IP, and Hotairm1 levels were determined by RT-qPCR. Values were normalized to the input samples and are presented relative to IgG IP. The results are representative of two immunoprecipitations.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Clinical Proteomics, Purification, Isolation, Magnetic Cell Separation, Selection, Quantitative RT-PCR, Control, Western Blot, Immunoprecipitation