Fig 2: 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.

Fig 3: 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.

Fig 4: 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.

Fig 5: 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 TNFa 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.