Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: Experimental layouts to determine the role of Nϕ in the A radioprotective and E radiomitigative effects of entolimod in BALB/c mice. Survival by Kaplan–Meier curves in mice treated with entolimod in the presence (rat IgG) or absence (α-Ly-6G) of Nϕ in the B radioprotective and F radiomitigative schemes. P -values were determined by Log-rank test. Measurement of total HPPs and granulocyte/macrophage (G/M) progenitors in BM by MethoCult in the C radioprotective ( n = 4–5 mice /group) and G radiomitigative ( n = 3–10 mice/group) schemes. Total HPPs were measured +1 h post-TBI for radioprotection and for radiomitigation, both total HPPs and G/M progenitors +3d post-treatment with vehicle or entolimod. D Absolute number of stem cell populations in BM by flow cytometry in the presence (rat IgG) and absence (α-Ly-6G) of Nϕ on day 8 post-TBI in the radioprotection scheme ( n = 3–5 mice/group). Stem cell populations were defined as follows: HSC (Lineage − Flt3 − c-kit + Sca-1 + ), MMP (Lineage − Flt3 + c-kit + Sca-1 + ), CLP (Lineage − Flt3 − IL-7R + c-kit + Sca-1 + ), Myeloid (Lineage − Flt3 − IL-7R − c-kit − Sca-1 − ), CMP (Lineage − Flt3 − IL-7R − CD34 + CD16/32 − ), MEP (Lineage − Flt3 − IL-7R − CD34 − CD16/32 − ), and GMP (Lineage − Flt3 − IL-7R − CD34 + CD16/32 + ). Error bars represent mean ± SEM; P -values were determined by Student’s t -test.

Article Snippet: The TLR5 agonist entolimod (CBLB502) is a cGMP-manufactured drug product that was obtained from Cleveland BioLabs, Inc. [ ].

Techniques: Flow Cytometry

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: A Fold change in the Nϕ response in the indicated organs calculated from the absolute number of Nϕ in entolimod-treated (2 h and 5 h) versus vehicle-treated mice ( n = 3–5 mice /group). Flow cytometry was used to identify Nϕ (CD45 + CD11b + Ly-6C + Ly-6G hi ). B Absolute number of endogenous and adoptively transferred Nϕ in the BM, liver, and lungs 2 h post-treatment with vehicle or entolimod ( n = 6–8 mice/group). Adoptive transfers were done by i.v. injection of GFP-expressing BM immediately before treatment. Endogenous versus transferred Nϕ were distinguished by flow cytometry for GFP among the total Nϕ population (CD45 + CD11b + Ly-6C + Ly-6G hi ). C Absolute number (left) and composition (right) of Nϕ populations in the bone marrow at the indicated time points post-treatment by flow cytometry as defined by: pre-Nϕ (Lineage − c-kit int CD115 − CD11b + Gr-1 + CXCR4 + ), immature Nϕ (Lineage − c-kit int CD115 − CD11b + Gr-1 + CXCR4 − CXCR2 − Ly-6G + ), and mature Nϕ (Lineage − c-kit int CD115 − CD11b + Gr-1 + CXCR4 − CXCR2 + Ly-6G + ) where lineage was defined as CD3 − and B220 − ( n = 3–9 mice/group). D Flow cytometry-based staining for TLR5 using biotinylated-entolimod plus streptavidin fluorochrome “sandwich” platform on Nϕ (CD45 + CD11b + Ly-6C + Ly-6G hi ), NK cells (CD45 + CD3ε − NKp46 + ), DCs (CD45 + Ly-6G − B220 − F4/80 − CD11c + ), monocytes (CD45 + Ly-6G − B220 − CD11c − F4/80 + ), B cells (CD45 + Ly-6G − CD11c − F4/80 − B220 + ), CD8 + T cells (CD45 + CD3ε + CD8 + ), CD4 + T cells (CD45 + CD3ε + CD4 + ), platelets (CD45 − CD41 + ), and RBCs (CD45 − Ter119 + ) in the blood of BALB/c and Tlr5 −/− mice. Error bars represent mean ± SEM. * P < 0.04; P -values were determined by Student’s t -test.

Article Snippet: The TLR5 agonist entolimod (CBLB502) is a cGMP-manufactured drug product that was obtained from Cleveland BioLabs, Inc. [ ].

Techniques: Flow Cytometry, Injection, Expressing, Hi-C, Staining

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: Tlr5 −/− bone marrow chimeric mice received an adoptive transfer of either Tlr5 WT/WT or Tlr5 −/− Nϕ (10 million /mouse) followed immediately by vehicle or entolimod 24 h post-TBI. A , B Survival was measured by Kaplan–Meier curves. P -values were determined by Log-rank test; n = 10–16 mice/group. C Measurement of total HPPs by MethoCult in BM on day 7 post-treatment using the same experimental setup as A . Error bars represent mean ± SEM; P -values were determined by Student’s t -test; n = 4–7 mice/group. In all instances, Nϕ purity was routinely >98%.

Article Snippet: The TLR5 agonist entolimod (CBLB502) is a cGMP-manufactured drug product that was obtained from Cleveland BioLabs, Inc. [ ].

Techniques: Adoptive Transfer Assay

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: Protein microarray analysis of A mouse serum using R&D Systems Proteome Profiler Mouse XL Cytokine Array in the presence (rat IgG) and absence (α-Ly-6G) of Nϕ 24 h post-TBI and B Nϕ isolated from BM that were ex vivo treated with vehicle or entolimod for 30 min. Measurement by ELISA of MMP-9 in C serum from mice ( n = 5/group) in the presence (rat IgG) and absence (α-Ly-6G) of Nϕ 2 h post-treatment with vehicle or entolimod (TBI was delivered 24 h prior to vehicle or entolimod) and D supernatants from Tlr5 WT/WT and Tlr5 −/− Nϕ isolated from BM ( n = 6 mice/group) and ex vivo treated with vehicle or entolimod for 30 min.

Article Snippet: The TLR5 agonist entolimod (CBLB502) is a cGMP-manufactured drug product that was obtained from Cleveland BioLabs, Inc. [ ].

Techniques: Microarray, Isolation, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: A BALB/c mice ( n = 5/group) were given 5 Gy TBI followed 24 h later by treatment with vehicle, entolimod, or the indicated doses of recombinant pro-MMP-9 (rMMP-9). Measurement of total HPPs in BM by MethoCult was done on day 3 post-treatment. B Survival by Kaplan–Meier curve in BALB/c mice treated with vehicle, entolimod, or 10 μg/kg rMMP-9 24 h post 7.5 Gy TBI ( n = 9–10 mice /group). P -values were determined by the Log-rank test. C BALB/c mice ( n = 5/group) were given 5 Gy TBI followed 6 h later by rat IgG or α-Ly-6G. Twenty-four hours later mice were treated with vehicle, entolimod, rMMP-9 (10 μg/kg), or rMMP-9 plus entolimod. Measurement of total HPPs in BM by MethoCult was done on day 3 post-treatment. For A and C error bars represent mean ± SEM; P -values were determined by Student’s t -test.

Article Snippet: The TLR5 agonist entolimod (CBLB502) is a cGMP-manufactured drug product that was obtained from Cleveland BioLabs, Inc. [ ].

Techniques: Recombinant

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: Following lethal radiation damage, entolimod stimulates Nϕ to release MMP-9 which, in cooperation with G-CSF, accelerates the recovery of hematopoiesis.

Article Snippet: The TLR5 agonist entolimod (CBLB502) is a cGMP-manufactured drug product that was obtained from Cleveland BioLabs, Inc. [ ].

Techniques: