Fig 1.
Gamma radiation enhances expression of activation markers in primary human MDMs and THP1 monocytes.
(A-C) Expression of macrophage anti-inflammatory markers (A), pro-inflammatory markers (B), and major cytokines (C) in primary human MDMs, 48h post-irradiation. MDMs were differentiated from PBMCs, isolated from 6 donors, by treatment with M-CSF and exposed to 5Gy γIR; box plots of RT-qPCR measured expression of n = 6 is shown. * p<0.05, paired Wilcoxon test. (D and E) Protein concentrations of soluble macrophage activation markers CXCL10, CCL2 and sCD163 (D) and cytokines IL-6, TNF-α, and IL-10 (E) in culture medium of primary MDMs, 48h post-irradiation. Error bars indicate ±SD of three independent biological replicates; * p<0.05, ** p<0.01, panel D and below–paired t test. (F and G) Expression of macrophage pro-inflammatory (F) and anti-inflammatory (G) markers in human monocytic cells THP1 48h after exposure to γIR. Error bars indicate ±SD of four independent biological replicates. (H) Expression of cytokines in THP1 cells, 48h after γIR. Cells were exposed to indicated γIR doses; RT-qPCR analysis was performed with primers specific for indicated cytokines. Error bars indicate ±SD of 4 independent biological replicates. (I) Quantitation of proinflammatory (IL-1β, IL-6, TNF-α, CCL2) and anti-inflammatory (IL-10) cytokines in culture media of γIR-exposed THP1 cells using Luminex multiplex immunoassay, 48h after γIR, PMA (20 nM) or poly(I:C) (2 μg/ml) treatment. Error bars indicate ±SD of three independent biological replicates; ** p<0.01 vs 0 Gy. For panels F to I: * p<0.05, ** p<0.01, NS non-significant.
Fig 2.
Secretory profile and macrophage phenotype of γIR-exposed monocytes and MDMs are related in part to radiation-induced senescence.
(A) Expression of intracellular senescence markers (CDKN1A and GLB1) in THP1 cells (blue columns) and primary MDMs (red columns) 48h after irradiation. Error bars: ±SD of three independent biological replicates. (B) Western blot analysis of senescence marker p21Waf1 (CDKN1A) protein in THP1 lysates, 48 h after irradiation or treatment with PMA or poly(I:C). Forty μg of total protein were loaded. (C) Expression of indicated macrophage SASP markers in human monocytic cells THP1 (blue columns) and primary MDMs (red columns) 48h after irradiation. Error bars indicate ±SD of three independent biological replicates. In all panels: * p<0.05, ** p<0.01, NS non-significant. (D) Quantitation of indicated macrophage SASP markers in culture media of irradiated THP1 cells (n = 6) using Luminex multiplex immunoassay, 48h after γIR. Error bars indicate ±SD; * p<0.05, paired Wilcoxon test. (E) Microscope images of untreated, irradiated, and PMA-treated THP1 monocytes, 48h incubation. Cells were fixed with paraformaldehyde, labeled for F actin with phalloidin-rhodamine conjugate and for nuclear DNA with DAPI (upper panels) or vital images were obtained (lower panels). Scale bars: 75 μm. (F) Proportion of CD11b-positive THP1 cells in irradiated or control cells detected by flow cytometry of viable (DAPI-) cells, 72h after γIR (graphs represent median fluorescence intensity data of 4 independent experiments). (G) Quantitation (flow cytometry) of CD11b-positive populations in cells exposed to increasing doses of γIR, or treated with PMA or poly(I:C), 72h post-exposure (only viable, DAPI- cells were analyzed; box plot of n = 4 is shown. ** p<0.01.
Fig 3.
Gamma radiation induces expression of type I interferons and inflammation-related genes in THP1 monocytes and MDMs.
(A and B) Box plots showing expression, measured by RT-qPCR, of IFNα (A) and IFNβ (B) in MDMs, 48h after irradiation (n = 15). M-CSF-differentiated MDMs from PBMCs isolated from 5 donors were exposed to indicated doses of γIR. Error bars: ±SD, * p<0.05, NS non-significant, paired Wilcoxon test. (C) Expression of IFNα and IFNβ in THP1 cells, 48h post-irradiation, measured by RT-qPCR. Cells were exposed to indicated doses of γIR or treated with PMA or poly(I:C) with or without indicated transfection reagents. Error bars: ±SD of four independent biological replicates; * p<0.05, ** p<0.01. (D) Immunoblotting of phosphorylated STAT1 at Ser727 and Tyr701 (40 μg of total protein) in THP1 cells, 48h post-irradiation. (E) Volcano plot showing ISG expression in irradiated vs. control THP1 cells, measured by PCR array of total cellular RNA samples, 48h post-irradiation. Red dots indicate significant (above blue line) or insignificant (below blue line) increase in gene expression in irradiated cells (p<0.05, fold change >2.0). (F and G) Activation of NF-κB-dependent transcription (F) and IFNAR signaling (G) by culture media from irradiated (5 Gy) or non-irradiated THP1 in the reporter THP1-Dual cells (pink columns) expressing SEAP gene driven by an IFN-β minimal promoter activated by NF-κB and secreted Lucia luciferase gene under the control of an IFNAR signaling-activated ISG54 minimal promoter, and THP1-Dual KO-IFNAR2 cells (blue columns), generated from THP1-Dual cells by stable knockout of the IFNAR2 receptor. Similarly, the culture supernatant from Poly(I:C)-transfected THP1 was used as a positive control. After 18 h of incubation and staining with SEAP-sensitive QUANTI-Luc dye, the absorbance (600 nm) (F) or luminescence (G) of each sample were measured and normalized to fresh RPMI media controls. Error bars indicate ±SD of a minimum of 3 independent biological replicates; p<0.01. (H) Immunoblotting to assess STAT1 Ser727 and Tyr701 phosphorylation (20 μg of total protein) in THP1 cells incubated for 24h with filtered culture media from irradiated or non-irradiated THP1, with or without interferon-α/β receptor inhibitor IFNAR-IN-1. (I) Expression ratio of IFNα and indicated cytokines (5 Gy-to-0 Gy ratio of RNA count) in THP1 cells transfected with indicated siRNA (24 h before irradiation), with or without exposure to 5 Gy γIR for 48 hours. RNA was quantified by RT-qPCR. Error bars: ±SD of at least four independent biological replicates; * p<0.05. (J) Immunoblot of ssRNA sensor RIG-I and dsRNA sensors MDA-5 and TLR3 (40 μg of total protein) in THP1 cells, 48h post-irradiation. (K) Western blot of downstream markers of the activation of RIG-I/MDA-5 (MAVS, phospho-IKKε, and phospho-IRF3) and TLR3 (phospho-IKKε, and phospho-IRF3) pathways. THP1 lysates (40 μg total protein), harvested 48h post-exposure.
Fig 4.
Gamma radiation activates HERV transcription in primary human MDMs and THP1 cells.
(A) Top: Venn diagram of differentially expressed retroelements and HERVs identified between irradiated vs. control THP1 cells or MDMs. Bottom: expression fold changes detected for the common set (626 features) shown on the upper Venn diagram. (B) Expression of six HERV clades, identified upregulated by RNA-Seq in both irradiated THP1 cells and MDMs (panel A, red circles), measured by RT-qPCR at 48h post-exposure to 5 Gy γIR dose. Primers were designed for the alignments within the clusters identified across gag, pol or env genes of the sequences within the clades. Error bars: ±SD of three independent biological replicates. (C) Expression, measured by RT-qPCR, of randomly selected differentially expressed HERVK in THP1 cells (grey columns) and primary MDMs (red columns), 48h post-irradiation. Error bars: ±SD of three independent biological replicates. (D) Transcription of HERVK HML-2 env in response to 5 Gy γIR dose, measured by RT-qPCR at 48h post-exposure in CD11b-positive and negative THP1 cells, separated by sorting flow cytometry. Only viable, DAPI-negative cells were analyzed; box plot of n = 3 is shown. Error bars: ±SD of three independent biological replicates. (E) Ratio of HERV RNA bound to anti-dsRNA antibodies to total RNA input: HML-2 (left) and HERV9NC Cl.1 pol (right) from 5 Gy γIR exposed and unexposed THP1 cells. RT-qPCR of RNA-IP (RIP) complexes with rJ2 and 9D5 antibodies, 48h post-irradiation. Error bars: ±SD of five (HML-2) or four (HERV9NC) independent biological replicates; in panels B-E,* p<0.05, ** p<0.01. (F) Relative count of HERVK HML-2 antisense RNA identified with antisense strand-specific reverse transcription primers targeting regions in the env, pol and gag genes, and quantitated by qPCR. Error bars: ±SD of at least 6 independent biological replicates. (G) Relative count of the sense and antisense transcripts of HERVK HML-2 measured by RT-qPCR using env-specific primers, 48h after irradiation of MDMs. MDMs were differentiated with M-CSF from PBMCs isolated from 5 donors. Box plot of n = 15 is shown. In panels F and G, * p<0.05, NS non-significant, paired Wilcoxon test.
Fig 5.
HERVK HML-2 env knockdown decreases IFN-I expression and alters the secretory profile of THP1 monocytes and primary MDMs.
(A) Transcription of HERVK HML-2 and five other HERV clades, identified upregulated by RNA-Seq in both irradiated THP1 cells and MDMs (Fig 4A), measured by RT-qPCR in THP1 cells expressing control (grey) or shRNA-Env (red), 48h post-irradiation. Error bars: ±SD of three independent biological replicates. (B) Expression, measured by RT-qPCR, of macrophage activation markers in human monocytic THP1 cells, expressing shRNA-Env (red) or control shRNA (grey), 48h post-irradiation. Error bars: ±SD of three independent biological replicates. In panels A and B, * p<0.05, ** p<0.01; A and B, genes, whose expression changed significantly in response to shRNA-Env are indicated by red symbols. (C) Immunoblot against intracellular senescence marker p21Waf1 (CDKN1A) in lysates (40 μg total protein) of THP1 cells, expressing either shRNA-Env or control shRNA, 48h after irradiation. (D) Phosphorylation of STAT1 Ser727 and Tyr701 (40 μg total protein) in THP1 cells, expressing control shRNA or shRNA-Env, 48h post-irradiation. (E) Volcano plot depicting ISG expression in THP1 cells expressing shRNA-Env or control shRNA, 48h post-IR: measured by PCR array of total cellular RNA samples. Green dots represent significant (above blue line) or non-significant decrease in expression of indicated genes in shRNA-Env vs. control shRNA expressing cells (p<0.05, fold change >2.0). (F and G) Soluble macrophage activation markers (F) and key cytokines (G) in culture medium of THP1 cells, 48h post-irradiation, determined by multiplex immunoassay (Luminex). Error bars: ±SD of three independent biological replicates. * p<0.05, ** p<0.01, NS non-significant. (H) Macrophage SASP markers in culture media of irradiated THP1 cells (n = 6), expressing shRNA-Env (yellow) or control shRNA (blue), using multiplex immunoassay (Luminex), 48h after γIR. Error bars indicate ±SD; p value of all differences is <0.05, unpaired Mann-Whitney test. (I) Transcription levels, measured by RT-qPCR, of HERVK HML-2 env, IFN-I (IFNα, IFNβ), inflammation (IL-1β, TNF-α) and anti-inflammatory (TGF-β, IL-10) markers in irradiated primary human MDMs, expressing control or shRNA-Env, 48h post-irradiation. M-CSF-differentiated MDMs from n = 6 donors. * p<0.05, NS non-significant, paired Wilcoxon test. (J) Soluble macrophage activation markers in culture medium of primary human MDMs, 48h post-irradiation assessed by multiplex immunoassay. Error bars: ±SD of three independent biological replicates; ** p<0.01, two-tailed paired t test.
Fig 6.
HERVK env RNA is associated with cytoplasmic dsRNA receptors MDA-5 and TLR3 in irradiated monocytes THP1.
(A and B) Immunoblot of MDA-5 (A) and TLR3 (B) with mouse monoclonal antibodies in immunocomplexes after IP of these proteins with rabbit polyclonal antibodies from THP1 lysates, exposed to indicated doses of γIR or treated with PMA or poly(I:C); 48 h post-irradiation. β-Actin in cell lysates, utilized for IP, was used as a reference. (C and D) Relative abundance of sense and antisense HERVK HML-2 env RNA in immunocomplexes after RNP-immunoprecipitation (RIP) of MDA-5 (C) and TLR3 (D) from THP1 lysates, shown on panels A and B. The fold change RNA count (ΔΔCt) was calculated in relation to β-actin reference RNA, detected in the lysates used for RIP. Error bars: ±SD of three independent replicates; * p<0.05, ** p<0.01, NS non-significant. (E) PLA assay visualizing association of HML-2 env RNA with MDA-5 receptor in THP1 in irradiated or control cells; 48h post-IR. Nuclei–DAPI staining, MDA-5/RNA complexes–Texas Red. Scale bars: 30 μm.
Table 1.
Expression and secretion of pro-inflammatory and anti-inflammatory macrophage markers in THP1 monocytes and primary monocyte-derived macrophages in response to gamma radiation.
Table 2.
Key resources.