Senescent cells suppress macrophage-mediated corpse removal via upregulation of the CD47-QPCT/L axis

Abstract

Progressive accrual of senescent cells in aging and chronic diseases is associated with detrimental effects in tissue homeostasis. We found that senescent fibroblasts and epithelia were not only refractory to macrophage-mediated engulfment and removal, but they also paralyzed the ability of macrophages to remove bystander apoptotic corpses. Senescent cell-mediated efferocytosis suppression (SCES) was independent of the senescence-associated secretory phenotype (SASP) but instead required direct contact between macrophages and senescent cells. SCES involved augmented senescent cell expression of CD47 coinciding with increased CD47-modifying enzymes QPCT/L. SCES was reversible by interfering with the SIRPα-CD47-SHP-1 axis or QPCT/L activity. While CD47 expression increased in human and mouse senescent cells in vitro and in vivo, another ITIM-containing protein, CD24, contributed to SCES specifically in human epithelial senescent cells where it compensated for genetic deficiency in CD47. Thus, CD47 and CD24 link the pathogenic effects of senescent cells to homeostatic macrophage functions, such as efferocytosis, which we hypothesize must occur efficiently to maintain tissue homeostasis.

Figure 1.

Chemical treatment induces a senescent cellular phenotype. Senescence was induced by palbociclib, except for A549 cells where etoposide was used. (A) Representative β-galactosidase staining was performed to mark senescent cells from indicated cell types after chemical induction of senescence. Scale bars indicate 50 µm. Images are representative of at least three independent experiments. (B) Representative immunofluorescence images from indicated cell types stained for Phalloidin (green) and Hoechst (blue). Scale bars indicate 20 µm. n = 2. (C) qPCR was performed to test CDKN1A gene expression in proliferating or senescent cells. Relative mRNA levels were normalized to the respective proliferating controls. Data are representative of three independent experiments. All values are means ± SEM. **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test. (D) Proliferation was quantified in the indicated proliferating or senescent cells using a BrdU Assay. Proliferation rate was normalized to the respective proliferating control. Data are representative of three independent experiments. All values are means ± SEM. **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test. (E and F) qPCR was performed to test CXCL1 (E) and IL6 (F) gene expression in proliferating or senescent cells. Relative mRNA levels were normalized to the respective proliferating controls. Data are representative of three independent experiments. All values are means ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test. (G and H) CXCL1 (G) and IL6 (H) secretion of the indicated proliferating or senescent cells was quantified in the supernatant by ELISA. Data are representative of at least two independent experiments. All values are means ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test.

Figure 2.

Macrophages interact with but do not engulf or remove senescent cells. (A) Schematic representation of senescence induction in murine and human primary and transformed cells. (B) Senescence was induced in actin-GFP mouse embryonic fibroblasts (MEFs) by either passaging stress until the Hayflick limit was reached by γ-irradiation or by treatment with palbociclib. Senescent MEFs were co-cultured with BMDMs, isolated from tdTomato⁺ mice, in a ratio of 10 BMDMs to 1 senescent cell and imaged over 48 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 100 µm. (C) Quantification of green fluorescent area signal of senescent cells in co-culture with different ratios of BMDMs as indicated. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by two-way ANOVA with Bonferroni correction. (D) Senescent NHLF (upper panel) or senescent human pancreatic epithelial cancer cells (Panc1; lower panel) stained with green Cytolight dye and co-cultured with human MDMs (unstained). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm.

Figure S1.

Impairment of efferocytosis by senescent cells is independent of macrophage polarization. (A) Senescent 3T3 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve BMDMs. Co-cultures were either stimulated with LPS (upper panel) or IL4 and IL13 (lower panel). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (B) Senescent Panc1 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve MDMs. Co-cultures were either stimulated with LPS (upper panel) or IL4 and IL13 (lower panel). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (C) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of proliferating (black line) or senescent (red line) A549 cells over time. First, co-cultured macrophages were exposed to unlabeled apoptotic corpses. After 24 h, pHrodo labeled apoptotic corpses were added to the co-culture and efferocytotic activity of the macrophages was monitored over time by the IncuCyte S3 system. (D) Efferocytosis of apoptotic corpses by differently stimulated macrophages in the presence of senescent cells (induced by palbociclib). Unstimulated macrophages were polarized to a M1 phenotype by the addition of LPS or to a M2 phenotype by the addition of IL4 and IL13. Data are representative of three independent experiments. All values are means ± SEM; ***P < 0.001; ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates. (E) Quantification of efferocytosis of apoptotic corpses by MDMs that were co-cultured with NHLF, IPF or Panc1 cells and then further stimulated with either LPS (upper row) or IL4 and IL13 (lower row) Efferocytotic capability of macrophages in single (yellow line) or co-culture with proliferating (black line) or senescent (red line) was monitored over time.

Figure 3.

Senescent cells impair macrophages’ ability to remove corpses. (A) Schematic overview of the experimental design using direct co-cultures between proliferating or senescent cells and primary macrophages. Cells were co-cultured for 16 h and then exposed to pHrodo labeled apoptotic corpses (Raji cells; 2.5:1 ratio). Corpse removal was monitored by live cell imaging (IncuCyte) for 24 h. (B and C) Quantification of efferocytosis of apoptotic corpses in co-cultures between human MDMs and senescent or proliferating primary lung fibroblasts (B; NHLF or IPF-derived fibroblasts [IPF]), primary liver stellate cells, primary lung small airway epithelial cells (SAEC), or transformed epithelial cell lines (C; Panc1, A549). Efferocytotic capability of macrophages in co-culture with proliferating (black line) or senescent (red line) cells was monitored over time using the IncuCyte S3 system. Data are representative of at least three independent experiments. All values are means ± SEM.

Figure 4.

Senescent cells reduce the uptake of apoptotic corpses by macrophages. (A) Schematic overview of the experimental design using direct co-cultures between proliferating or senescent fibroblasts (normal healthy or IPF-derived) and primary macrophages. Cells were co-cultured for 16 h, and then exposed to Cell tracker green and CypHer5E co-labeled apoptotic corpses (Raji cells; 2.5:1 ratio). Co-cultures were washed 1, 2 or 4 h post feeding and subsequently fixed to perform immunofluorescence staining. (B) Immunofluorescence microscopy of MDM in indicated co-cultures (either proliferating or senescent primary IPF-derived fibroblasts) exposed to apoptotic Rajis (labeled with Cell tracker green [green] and the pH-sensitive dye CypHer5E [white]) for 1, 2 or 4 h were then washed, fixed, and stained for CD45 (red) and Hoechst (blue). Scale bar: 20 µm. Representative images are from two independent experiments. (C and D) Quantification of apoptotic corpse uptake by macrophages in co-culture with human MDMs and senescent or proliferating primary IPF-derived (C) or normal healthy fibroblasts (D). Measurements and analysis of the number of green events per image (cell tracker green labeled Raji cells) were performed using the IncuCyte S3 system. All values are means ± SEM. Statistically significant differences were determined by two-way ANOVA with Tukey correction; n = 6 biological replicates. *P < 0.05, **P < 0.001. (E and F) Quantification of the ratio between red (CypHer5E)⁺ green (Cell tracker)⁺ (overlap) events and the total number of green events in the same field of view. Data are obtained from co-cultures between MDMs and proliferating (black) or senescent (red) IPF-derived (E) or normal healthy fibroblasts (F) exposed to apoptotic Rajis as shown in A. All values are means ± SEM. n = 6 biological replicates.

Figure 5.

Senescent cells inhibit corpses removal through direct cell-cell contact. (A) Quantification of efferocytosis of apoptotic corpses by BMDMs in the presence of different types of murine 3T3 senescent cells (senescence induction by Hayflick limit, γ-irradiation or Palbociclib). BMDMs were primed in co-culture for 6 or 24 h. Apoptotic corpses (Jurkat cells) were labeled with PKH26 and added to macrophages in a 5:1 ratio. Samples were analyzed by flow cytometry 1 h post corpse exposure. All values in are means ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates. (B) Quantification of efferocytosis of apoptotic eosinophils by BMDMs in the presence of senescent 3T3 cells (induced by Palbociclib). All values in are means ± SEM; ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates. (C) Conditioned media (CM) derived from either proliferating (black line) or senescent cells (red line) was transferred to naïve human MDMs. pHrodo labeled apoptotic corpses (AC, Raji cells) were added to MDMs and efferocytosis was monitored over time using the IncuCyte S3 System. Data are representative of three independent experiments. All values are means ± SEM. (D) Senescent cells (3T3, Palbociclib) were seeded in a transwell insert, which was placed into a well containing BMDMs. Efferocytosis of apoptotic corpses (Jurkat cells) by macrophages co-cultured with senescent cells in a transwell was analyzed by flow cytometry. Apoptotic corpses were labeled with PKH26 and added in a 5:1 ratio to the part of the well containing BMDMs. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates.

Figure S2.

Senescent cells inhibit macrophages’ ability to phagocytose. (A) Schematic overview of the experimental design to monitor phagocytosis by macrophages in co-culture with proliferating or senescent cells. 16 h after the assembly of the co-cultures, either Escherichia coli, secondary necrotic corpses or inert silicon beads were added. Uptake by macrophages was monitored over time using the IncuCyte S3 system. (B) Removal of the labeled phagocytotic prey by macrophages was quantified over time. Macrophages were in co-culture with either proliferating (black line) or senescent cells (red line) of the indicated origin.

Figure 6.

Senescent cells exhibit increased CD47 expression in vivo and in vitro. (A) Violin plots showing Cd47, SIRPα and Cdkn1a expression in lung tissue as assayed by the Tabula Muris Senis dataset. *P < 0.05, ****P < 0.0001. Statistically significant differences were determined by Wilcoxon test. Expression levels obtained from RNA-sequencing data for liver fibrosis rat models (Wang et al. 2021). (B and C) Transcripts per million (tpm) expression values for Cd47, Sirpα, and Cdkn1a in rat livers derived frombile duct ligation (BDL; B) or thioacetamide (TAA)-induced liver fibrosis (C). Data are represented as mean ± SEM. Significant de-regulation (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001) was determined by limma/voom based on counts derived from featureCounts. (D) Representative immunofluorescence images showing that p21 staining (red) co-localized with enhanced CD47 expression (green) in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in mice upon feeding with a high fat diet. Scale bar: 50 µm. n = 3–4 mice per group. (E) Quantification of p21-positive cells per field and the percentual area of CD47 staining in SAT and VAT from animals fed a high fat diet (HFD) compared to normal diet (ND). All values are means ± SEM; *P < 0.05. Statistically significant differences were determined by t test or Mann-Whitney test, as appropriate; n = 3–4 mice per group.

Figure 7.

Increased CD47 expression in aging and replicative senescence. (A) Quantification of relative CD47 mRNA levels by qPCR. Expression levels of senescent cells were normalized to the respective proliferating control (white bar). Data are representative of three independent experiments. All values are means ± SEM. **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test. (B) Representative immunofluorescence images from indicated cell types stained for CD47 (green) and Hoechst (blue). Scale bar: 20 µm, except for 3T3 cells: 50 µm. (C) Whole-cell lysates from indicated cell types were isolated and analyzed by Western blotting for the indicated proteins. Senescence was induced by chemical treatment (palbociclib, except for A549: etopsoside). Shown are representative blots of three independent experiments. GAPDH images are derived from the same blot as Fig. S3 (for Panc1 cells the same GAPDH loading control is additionally used in Fig. 8 G). (D) Representative immunofluorescence images of proliferating or senescent small epithelial cells (SAEC) stained for CD47 (green) and Hoechst (blue). scale bars indicate 20 µm. n = 2–3. (E) Western blot analysis of CD47 expression in SAEC cells. Senescence was induced by chemical treatment (palbociclib). GAPDH was used as loading control. n = 2. GAPDH images are derived from the same blot as Fig. S3. (F) Representative immunofluorescence images of primary lung fibroblast (proliferative or replicative senescent by serially passaging; normal healthy [NHLF] or IPF-derived [IPF]) showing CD47 (green) and Hoechst (blue) staining. scale bars indicate 20 µm. n = 2. (G) Whole-cell lysates from primary NHLF or IPF lung fibroblasts were isolated and analyzed by Western blotting for the indicated proteins. Senescence was induced by replicative passing. Shown are representative blots of three to four independent experiments. Source data are available for this figure: SourceData F7.

Figure 8.

Impairment of efferocytosis by senescent cells is mediated by CD47 and CD24 expression. (A) CD47 loss-of-function in 3T3 cells was verified by CD47 staining in flow cytometry. (B) Representative immunofluorescence images from senescent 3T3 cells (WT or CD47 KO) showing CD47 (green) and Phalloidin (blue) staining (scale bar: 50 µm). n = 2. (C) Quantification of efferocytosis of apoptotic corpses by BMDMs in the presence of CD47 KO senescent cells (induced by palbociclib). Samples were analyzed by flow cytometry. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates. (D) Schematic overview of the experimental design. Senescent human fibroblasts were incubated with neutralizing anti-CD47 FAB fragments for 1 h, then direct co-cultures with primary macrophages were assembled for 6 h, followed by exposure to pHrodo labeled apoptotic corpses. Corpse removal was monitored by live cell imaging (IncuCyte). (E and F) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and senescent primary NHLF (E) or IPF-derived fibroblast (IPF; F). Efferocytotic capability of macrophages in co-culture with senescent fibroblast in the presence (blue bar) or absence of FAB fragments (gray bar) was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 20 h was calculated and plotted. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by unpaired Student’s t test. (G) Whole-cell lysates derived from Panc1 cells (WT and CD47 KO) were analyzed by Western blotting for the indicated proteins. GAPDH was used as loading control. Senescence was induced by chemical treatment (Palbociclib). Blots are representative of three independent experiments. GAPDH image is derived from the same blot as Fig. 7 and Fig. S3. CD24 image is derived from the same blot as in Fig. S3. (H) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of senescent Panc1 cells. Efferocytotic capability of macrophages in co-culture with WT (red line) or CD47 KO (green line) Panc1 cells was monitored over time. Data are representative of at least three independent experiments. All values are means ± SEM. (I) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and proliferating (black bar) or senescent CD47 KO Panc1 cells. Senescent CD47 KO cells were treated with (orange bar) or without anti-CD47 FAB fragments (green bar) prior to the assembly of the co-culture with MDMs. Efferocytotic capability of macrophages was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 22 h was calculated and plotted. Shown values are means of at least technical triplicates ± SEM in one representative experiment. Data are representative of three independent experiments. **P < 0.01. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction. (J) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and proliferating (gray bar) or senescent primary small airway cells (SAEC). Senescent SEAC cells were either untreated (green bar) or treated with anti-CD47 (blue bar) or anti-CD24 FAB fragments (orange bar) or the combination of both FAB fragments (violet bar) prior to the assembly of the co-culture with MDMs. Efferocytotic capability of macrophages was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 22 h was calculated and plotted. Shown values are means of technical quadruplicates ± SEM in one representative experiment. Data are representative of two independent experiments *P < 0.05, **P < 0.005. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction. Source data are available for this figure: SourceData F8.

Figure S3.

Increased CD24 expression in senescent epithelial cells. (A) Senescent CD47 KO 3T3 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve BMDMs (unstained). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (B) Senescent CD47 KO Panc1 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve MDMs (unstained). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (C) Whole-cell lysates from indicated cell types were isolated and analyzed by Western blotting for the indicated proteins. Senescence was induced by chemical treatment. Data are representative of at least two independent experiments. GAPDH images are derived from the same blot as Fig. 7, C and E (SAEC) and Fig. 8 G. CD24 image is derived from the same blot as in Fig. 8 G. (D) Representative immunofluorescence images from indicated cell types (proliferating or senescent) stained for CD24 (red) and Hoechst (blue). Scale bars indicate 20 µm. n = 2. (E and F) Expression levels obtained from RNA-sequencing data for liver fibrosis rat models (Wang et al. 2021). Transcripts per million (tpm) expression values for Cd24 in rat livers derived from bile duct ligation (BDL; E) or thioacetamide (TAA)-induced liver fibrosis (F). Data are represented as mean ± SEM. Significant de-regulation (***P < 0.0005, ****P < 0.0001) was determined by limma/voom based on counts derived from featureCounts. Source data are available for this figure: SourceData FS3.

Figure 9.

SHP-1 mediates efferocytosis paralysis. (A) Representative immunofluorescence images from direct co-cultures of senescent Panc1 cells and human MDMs. Cells were specifically stained for SHP-1 (green), macrophage marker CD45 (red), Phalloidin (magenta), and Hoechst (blue); scale bar: 20 µm. n = 3. (B) BMDMs from tdTomato⁺(orange) mice were co-incubated with senescent WT or Cd47-deficient 3T3 cells for the times shown on the left of the images. Fixed samples were stained for SHP-1 (green) and Phalloidin (blue). Scale bars indicate 50 µm (main) or 12.5µm (inset). n = 2. (C) Schematic overview of the intensity profile measurement of SHP-1 in BMDMs. Intensity was measured across the widest point of each cell. Intensity profiles were generated by measuring the distance in µm and the gray value intensity. (D) Quantification of intensity profile measurement of SHP-1 in BMDMs. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; ****P < 0.0001. n = 20 biological replicates. (E) Quantification of PHK26-labelled corpse efferocytosis in the presence of absence of NSC-87877 (SHP-1 and SHP-1 inhibitor). Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; *P < 0.05, ***P < 0.001, ****P < 0.0001; n = 3 biological replicates. (F and G) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of senescent NHLF (F) or Panc1 cells (G). Macrophages were pre-incubated for 1 h with NSC-87877, and then co-cultures were assembled prior to exposure to pHrodo labeled apoptotic corpses. Efferocytotic capability of macrophages with (blue line) or without inhibitor (black line) was monitored over time. Data are representative of three independent experiments. All values are means ± SEM.

Figure S4.

Increased QPCT/L expression in senescent cells. (A and B) Expression levels obtained from RNA-sequencing data for liver fibrosis rat models (Wang et al. 2021). Transcripts per million (tpm) expression values for Qpct and Qpctl in rat livers derived from BDL (A) or TAA-induced liver fibrosis (B). Data are represented as mean ± SEM. Significant de-regulation (*P < 0.05, **P < 0.005, ****P < 0.0001) was determined by limma/voom based on counts derived from featureCounts. (C and D) Quantification the relative QPCT (C) and QPCTL (D) mRNA levels by qPCR. Expression levels of senescent cells were normalized to the respective proliferating control (white bar). Data are representative of three independent experiments. All values are means ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistically significant differences were determined by unpaired Student’s t test. (E) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and proliferating (gray bar) or senescent IPF-derived lung fibroblasts. Senescent IPF-derived lung fibroblasts were treated with the QPCTL inhibitor SEN-177 (1, 5, 10 µM) prior the assembly of the co-culture with MDMs. Efferocytotic capability of macrophages was monitored over time using the IncuCyte S3 system. Data are representative of three independent experiments. All values are means ± SEM.

Figure 10.

QPCT/L is essential for mediating the suppression of efferocytosis through the CD47-SIRPα axis. (A and B) Proteomic analysis of proliferating versus senescent NHLF (A) and Panc1 cells (B). Volcano plot representing the negative log10-transformed P values vs. the log₂ fold changes in protein intensities of senescent compared to proliferating cells. Dotted lines depict a P value of 0.05 and a fold change of 1.5. Senescence was induced by chemical treatment. Relative QPCTL (red symbol) and CD47 (blue symbol) protein expression normalized to the proliferating control. n = 3 biological replicates. (C) Quantification the relative QPCTL enzyme activity. Enzyme activity of senescent cells was normalized to the respective proliferating control (white bar). Data are representative of two independent experiments. All values are means ± SEM. (D) SIRPα binding of proliferating (white bar) or senescent cells (orange bar) was quantified using a luminescent reporter assay. All values are means ± SEM. Statistically significant differences were determined by unpaired Student’s t test; n = 3–6 biological replicates. (E) Schematic overview of the overall experimental design. Senescent fibroblasts were incubated with neutralizing anti-SIRPα antibodies (mouse) or FAB fragments (human) for 1 h, then direct co-cultures with primary macrophages were assembled for 6 h, and then exposed to labeled apoptotic corpses. Corpse removal was monitored by flow cytometry (mouse) or live cell imaging (human). (F) Efferocytosis in the presence of senescent 3T3 cells (induced by palbociclib) and a SIRPα blocking antibody. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; ****P < 0.0001. n = 3 biological replicates. (G) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and senescent primary IPF-derived fibroblast (IPF). Efferocytotic capability of macrophages in co-culture with senescent fibroblast in the presence (magenta line) or absence of FAB fragments (black line) was monitored over time using the IncuCyte S3 system. Data are representative of three independent experiments. All values are means ± SEM. (H) QPCTL activity was quantified in proliferating or senescent primary IPF-derived lung fibroblasts in the presence (5, 10 µM) or absence of the QPCTL inhibitor SEN-177. Enzyme activity of senescent cells was normalized to the respective proliferating control (gray bar). Data are representative of four independent experiments. All values are means ± SEM. **P < 0.005. Statistically significant differences were determined by one-way ANOVA with Tukey correction. (I) SIRPα binding of proliferating or senescent primary IPF-derived lung fibroblasts in the presence (1, 5, 10 µM) or absence of the QPCTL inhibitor SEN-177 was quantified using a luminescent reporter assay. Data are representative of three independent experiments. All values are means ± SEM. ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction. (J) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and proliferating (gray bar) or senescent IPF-derived lung fibroblasts. Senescent IPF-derived lung fibroblasts were treated with the QPCTL inhibitor SEN-177 (1, 5, 10 µM) prior the assembly of the co-culture with MDMs. Efferocytotic capability of macrophages was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 22 h was calculated and plotted. Data are representative of three independent experiments. All values are means ± SEM. *P < 0.05, **P < 0.005. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction.