Journal: bioRxiv

Article Title: The phosphate exporter XPR1 regulates a gasdermin D–independent mature IL-1β secretion pathway in LPS-stimulated human monocytic cells

doi: 10.64898/2025.12.23.695886

Figure Lengend Snippet: (A) Experimental workflow of PMA differentiation and LPS stimulation of THP-1 cells. Cells were seeded at 1 x 10 5 cells per well (200 µL) and differentiated with PMA (300ng/mL) for 3 h. After three washes, cells were incubated in PMA-free medium for 18 h. Cells were then stimulated with 10 ng/mL ultrapure LPS from E. coli for 24 h. As a positive control for cell death, pyroptosis was induced by nigericin (Nig.) (10 µM) added 4 h after LPS. See also Figure S1. (B) Secretion of mature IL-1β and assessment of cell viability following LPS stimulation of wild-type (WT) or (C) GSDMD -/- THP-1 cells. PMA-differentiated THP-1 cells (WT of GSDMD -/- ) were stimulated with LPS (10 ng/mL) or not for 24 h. Secreted mature IL-1β was measured by ELISA. Cell death was evaluated by CellTox TM Green uptake and LDH release. Metabolic activity was assessed using a MTS/PMS assay. Nigericin (10µM), added 4 h after LPS, was used as a positive control for pyroptosis. Data are shown as means ± SEM of biological triplicates. Each panel is representative of at least three independent experiments. The results with GSDMD -/- THP-1 cells were reproduced with two different clones generated with distinct gRNAs (Table S1), and complete gene invalidation was verified by targeted sequencing (Table S1).

Article Snippet: We used a rabbit IgG Isotype Control (Alexa Fluor 488 conjugate) (Cell Signaling #4340) as negative control (Isoctrl), a rabbit Alexa Fluor 488 coupled antibody against cleaved IL-1β (Cell Signaling – custom) and a mouse FITC anti-human IL-1β antibody (BioLegend #508206, clone JK1B-1) which recognizes both pro- and mature IL-1β.

Techniques: Incubation, Positive Control, Enzyme-linked Immunosorbent Assay, CellTox Assay, Activity Assay, Clone Assay, Generated, Sequencing

Journal: bioRxiv

Article Title: The phosphate exporter XPR1 regulates a gasdermin D–independent mature IL-1β secretion pathway in LPS-stimulated human monocytic cells

doi: 10.64898/2025.12.23.695886

Figure Lengend Snippet: WT or GSDMD -/- PMA-differentiated THP-1 cells were stimulated with LPS (10 ng/mL) for 24 h. At 2, 4, 7, 10, and 24 h, supernatants were harvested and cells were resuspended in 200 µL of fresh medium (same volume as supernatants). Cells and supernatants were frozen at -20 °C for at least 18 h. After thawing, cell lysates were clarified by centrifugation to remove cellular debris. Mature IL-1β (A) and IL-1RA (B) were measured by ELISA in both supernatants and cell lysates. Secretion efficiency after 7 or 24 h of LPS stimulation was calculated as the ratio of the cytokine content measured in supernatant (200µL) to the total cytokine content measured in the supernatant plus the corresponding cell lysate (200µL). The results with GSDMD -/- THP-1 cells were reproduced with two different clones generated with distinct gRNAs (Table S1), and complete gene invalidation was verified by targeted sequencing (Table S1). See also Figure S3.

Article Snippet: We used a rabbit IgG Isotype Control (Alexa Fluor 488 conjugate) (Cell Signaling #4340) as negative control (Isoctrl), a rabbit Alexa Fluor 488 coupled antibody against cleaved IL-1β (Cell Signaling – custom) and a mouse FITC anti-human IL-1β antibody (BioLegend #508206, clone JK1B-1) which recognizes both pro- and mature IL-1β.

Techniques: Centrifugation, Enzyme-linked Immunosorbent Assay, Clone Assay, Generated, Sequencing

Journal: bioRxiv

Article Title: The phosphate exporter XPR1 regulates a gasdermin D–independent mature IL-1β secretion pathway in LPS-stimulated human monocytic cells

doi: 10.64898/2025.12.23.695886

Figure Lengend Snippet: (A) Schematic overview of the CRISPR/Cas9 library screening strategy. The genome-wide human library Brunello, encoding Cas9 and 77,441 single guide (sg)RNAs, was packaged into lentiviruses produced by HEK293 cells and immediately used to transduce GSDMD -/- THP-1 cells. Three independent batches of THP-1 cells were infected on the same day with the same pool of viruses. After 19 days of culture, transduced cells were differentiated with PMA and stimulated with LPS for 24 h. After intracellular labeling of mature IL-1β, the cells of each batch displaying the highest staining were sorted by flow cytometry. Genomic DNA of sorted cells, together with control DNA from unselected transduced cells, was used as a template for sgRNA PCR amplification, followed by high-throughput sequencing. sgRNA abundance and enrichment were analyzed with MAGeCK. See also Dataset S1 and S2 and Figure S4. (B) Volcano plot showing gene enrichment and depletion in the screen. For each gene, the x axis shows its enrichment or depletion, calculated as the mean of all four sgRNAs targeting the gene, in the three sorted mIL-1β positive populations relative to the corresponding unsorted populations (paired analysis). The y axis shows statistical significance. Significantly enriched genes are highlighted in red. (C) Representative immunofluorescence images of WT, GSDMD -/- , and GSDMD -/- XPR1 -/- THP-1 cells differentiated with PMA and labeled with anti-XPR1 (green) and DAPI (blue). Magnification 63×; scale bar, 10 µm. (D) Cell viability following LPS stimulation of XPR1-invalidated THP-1 cells. GSDMD -/- XPR1 -/- THP-1 cells were differentiated with PMA and stimulated or not with LPS for 24 h. Metabolic activity was assessed with the MTS/PMS assay. As a positive control for cell death, pyroptosis was induced by addition of nigericin (Nig) (10 µM) 4 h after LPS stimulation. Data are represented as means ± SEM of biological triplicates. Data shown are representative of at least three independent experiments. The results shown for gene-invalidated cells in panels C and D were reproduced with two different clones generated with distinct gRNAs (Table S1), and complete gene invalidation was verified by targeted sequencing (Table S1).

Article Snippet: We used a rabbit IgG Isotype Control (Alexa Fluor 488 conjugate) (Cell Signaling #4340) as negative control (Isoctrl), a rabbit Alexa Fluor 488 coupled antibody against cleaved IL-1β (Cell Signaling – custom) and a mouse FITC anti-human IL-1β antibody (BioLegend #508206, clone JK1B-1) which recognizes both pro- and mature IL-1β.

Techniques: CRISPR, Library Screening, Genome Wide, Produced, Transduction, Infection, Labeling, Staining, Flow Cytometry, Control, Amplification, Next-Generation Sequencing, Immunofluorescence, Activity Assay, Positive Control, Clone Assay, Generated, Sequencing

Journal: bioRxiv

Article Title: The phosphate exporter XPR1 regulates a gasdermin D–independent mature IL-1β secretion pathway in LPS-stimulated human monocytic cells

doi: 10.64898/2025.12.23.695886

Figure Lengend Snippet: (A) Mature IL-1β secretion efficiencies by indicated gene-edited THP-1 cells after 24 h of LPS stimulation, measured and calculated as indicated in the legend of . Data represent three biological replicates from each of three independent experiments. Statistical analysis was performed using a nested one-way analysis of variance with Tukey’s test; ****, P ≤ 0.0001; all other comparisons were not significant. (B) Cell-surface expression of XPR1. Cells were labeled with an anti-XPR1 immunoadhesin ligand comprising the receptor–binding domain of the xenotropic murine leukemia virus envelope glycoprotein (XRBD) fused to mouse IgG1 Fc, followed by a PE-conjugated anti-mouse IgG1 secondary antibody. Results were reproduced with two different clones generated with distinct gRNAs (Tables S1 and S2). For each clone, complete gene invalidation was verified by targeted sequencing (Tables S1 and S2). See also Figure S5. (C) Mature IL-1β secretion efficiencies by the indicated gene-edited THP-1 cells after PMA-differentiation and 24 h of LPS stimulation in RPMI without phosphate (0 mM Pi) or supplemented with 5mM Pi, measured and calculated as described in the legend of . Data represent two biological replicates from each of three independent experiments. Statistical analysis was performed using a nested one-way analysis of variance with Tukey’s test; **, P ≤ 0.01; ns, nonsignificant; Pi, inorganic phosphate.

Article Snippet: We used a rabbit IgG Isotype Control (Alexa Fluor 488 conjugate) (Cell Signaling #4340) as negative control (Isoctrl), a rabbit Alexa Fluor 488 coupled antibody against cleaved IL-1β (Cell Signaling – custom) and a mouse FITC anti-human IL-1β antibody (BioLegend #508206, clone JK1B-1) which recognizes both pro- and mature IL-1β.

Techniques: Expressing, Labeling, Binding Assay, Virus, Clone Assay, Generated, Sequencing

Journal: bioRxiv

Article Title: The phosphate exporter XPR1 regulates a gasdermin D–independent mature IL-1β secretion pathway in LPS-stimulated human monocytic cells

doi: 10.64898/2025.12.23.695886

Figure Lengend Snippet: In GSDMD -/- THP-1 cells phosphate homeostasis is regulated by the phosphate importers PiT1 and PiT2 together with the phosphate exporter XPR1. Increased extracellular inorganic phosphate (Pi) is associated with elevated intracellular phosphate levels, which may promote the synthesis of inositol pyrophosphates, which in turn activate XPR1-dependent phosphate export. In GSDMD -/- XPR1 -/- THP-1 cells, phosphate export is impaired, leading to intracellular phosphate accumulation and potentially increased levels of inositol pyrophosphates. Under these conditions, the secretion of mature IL-1β is inhibited. Under phosphate-deprivation conditions, intracellular phosphate levels are expected to decrease, which may lower inositol pyrophosphates levels and is associated with an increase of mature IL-1β secretion.

Article Snippet: We used a rabbit IgG Isotype Control (Alexa Fluor 488 conjugate) (Cell Signaling #4340) as negative control (Isoctrl), a rabbit Alexa Fluor 488 coupled antibody against cleaved IL-1β (Cell Signaling – custom) and a mouse FITC anti-human IL-1β antibody (BioLegend #508206, clone JK1B-1) which recognizes both pro- and mature IL-1β.

Techniques: