Journal: bioRxiv

Article Title: Th17 effector cytokines induce shared and distinct microglial and endothelial cell responses in post-streptococcal encephalitis

doi: 10.64898/2026.02.04.703836

Figure Lengend Snippet: a–c, Proposed model illustrating how effector cytokines produced by infiltrating Th1 and Th17 lymphocytes following repeated GAS infections may differentially shape transcriptional responses in brain endothelial cells (BECs) and microglia. a, IFNγ is predicted to contribute to the induction of interferon-response programs and antigen-presentation pathways in both microglia and BECs. b,c, IL-17A and GM-CSF appear to exert partially overlapping effects on microglial activation, including regulation of proliferative responses and induction of disease-associated microglial (DAM) genes, cytokines, and chemokines, while also displaying cytokine-specific contributions. Notably, IL-17A signaling may more strongly influence endothelial transcriptional alterations following GAS infections. In addition, antigen-presentation signatures remain elevated in conditions of Th17 deficiency or systemic IL-17A neutralization, suggesting that IL-17A may normally modulate these pathways in both microglia and BECs. Together, these models highlight shared and distinct roles for Th17-associated cytokines in shaping neuroimmune and vascular responses after recurrent GAS exposure.

Article Snippet: Starting 24 hours prior to the first GAS infection, mice were injected intraperitoneally twice weekly with 500 μg of either InVivoMAb anti-mouse IL-17A monoclonal antibody, clone 17F3 (Bio X Cell catalog, BE0173), or mouse IgG1 isotype control monoclonal antibody, clone MOPC-21 (Bio X Cell, catalog BE0083), in 100 μL of dilution buffer (Bio X Cell catalog IP0070 and IP0065, respectively).

Techniques: Produced, Immunopeptidomics, Activation Assay, Neutralization

Journal: bioRxiv

Article Title: Th17 effector cytokines induce shared and distinct microglial and endothelial cell responses in post-streptococcal encephalitis

doi: 10.64898/2026.02.04.703836

Figure Lengend Snippet: a, Timeline of recurrent intranasal GAS infections and administration of an α–IL-17A–neutralizing monoclonal antibody (mAb) or isotype control. b, Heat maps of differentially expressed genes (DEGs) related to BBB function, LPS response, interferon signaling, and antigen presentation in olfactory bulb (OB) brain endothelial cells (BECs) from GAS-infected mice treated with isotype control or α–IL-17A mAb. Values are shown as log(z-score); significantly altered genes are indicated in black (adjusted p < 0.05). c, Gene ontology (GO) pathway enrichment analysis of transcripts upregulated and downregulated in α–IL-17A mAb–treated versus isotype-treated BECs following GAS infections. d, Representative immunofluorescence (IF) images of endogenous IgG leakage (green) in the granular layer of the OB. Blood vessels are labeled with Glut1 (cyan). e, f, Quantification of IgG extravasation (relative fluorescence intensity) in the glomerular ( e ) and granular ( f ) OB layers from PBS- or GAS-infected mice treated with α–IL-17A mAb or isotype control. Comparisons were performed using two-way ANOVA with Šídák’s multiple comparisons test ( p < 0.05; ** p < 0.001; n = 3–7 mice per group). g–i, Representative IF images for IFITM3 (pink), Iba1 (yellow), and CD31 (blue) in the OB of GAS-infected mice treated with isotype or α–IL-17A mAb ( g,h ), and quantification of Ifitm3-positive area within CD31⁺ vasculature ( i ). j,k, Representative fluorescence in situ hybridization (FISH) images of Itm2a mRNA (pink) combined with Glut1 (blue) in the glomerular layer ( j ) and quantification of vascular Itm2a signal ( k ). l, Representative IF images of the tight junction proteins Claudin-5 (green) and ZO-1 (red) in the OB vasculature of GAS-infected mice treated with isotype or α–IL-17A mAb. m, Serum cytokine concentrations measured by multiplex immunoassay in PBS- or GAS-infected mice treated with isotype or α–IL-17A mAb. Data are mean ± SEM. Comparisons were performed using one-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05; p < 0.05; * p < 0.01; ** p < 0.001; *** p < 0.0001; n = 3 - 6 mice per group). n, Survival curves of GAS-infected mice treated with isotype control or α–IL-17A mAb (n = 13–48 mice per group). Statistical significance was assessed using the Mantel–Cox log-rank test.

Article Snippet: Starting 24 hours prior to the first GAS infection, mice were injected intraperitoneally twice weekly with 500 μg of either InVivoMAb anti-mouse IL-17A monoclonal antibody, clone 17F3 (Bio X Cell catalog, BE0173), or mouse IgG1 isotype control monoclonal antibody, clone MOPC-21 (Bio X Cell, catalog BE0083), in 100 μL of dilution buffer (Bio X Cell catalog IP0070 and IP0065, respectively).

Techniques: Control, Immunopeptidomics, Olfactory, Infection, Immunofluorescence, Labeling, Fluorescence, In Situ Hybridization, Multiplex Assay

Journal: The Journal of Clinical Investigation

Article Title: Pharmacological targeting of the IL-17/neutrophil axis attenuates calcific deposits in rat models of calciphylaxis

doi: 10.1172/JCI190369

Figure Lengend Snippet: ( A ) Immunofluorescence staining and quantification of IL-17a + cells in skin tissue from rats treated with DHT p.o. + FeCl 3 s.c. or vehicle p.o. + H 2 O s.c. (representative images; n = 5 animals per group). Scale bar: 50 μm. ( B ) Schematic of the IL-17a mAb experimental timeline. ( C ) Gross images of dorsal skin lesions. Black arrowheads show visible regions of calcification. ( D ) Representative von Kossa–stained images of rat dermal tissue highlighting calcified regions (arrowheads). Quantification of calcified percentage in the IL-17a mAb–treated group ( n = 12) compared with IgG control ( n = 8). ( E ) Top 5 KEGG pathways enriched in DEGs between IgG-treated ( n = 3 animals) and IL-17a mAb–treated rats following DHT p.o. + FeCl 3 s.c. ( n = 3 animals). ( F ) Enriched genes in IL-17 signaling pathway. Data are represented as mean ± SEM. *** P < 0.001, 2-tailed Student’s t test.

Article Snippet: To determine the specific role of IL-17 in mediating calcific deposits, we injected the animals with a monoclonal antibody targeting IL-17a (IL-17a mAb, Bio X Cell, BP0173).

Techniques: Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) qPCR of Pla2g12a in various tissues of 10-week-old C57BL/6 mice. (B) Procedure used for flow cytometry of CD4 + -gated TCRβ + IL-17A + Th17 cells from ex vivo Th17 differentiation culture. (C) qPCR of various PLA 2 enzymes in splenic CD4 + T cells cultured for 3 days under Th0 and Th17 differentiation conditions ( n = 3). (D) Strategy for gene targeting of Pla2g12a . Positions of PCR primers for genotyping are indicated by arrows. (E) PCR genotyping of Pla2g12a +/+ (+/+), Pla2g12a +/– (+/–), and Pla2g12a -/- (–/–) mice. (F) qPCR of Pla2g12a in the skin and spleen of Pla2g12a +/+ and Pla2g12a -/- mice. Values are mean ± s.e.m.. Representative data of two experiments (C) and combined results of two experiments (A, F) are shown. Statistical analysis was performed by Mann-Whitney U test (B, F, G). **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Flow Cytometry, Ex Vivo, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for ex vivo culture of splenic naïve CD4 + T cells with (Th17) or without (Th0) IL-1β, IL-6, IL-23 and TGF-β on anti-CD3ε/CD28-coated plates. (B) qPCR of Pla2g12a in splenic CD4 + T cells cultured for various periods under Th0 and Th17 differentiation conditions. (C) FACS of Th17 (CD4 + -gated TCRβ + IL-17A + ) cells after culture of naïve T cells prepared from Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion and number of Th17 cells ( right ) are shown. (D-F) Scatter plots (D) and heatmap (E) of genes expressed in CD4 + T cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days under Th0 and Th17 differentiation conditions. Cells from 6-7 mice of each genotype were pooled and subjected to microarray analysis. Colors in (E) show the z-score. (F) Pathway enrichment analysis of genes decreased (>2-fold) in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. (G) Heatmap of Th17-related genes in Th17 cells from Pla2g12a -/- mice relative to those from Pla2g12a +/+ mice. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (H) qPCR of Th17 signature genes in Th0 and Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice. (I) Heatmap of lipid-related genes in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (J) FACS of non-pathogenic Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days with IL-6 and TGF-β. Values are mean ± s.e.m.. Representative data of three experiments (C) and results of one experiment (B, D–I) are shown. Statistical analysis was performed by one way ANOVA with Tukey’s multiple comparisons test (C, J), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (H), and two-way ANOVA with Sidak’s multiple comparisons test (B). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Ex Vivo, Cell Culture, Microarray

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for IMQ-induced psoriasis. (B) qPCR of Pla2g12a in the LNs of WT mice after IMQ challenge over 3 days. (C) IMQ-induced ear swelling in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice over 6 days ( n = 6–18). (D, E) HE staining of ear skin sections (D) and quantification of epidermal and dermal thickness (E) in Pla2g12a +/+ and Pla2g12a -/- mice ( n = 35–44) (E). Scale bar, 100 μm. (F) qPCR of Il17a in the LNs and skin of Pla2g12a +/+ and Pla2g12a -/- mice. (G) FACS of Th17 (CD3ε + -gated TCRβ + IL-17A + ) cells in the spleen of Pla2g12a +/+ and Pla2g12a -/- mice on day 6. Representative FACS profiles ( left ) and the proportion and number of Th17 cells ( right ) are shown. (H, I) FACS of Th17 (CD45 + -gated TCRβ + IL-17A + ) cells (H) and γδ T (CD45 + -gated TCRγδ + IL-17A + ) cells (I) in the skin on day 6. Representative FACS profiles ( left ) and the proportions and numbers of Th17 cells (H) and γδ T cells (I) ( right ) are shown. Values are mean ± s.e.m.. Representative data of two (C, F) or four (G) experiments, results from one experiment (H, I), and combined results of two experiments (B, D, E) are shown. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (B), one-way ANOVA with Tukey’s multiple comparisons test (G), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (H, I), Mixed-effects model with Sidak’s multiple comparisons test (C), and two-way ANOVA with Sidak’s multiple comparisons test (E, F). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Staining

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A-C) qPCR of cytokines (A, B) and keratinocyte markers (C) in the LNs (A) and skin (B, C) of Pla2g12a +/+ and Pla2g12a -/- mice. (D, E) Procedures used for flow cytometry of CD3ε + -gated TCRβ + IL-17A + Th17 cells from the spleen (D) and CD45 + -gated TCRβ + IL-17A + Th17 cells and TCRγδ + IL-17A + γδ T cells from the skin (E). Values are mean ± s.e.m.. Results of one experiment (A, B) and combined results of two experiments (C) are shown. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (A–C). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) qPCR of Pla2g12a in the whole spleen and isolated CD4 - and CD4 + cells of Pla2g12a fl/fl (fl/fl) and Pla2g12a fl/fl Cd4 cre (T-KO) mice. (B) FACS of Th17 cells after culture of naïve T cells from fl/fl and T-KO mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (C) IMQ-induced ear swelling in fl/fl and T-KO mice over 6 days ( n = 20–30). (D) HE staining of ear skin sections from IMQ-treated fl/fl and T-KO mice on day 6. Scale bar, 100 μm. (E, F) FACS of Th17 cells in the spleen (E) and Th17 and γδ T cells in the skin (F) of IMQ-treated fl/fl and T-KO mice on day 6. (G) qPCR of Il17a in the skin of IMQ-treated fl/fl and T-KO mice on day 6. (H) qPCR of Pla2g12a in the whole LNs and isolated CD45 + hematopoietic cells and CD140a + fibroblasts of fl/fl and Pla2g12a fl/fl Col1a2 cre (F-KO) mice. (I) FACS of Th17 cells after culture of naïve T cells from fl/fl and F-KO mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (J) IMQ-induced ear swelling in fl/fl and F-KO mice over 6 days ( n = 4–6). (K) FACS of splenic Th17 cells in IMQ-treated fl/fl and F-KO mice on day 6. A representative histogram ( left ) and the number of Th17 cells ( right ) are shown. Values are mean ± s.e.m.. Representative data of two (A, F) or three (E) experiments, results of one experiment (B, I–K), and combined results of two (C, G, H) are shown. Statistical analysis was performed using unpaired t test (G, K), Mann-Whitney test (A, E, F, H), one-way ANOVA with Tukey’s multiple comparisons test (B, I), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (C, J). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Isolation, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure used for BM transfer. (B) IMQ-induced psoriasis in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice adoptively transferred with BM cells from Pla2g12a +/+ or Pla2g12a -/- mice ( n = 8-18). (C, D) FACS of cutaneous Th17 cells (C) and γδ T cells (D) on day 5 in (B). Representative FACS profiles ( left ) and the proportion and number of Th17 (C) or γδ T (D) cells ( right ) are shown. Values are mean ± s.e.m.. Representative data of two experiments (B) and results of one experiment (C, D) are shown. Statistical analysis was performed using one-way ANOVA Dunnett’s multiple test (C, D) and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (B). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques:

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for IMQ-induced psoriasis. (B, C) Hindlimb swelling ( n = 8–18) (B) and clinical score (n = 4–9) (C) in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice over 42 days after immunization with type II collagen. (D) Representative photos of the hindlimbs of Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. (E) HE and TRAP staining of the knee joints from Pla2g12a +/+ and Pla2g12a -/- mice with (+) or without (–) CIA on day 42. Yellow arrowheads, bone erosion; white arrowheads, inflammatory cell infiltration. Scale bar, 100 µm. (F, G) μCT analysis of the hindlimb bone from Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. Yellow arrowheads, bone erosion; white arrowheads, cortical bone; asterisk, trabecular bone. Scale bar, 1 mm. Representative μCT images (F) and tissue mineral density of the cortical bone (G) are shown. (H) FACS of splenic Th17 cells in Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (I, J) ELISA of IL-17A (I) and anti-type II collagen (J) in sera of Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. Values are mean ± s.e.m.. Representative data of three (B, C, G) or two (H) experiments and combined results of two experiments (I, J) are shown. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test (G, H), Kruskal-Wallis with Dunn’s multiple comparisons test (I, J), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (B, C). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure used for K/BxN serum transfer arthritis. (B, C) Ankle swelling (B) and arthritis score (C) in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice over 11 days. (D) qPCR of Pla2g12a and arthritis-related genes in the joints of Pla2g12a +/+ and Pla2g12a -/- mice on day 4 in (B, C). Values are mean ± s.e.m. Combined results of two experiments are shown (B–D). Statistical analysis was performed using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (D) and two-way ANOVA with Tukey’s multiple comparisons test (B, C). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques:

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: Skin-extracted phospholipids were incubated with various concentrations of recombinant human PLA2G12A for 30 min and the generation of lysophospholipids (A) and free fatty acids (B) was analyzed by LC-ESI-MS/MS ( n = 6). Values are mean ± s.e.m.. Combined results of two experiments are shown. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; **, P < 0.01 versus without PLA2G12A.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Incubation, Recombinant, Tandem Mass Spectroscopy

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A, B) Lipidomics analysis of various lysophospholipids in the LNs of Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice with (+) or without (–) IMQ treatment for 1 day. Heatmap of individual lysophospholipids, with their levels in Pla2g12a +/+ mice without IMQ treatment as 1 (A), and their quantified values (pmol per mg tissue) (B) are shown. (C, D) FACS of Th17 cells after culture of Pla2g12a +/+ and Pla2g12a -/- naïve T cells for 3 days with various concentrations of LPE(1-18:1) (C) or various lysophospholipids at 1 μM (D). (E) FACS of Th17 cells after culture of Pla2g12a +/+ and Pla2g12a -/- naïve T cells for 3 days with the ATX inhibitor PF-8380 at 1 nM in the presence or absence of LPE(1-18:1). (F) qPCR of Enpp2 in Th0 and Th17 cells of Pla2g12a +/+ and Pla2g12a -/- mice on day 3. (G) qPCR of LPA receptors in Th0 and Th17 cells after culture for 3 days. (H) A list of LPA receptor agonists used in (I). (I) FACS of Th17 cells after culture of Pla2g12a +/+ and Pla2g12a -/- naïve T cells for 3 days with various LPA receptor agonists at 10–100 nM. (J–L) FACS of Th17 cells after culture of naïve T cells prepared from Lpar1 +/+ and Lpar1 -/- mice (J), Lpar2 +/+ and Lpar2 -/- mice (K), and Lpar6 +/+ and Lpar6 -/- mice (L) for 3 days under Th0 and Th17 differentiation conditions. (M) A schematic illustration of LPA 1 and LPA 2 signaling. Both LPA 1 and LPA 2 can be coupled with G 12/13 , G q , and G i signaling. (N) FACS analysis of Th17 cells after culture of WT naïve T cells for 3 days in the presence of inhibitors for G 12/13 , G q , G i and G s signaling. (O) Immunoblotting of STAT3, p-STAT3, and β-actin. Representative immunoblots ( upper ) and quantification of the p-STAT3/STAT3 ratio ( lower ) are shown. Values are mean ± s.e.m.. A result of one experiment (L), representative data of two experiments (A–G, J, K, N, O), and combined results of two experiments (I) are shown. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test (E, L), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (C, D, F, G, I–K, L), and two-way ANOVA with Sidak’s multiple comparisons test (B). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) LC-ESI-MS/MS of PUFA metabolites in the LNs of Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice with (+) or without (–) IMQ treatment for 1 day. (B) LC-ESI-MS/MS of lysophospholipids in the skin of Pla2g12a +/+ and Pla2g12a -/- mice treated for the indicated periods with IMQ. Values are mean ± s.e.m.. Results from one (A) or two (B) experiments are shown. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (A) and two-way ANOVA with Sidak’s multiple comparisons test (B).

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Tandem Mass Spectroscopy

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) FACS of Th17 cells after culture of naïve T cells for 3 days in the presence of the indicated concentrations of the LPA 1/3 antagonist Ki16425. (B) qPCR of Lpar1 in Th0 and Th17 cells after culture for the indicated periods. (C) qPCR of Lpar1 in the LNs of Lpar1 +/+ and Lpar1 -/- mice. (D, E) IMQ-induced ear swelling over 6 days ( n = 8–12) (D) and ear histology on day 6 (E) of Lpar1 +/+ and Lpar1 -/- mice. (F, G) IMQ-induced ear swelling over 6 days ( n = 6) (F) and the number of splenic Th17 cells (G) of Lpar1 +/+ mice that had been transferred with Lpar1 +/+ or Lpar1 -/- BM cells. (H, I) FACS of splenic CDε+ T cells in Lpar1 +/+ mice that had been transferred with Lpar1 +/+ or Lpar1 -/- BM cells (H) or in Pla2g12a +/+ mice that had been transferred with Pla2g12a +/+ or Pla2g12a -/- BM cells (I). Representative FACS profiles ( left ) and the proportion of CD3ε + T cells ( right ) are shown. (J) FACS of CD4 + Th0 cells from Pla2g12a -/- , Lpar1 -/- , and Lpar2 -/- mice in comparison with those from respective control mice. Representative FACS profiles ( upper ) and the proportion of CD4 + T cells ( lower ) are shown. (K) FACS of splenic CD4 + -gated CD44 - CD62L + naïve T cells and CD44 + CD62L - effector memory T cells in Pla2g12a -/- , Lpar1 -/- , and Lpar2 -/- mice in comparison with WT mice. Values are mean ± s.e.m.. Representative data of two experiments (B, J) and results of one experiment (A, C–I, K) are shown. Statistical analysis was performed using unpaired t test (C, I, J), Mann-Whitney U test (G, H), ordinary one-way ANOVA with Dunnett’s multiple comparisons test (A, K), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (B), and two-way ANOVA with Sidak’s multiple comparisons test (D, F). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Comparison, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A, B) Secretion of EVs from Th0 and Th17 cells prepared from WT, Pla2g12a -/- , Lpar1 -/- , and Lpar2 -/- mice after culture for 2 days, as evaluated by protein amounts (μg/ml of culture medium) (A) and counts of CD9 + CD63 + EVs (B). In (B), EVs from six mice were pooled and subjected to the analysis. (C, D) TEM (C) and cryo-EM (D) of EVs secreted from Pla2g12a +/+ and Pla2g12a -/- Th17 cells before or after treatment with PLA2G12A. (E) Diameters of Th17-derived EVs from Pla2g12a +/+ and Pla2g12a -/- mice before or after treatment with PLA2G12A. (F) Heatmap of exosome-related genes in Th0 and Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice, with expression levels (fold values) in Pla2g12a +/+ -derived Th0 cells regarded as 1. (G) Phospholipid composition in Th17-derived EVs from Pla2g12a +/+ and Pla2g12a -/- mice. Values are mean ± s.e.m.. Representative data of two experiments (C, G) and results of one experiment (A, B, D–F) are shown. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (A) and Kruskal-Wallis test with Dunn’s multiple comparisons test (E). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Cryo-EM Sample Prep, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Lysophospholipid levels in EVs isolated from Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) Th17 cells after culture for 3 days. (B) LPE and LPC levels after incubation of Pla2g12a +/+ Th17-derived EVs (+/+ EVs) for 4 h with the indicated concentrations of recombinant mouse PLA2G12A. (C) FACS of Th17 cells after culture of Pla2g12a +/+ naïve T cells for 3 days in the presence or absence of +/+ EVs that had been treated with or without 1 μg/ml PLA2G12A. (D) FACS of Th17 cells after culture of naïve T cells from Pla2g12a +/+ and Pla2g12a -/- mice for 3 days in the presence or absence of +/+ EVs or Pla2g12a -/- Th17-derived EVs (–/– EVs). (E) FACS of Th17 cells after culture of naïve T cells from Pla2g12a +/+ and Pla2g12a -/- mice for 3 days in the presence or absence of –/– EVs that had been treated with or without the indicated concentrations of PLA2G12A. (F, G) Immunofluorescence microscopy of WT and Pla2g12a -/- Th17 cells cultured for 4 h with APC-labelled +/+ EVs or –/– EVs. Representative images (F) and the ratio of EV-captured (APC + ) cells to all (DAPI + ) cells (G) are shown. Scale bar, 50 µm. (H) Heatmap of representative miRNAs whose levels were altered in –/– EVs relative to +/+ EVs. Colors indicate fold changes in –/– EVs relative to +/+ EVs. (I–K) Proteome analysis of EVs. Volcano plot (I), pathway enrichment analysis (J), and heatmap (K) of representative proteins whose levels were altered in +/+ EVs relative to –/– EVs. Colors in (K) indicate fold changes in –/– EVs relative to +/+ EVs. Values are mean ± s.e.m.. Representative data of two experiments (A) and results of one experiment (B–K) are shown. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (A, B) and ordinary one-way ANOVA with Tukey’s multiple comparisons test (C-E, G). *, P < 0.05; **, P < 0.01.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Isolation, Incubation, Derivative Assay, Recombinant, Immunofluorescence, Microscopy, Cell Culture

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure used for antibody treatment in IMQ-induced psoriasis. (B) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A, -IL-17A, or control IgG 1 antibody over 6 days. Antibody treatment started on day 0 (arrow). (C) FACS of splenic Th17 cells in (B) on day 6. (D) IMQ-induced ear swelling in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice treated with anti-PLA2G12A or control antibody over 6 days. Antibody treatment started on day 0 (arrow). (E) FACS of splenic Th17 cells in (D) on day 6. (F) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A or control antibody. Antibody treatment started on day 3 (arrow). (G) FACS of splenic Th17 cells in (F) on day 6. (H) Schematic representation of the procedure used for antibody treatment in CIA. (I, J) Paw thickness (I) and clinical sore (J) in WT mice treated with anti-PLA2G12A, - IL-17A, or control antibody over 42 days. (K) FACS of splenic Th17 cells in (I, J) on day 42. (L) Schematic diagram showing PLA2G12A-driven regulation of Th17 differentiation. PLA2G12A is induced in CD4 + T cells after TCR stimulation and is also supplied from stromal fibroblasts. EV secretion is facilitated by Th17 differentiation driven by Th17-inducing cytokines. PLA2G12A hydrolyzes PE and PC to generate LPE and LPC in EV membranes. The EVs are taken up by Th17-differentiating cells and deliver LPE(1-18:1) intracellularly, which then transactivates RORγt. LPE and LPC are converted to LPA by ATX, which is supplied from Th17 cells, stromal cells ( e.g ., fibroblasts and endothelial cells), and serum. LPA acts mainly on LPA 2 (and to a lesser extent LPA 1 ) on Th17-differentiating cells to augment IL-6/IL23-mediated STAT3 phosphorylation via G 12/13 -Rho-ROCK2 signaling, assisting pathogenic Th17 differentiation in autocrine and paracrine fashions. Gene disruption of PLA2G12A dampens this lipid-orchestrated amplification loop and decreases EV secretion, uptake, and transfer of lipid, miRNA, and protein cargos. Accordingly, antibody-mediated neutralization of PLA2G12A efficiently attenuates psoriasis, arthritis, and probably other Th17-related pathologies such as EAE. Values are mean ± s.e.m.. Results are from one experiment (B–G, I–K). Statistical analysis was performed using unpaired t test (G), ordinary one-way ANOVA with Dunnett’s multiple comparisons test (C, E, K), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (B, D, F, I, J). *, P < 0.05; **, P < 0.01 versus control IgG 1 .

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Control, Phospho-proteomics, Disruption, Amplification, Neutralization

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Scatter plots of various biotypes of RNA, including protein-coding (messenger) RNAs, miRNAs, small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs (scaRNAs), ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs), and miscellaneous RNAs (misc_RNAs) in Th17-derived EVs from Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice. (B) Abundances of individual RNA biotypes in Th17-derived EVs from Pla2g12a -/- mice (–/– EVs) relative to those from Pla2g12a +/+ mice (+/+ EVs). (C) Heatmap of RNA biotypes whose levels were altered in Th17-derived EVs from Pla2g12a -/- mice relative to those from Pla2g12a +/+ mice. Colors indicate fold changes in –/– EVs relative to +/+ EVs. One experimental data set, in which EVs from six mice were pooled, is shown.

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) PLA 2 enzyme assay using human and mouse PLA2G12A proteins after incubation with several anti-PLA2G12A antibody clones (100 ng/ml), with the activity in the presence of a control antibody being regarded as 1. *, P < 0.05; **, P < 0.01 versus control. (B) FACS of Th17 cells after culture of WT naïve T cells for 3 days with several anti-PLA2G12A antibody clones (100 ng/ml). *, P < 0.05; **, P < 0.01 versus control. (C) Immunoblotting of human and mouse PLA2G12As and human PLA2G3 (50 ng/lane for each) with an anti-PLA2G12A antibody (clone #44). (D) ELISA of various sPLA 2 s with an anti-PLA2G12A antibody (clone #44). hIIA, human PLA2G2A; hIID, human PLA2G2D; hIIE, human PLA2G2E; hIIF, human PLA2G2F; hIII, human PLA2G3; hV, human PLA2G5; hX, human PLA2G10; hXIIA, human PLA2G12A; mXIIA, mouse PLA2G12A. *, P < 0.05; **, P < 0.01. (E) Immunohistochemistry of mouse skin sections with or without IMQ treatment with an anti-PLA2G12A antibody (clone #44). Red, PLA2G12A; green, CD4 + T cells. Scale bar, 100 µm. Values are mean ± s.e.m.. Results of one experiment is shown (A–D). Statistical analysis was performed using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (A) and ordinary one-way ANOVA with Dunnett’s multiple comparisons test (B, D).

Article Snippet: In the IMQ-induced psoriasis model, mouse anti-PLA2G12A monoclonal antibody (clone #44), InVivoMAb anti-mouse IL-17A antibody (clone 17F3; Bio X Cell), or GoInVivo purified mouse IgG 1 , κ isotype control antibody (BioLegend) was administrated intraperitoneally at 1 mg/kg every day from day 0 to day 5 of IMQ application (Figure 8A).

Techniques: Enzymatic Assay, Incubation, Clone Assay, Activity Assay, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry