Journal: Science Advances

Article Title: Galectin-3 exacerbates autoimmune diabetes by limiting regulatory T cell differentiation and function

doi: 10.1126/sciadv.adz7916

Figure Lengend Snippet: ( A ) Schematic diagram showing the protocol of TD139 treatment. ( B ) Incidence of diabetes expressed as percentage of diabetic mice at different ages ( n = 20). ( C ) Glucose excursion curve after receiving IPGTT ( n = 8). ( D ) Serum insulin concentration at 0 and 15 min during the IPGTT in 12-week-old TD139 or vehicle-treated NOD mice ( n = 7 to 8). ( E ) Representative H&E staining of pancreas from 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). ( F ) Insulitis scores calculated on the basis of histological evaluation of pancreatic section as in (E). ( G ) Frequencies of CD4 + and CD8 + T cells in total CD3 + T cells from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). ( H ) Frequencies of T reg cells (CD4 + CD25 + FOXP3 + ) from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). ( I to K ) Frequencies of perforin + CD8 + T, Tc1 (CD8 + IFN-γ + ), Tc2 (CD8 + IL-4 + ), Tc17 (CD8 + IL-17 + ), and granzyme B + CD8 + T subsets from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 3 to 4). ( L ) Frequencies of T H 1 (CD4 + IFN-γ + ), T H 2 (CD4 + IL-4 + ), and T H 17 (CD4 + IL-17 + ) from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01.

Article Snippet: Our findings suggest a promising avenue for potential therapeutic targets of T1D and introduce the Galectin-3 inhibitor TD139, as a potent antidiabetic drug, to prevent and treat T1D.

Techniques: Concentration Assay, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 1. Identification of galectin-3 as an inflammasome-regulated secretory protein. (A) Diagram of secreted proteins in CM from LPS/ATP–treated WT and Nlrp32/2 BMDMs. (B) Two-dimensional PAGE analysis of supernatant from WT and Nlrp3+/R258W BMDMs treated with 500 ng/ml LPS for 4 h. The labeled dots were dug out for MS analysis. (C) Protein list of identified proteins from (B) by MS. The blue-labeled proteins are at least 2-fold lower in Nlrp32/2 CM than that in WT CM of (A). p # 0.05. (D) Volcano plot of secreted proteins in (A). The pink circles represent individual protein at least 2-fold higher or lower in WT CM than that in Nlrp32/2 CM, with p # 0.05. (E) ELISA detection of serum galectin-3 level in WT mice (n = 5), which received i.p. injection of Ac-YVAD (20 mg/kg) or vehicle daily for 3 d. Data are shown as mean 6 SEM. ***p # 0.001. (F) ELISA detection of serum galectin-3 level in WT and Casp1/112/2 mice (n = 4). Data are shown as mean 6 SEM. ***p # 0.001.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 2. The inflammasome activation drives cellular and systemic secretion of galectin-3. (A) BMDMs from WT or Nlrp3+/R258W mice were stimulated with 500 ng/ml LPS for the indicated times; supernatant (SUP) and whole cell lysate (WCL) were collected for immunoblotting of the indi- cated proteins. Data are representative of at least three independent experiments. (B) LPS-primed WT BMDMs were treated with ATP (5 mM) or nigericin (5 mg/ml) for 30 min, and immunoblotting detection of indicated proteins is shown. Data are representative of at least three independent experiments. (C) LPS-primed peritoneal macrophages were stimulated with ATP (5 mM) for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least two independent experiments. (D) LPS-primed BMDC were stimulated with ATP (5 mM) for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least three independent experiments. (E) BMDM were primed with LPS for 10 min, followed by ATP (5 mM) challenge for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least three in- dependent experiments. (F) Western blotting analysis of SUP and WCL from WT, Nlrp32/2, Asc2/2, or Casp1/112/2 BMDMs stimulated with LPS/ATP. Data are representative of at least three independent experiments. (G) Western blotting analysis of SUP and WCL from LPS-primed THP-1 macrophages stimulated with ATP. Data are representative of at least three independent experiments. (H) Western blotting analysis of SUP and WCL from unprimed WT or Aim22/2 BMDMs transfected with poly(dA:dT) for 2 h. Data are representative of at least two independent experiments. (I) Western blotting analysis of SUP and WCL from unprimed WT or Nlrc42/2 BMDMs infected with S. typhimurium (multiplicity of infection = 10) for 3 h. Data are representative of at least two independent experiments. (J) Western blotting analysis of SUP and WCL from unprimed THP-1 macrophages transfected with poly(dA:dT) for 4 h. Data are representative of at least three independent experiments. (K and L) ELISA analysis of serum galectin-3 (K) and IL-18 (L) level in WT and Nlrp32/2, Aim22/2, Nlrc42/2, and Asc2/2 mice (n = 4); the WT group is the same one used in Fig. 1F. Data are shown as mean 6 SEM. ns: p . 0.05, **p # 0.01, ****p , 0.0001.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Activation Assay, Western Blot, Transfection, Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 3. The inflammasome-driven galectin-3 secretion bypasses the exosome pathway. (A) Transmission electron microscope imaging of exosomes isolated from supernatants of LPS/ATP–stimulated WT BMDMs. (B) Immunoblot detection of galectin-3 in whole cell lysate (WCL), supernatant (SUP), and exosomes (EXO) from LPS/ATP–stimulated BMDMs. The proteins in SUP group were precipitated from 0.5 ml of medium, the proteins in EXO group were enriched from 24.5 of ml medium. Data are representative of at least three independent experiments. (C) Western blotting analysis of trypsin (0.05%)- digested exosomes at room temperature for 10 min. The exosome was pretreated with 1% Triton X-100 for 20 min at 4˚C or not. Data are representative of at least three independent experiments. (D) Western blotting analysis of SUP from LPS/ATP–stimulated BMDMs. The SUP was pretreated with 50 mg/ml of proteinase K at 4˚C for 30 min or not. Data are representative of at least two independent experiments. (E) ELISA quantification of galectin-3, IL-1b, and TNF-a in SUP or EXO from LPS/ATP–stimulated BMDMs. The exosomes were pretreated with 1% Triton X-100 to release their contents. Data are representative of at least three independent experiments. **p , 0.01, ***p , 0.001, ****p , 0.0001. (F) Western blotting analysis of SUP and WCL from J774 cells stimulated with LPS and ATP is shown. Data are representative of at least three independent experiments. (G) Immunoblot detection of galectin-3 in SUP or EXO from LPS/ATP–treated shAlix J774 cells. LE, long-time exposure; SE, short-time exposure. Data are representative of at least three in- dependent experiments.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Transmission Assay, Microscopy, Imaging, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 4. Inflammasome-activated GSDMD drives galectin-3 secretion. (A) LPS-primed WT or Gsdmd2/2 BMDMs were stimulated with ATP. Su- pernatant (SUP) and whole cell lysate (WCL) were collected for immunoblotting of indicated proteins. Data are representative of at least three independent experiments. (B and C) Pam3CSK4 (1 mg per 1 3 106 cells) or LPS (1 mg per 1 3 106 cells) was electroporated into WT and Gsdmd2/2 BMDMs (B) or J774 cells (C). Cells were incubated for 30 min, followed by washout of dead cells, then the cells were incubated for another 60 min. SUP was collected and subjected to immunoblotting detection of galectin-3. Data are representative of at least two independent experiments. (D) Gsdmd2/2 BMDMs were reconstituted with vector, GSDMD (WT or uncleavable D276A mutant), followed by LPS priming and ATP pulse. Western blotting analysis of indi- cated proteins from SUP and WCL is shown. Data are representative of at least three independent experiments. (E) HEK293 cells were transfected with V5–galectin-3 and GSDMD-expressing plasmids. SUP and WCL were collected for immunoblotting of indicated proteins. Data are representative of at least three independent experiments. (F and G) ELISA detection of serum galectin-3 (F) and IL-18 (G) level in WT and Gsdmd2/2 mice (n = 5) is shown. Data are shown as mean 6 SEM. **p , 0.01, ***p , 0.001. FL-GSDMD, full-length GSDMD; N-GSDMDE15K/L193D, N-terminal GSDMD with Glu15 and Lys193 mutated to lysine and aspartic acid, respectively.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Western Blot, Incubation, Plasmid Preparation, Mutagenesis, Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 5. GSDMD perforates the plasma membrane to enable galectin-3 secretion. (A and B) Primed WT BMDMs were stimulated with ATP in the presence or absence of glycine (10 mM) for indicated times. Immunoblotting (A), and LDH release assay (B) of supernatant (SUP) and/or whole cell lysate (WCL) are shown. Data are representa- tive of at least three independent experiments. ****p , 0.0001. (C) HEK293 cells were transfected with mouse V5–galectin-3 and N-GSDMD–expressing plasmids, fol- lowed by incubation with/without glycine for 12 h. Data are representative of at least three independent experi- ments. (D and E) Primed BMDMs were pretreated with PUN for 10 min, followed by ATP challenge. Immuno- blot (D), and LDH release assay (E) of SUP and/or WCL are shown. The MOCK and LPS/ATP group are the same ones used in (B). Data are representative of at least three independent experiments. ****p , 0.0001. (F and G) LPS-primed BMDMs were transfected with poly(dA:dT) for 2 h in the presence of 2 mM Ca2+ or Mg2+

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Clinical Proteomics, Membrane, Western Blot, Lactate Dehydrogenase Assay, Transfection, Expressing, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 1. Identification of galectin-3 as an inflammasome-regulated secretory protein. (A) Diagram of secreted proteins in CM from LPS/ATP–treated WT and Nlrp32/2 BMDMs. (B) Two-dimensional PAGE analysis of supernatant from WT and Nlrp3+/R258W BMDMs treated with 500 ng/ml LPS for 4 h. The labeled dots were dug out for MS analysis. (C) Protein list of identified proteins from (B) by MS. The blue-labeled proteins are at least 2-fold lower in Nlrp32/2 CM than that in WT CM of (A). p # 0.05. (D) Volcano plot of secreted proteins in (A). The pink circles represent individual protein at least 2-fold higher or lower in WT CM than that in Nlrp32/2 CM, with p # 0.05. (E) ELISA detection of serum galectin-3 level in WT mice (n = 5), which received i.p. injection of Ac-YVAD (20 mg/kg) or vehicle daily for 3 d. Data are shown as mean 6 SEM. ***p # 0.001. (F) ELISA detection of serum galectin-3 level in WT and Casp1/112/2 mice (n = 4). Data are shown as mean 6 SEM. ***p # 0.001.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 2. The inflammasome activation drives cellular and systemic secretion of galectin-3. (A) BMDMs from WT or Nlrp3+/R258W mice were stimulated with 500 ng/ml LPS for the indicated times; supernatant (SUP) and whole cell lysate (WCL) were collected for immunoblotting of the indi- cated proteins. Data are representative of at least three independent experiments. (B) LPS-primed WT BMDMs were treated with ATP (5 mM) or nigericin (5 mg/ml) for 30 min, and immunoblotting detection of indicated proteins is shown. Data are representative of at least three independent experiments. (C) LPS-primed peritoneal macrophages were stimulated with ATP (5 mM) for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least two independent experiments. (D) LPS-primed BMDC were stimulated with ATP (5 mM) for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least three independent experiments. (E) BMDM were primed with LPS for 10 min, followed by ATP (5 mM) challenge for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least three in- dependent experiments. (F) Western blotting analysis of SUP and WCL from WT, Nlrp32/2, Asc2/2, or Casp1/112/2 BMDMs stimulated with LPS/ATP. Data are representative of at least three independent experiments. (G) Western blotting analysis of SUP and WCL from LPS-primed THP-1 macrophages stimulated with ATP. Data are representative of at least three independent experiments. (H) Western blotting analysis of SUP and WCL from unprimed WT or Aim22/2 BMDMs transfected with poly(dA:dT) for 2 h. Data are representative of at least two independent experiments. (I) Western blotting analysis of SUP and WCL from unprimed WT or Nlrc42/2 BMDMs infected with S. typhimurium (multiplicity of infection = 10) for 3 h. Data are representative of at least two independent experiments. (J) Western blotting analysis of SUP and WCL from unprimed THP-1 macrophages transfected with poly(dA:dT) for 4 h. Data are representative of at least three independent experiments. (K and L) ELISA analysis of serum galectin-3 (K) and IL-18 (L) level in WT and Nlrp32/2, Aim22/2, Nlrc42/2, and Asc2/2 mice (n = 4); the WT group is the same one used in Fig. 1F. Data are shown as mean 6 SEM. ns: p . 0.05, **p # 0.01, ****p , 0.0001.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Activation Assay, Western Blot, Transfection, Infection, Enzyme-linked Immunosorbent Assay