Fig 2: AG induces expression of MMPs Heat map showing RPKM (Reads Per Kilobase per million of mapped reads) mean values of Mmps from mouse peritoneal macrophages (MPM) stimulated with AG (1 µg/ml) for 24 h or left untreated (NT).Quantitative polymerase chain reaction (qPCR) analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12, and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG (1 µg/ml) for indicated times.Immunoblots of cell supernatants to analyze secreted MMP2, MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages stimulated with AG (1 µg/ml) for indicated times; GADPH of cell lysates served as a loading control.qPCR analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12, and Mmp13 from the lungs of mice at indicated days post-intraperitoneal administration of AG (100 µg).qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages stimulated with AG (1 µg/ml) for 24 h in the absence or presence of AG aptamers (0.5 µg/ml).Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages stimulated with AG (1 µg/ml) for indicated times in the absence or presence of AG aptamers (0.5 µg/ml); GADPH of cell lysates served as the loading control.qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from the lungs of mice at 3 days post-intraperitoneal administration of AG (100 µg) in the absence or presence of AG aptamers. AG aptamers (1 µg/mouse) were intranasally administrated per day.qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24 h (MOI = 5) in the absence or presence of AG aptamers (1 µg/ml).Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI = 5) in the absence or presence of AG aptamers (0.5 µg/ml); GADPH of cell lysates served as the loading control.qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from lungs of mice intranasally infected with H37Rv (2 × 106 cfu/mouse) for 4 weeks in the absence or presence of AG aptamers. AG aptamers (1 µg/mouse) were intranasally administrated once at a 1-week interval. Data information: Data in (B, E, H) are means ± SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Data in (D, G, J) are means ± SD of indicated numbers of mice from 1 of at least n = 2 independent experiments, and each symbol represents data from 1 mouse. Data (D, G, J) shown are representative of n = 2 (D) or n = 3 (G, J) independent experiments. Two-way ANOVA followed by Tukey's post hoc test (B, D, E, G, H, J) was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.

Fig 3: AG activates ERK to induce MMPs AImmunoblots of cell lysates were performed to analyze p-ERK1/2, p-JNK, p-P38, and p-P65 by THP-1 cells stimulated with AG (1 µg/ml) for indicated times, and GADPH as a loading control.BImmunoblots of cell lysates were performed to analyze p-ERK1/2, p-JNK, p-P38, and p-P65 by mouse peritoneal macrophages stimulated with AG (1 µg/ml) for indicated times left untreated or pretreated with AG aptamer (1 µg/ml), and GADPH as a loading control.C–EqPCR analysis of Mmps including Mmp9 (C), Mmp10 (D), and Mmp12 (E) from THP-1 cells stimulated with AG (1 µg/ml) for 24 h in the absence or presence of different inhibitors targeting NF-?B (PDTC), ERK (PD98059), JNK (SP600125), and p38 (SB203580) at the concentration of 10 µM.FImmunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages stimulated with AG (1 µg/ml) for indicated times in the absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 µM; GADPH of cell lysates served as the loading control.GImmunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI = 5) in the absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 µM; GADPH of cell lysates served as the loading control. Data information: Data in (A and B) are representative of n = 3 independent experiments. Data in (C–E) are means ± SD averaged from n = 3 independent experiments performed with technical triplicates, and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test (C–E) were used for statistical analysis. ns, not significant; *P < 0.05; ****P < 0.0001. Source data are available online for this figure.

Fig 4: Galectin-9 mediates TAK1 recruitment to induce production of MMPs A, BImmunoblots and immunoprecipitation of cell lysates to analyze endogenous interaction of galectin-9 with TAK1 by human THP-1 cells (A) or mouse peritoneal macrophages (B) left unstimulated or stimulated with AG (1 µg/ml) for 1 h.C In vitro glutathione S-transferase (GST) precipitation assay purified histidine (His)-tagged Galectin-9 (+) with GST alone or GST-tagged TAK1.DConfocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 µg/ml) for 2 h (middle row) or infected with H37Rv for 3 h (MOI = 5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 µm. Data in the right graph show mean ± SD of n = 12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field.EImmunoblots of cell lysates to analyze phosphorylated TAK1 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 µg/ml) for indicated times; GADPH of cell lysates served as the loading control. Data are representative of at least n = 3 independent experiments.FImmunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 µg/ml) in the absence or presence of TAK1 inhibitor 5Z-7-OZ (1 µM) for indicated times. Data are representative of n = 3 independent experiments.G–JqPCR analysis of Mmps including Mmp9 (G), Mmp10 (H), Mmp12 (I), and Mmp13 (J) from WT or Galectin-9 KO mouse peritoneal macrophages stimulated with AG (1 µg/ml) for 24 h in the absence or presence of TAK1 inhibitor 5Z-7-OZ (1 µM). Data information: Data in (G to J) are means ± SD averaged from 3 independent experiments performed with technical triplicates, and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 ****P < 0.0001. Source data are available online for this figure.

Fig 5: Galectin-9 is essential for the in vivo effects of AG AqPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from the lungs of WT or Galectin-9 KO mice at 3 days post-intraperitoneal administration of AG (100 µg).B, CWT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 days in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (B) and quantification of lung lesion burden from H&E-stained sections (C).D, EWT or Galectin-9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 µg) once at a 1-week interval. Lung sections stained with H&E (D) and quantification of lung lesion burden from H&E-stained sections (E).FCFU quantification of the bacterial titers of lung tissue homogenates from WT or Galectin-9 KO mice intranasally infected with H37Rv for 4 weeks in the absence or presence of intranasally administrated AG aptamers (1 µg) once at a 1-week interval. Data information: Data in (B and D) are representative of n = 3 independent experiments. Data in (A, C, E, F) are means ± SD of the indicated number of mice from 1 of n = 3 independent experiments and each symbol represents data from 1 mouse. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Scale bar, 200 µm.