Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: Galectin-9 inhibits mycobacterial growth directly . A. Profile of Mtb H37Rv (Rv) grown at 37°C in Middlebrook 7H9 liquid medium with different concentration of Galectin-9 (Gal9, 0, 0.01, 0.1, 1, 10 μg/mL). Growth curve was measured using a Bioscreen Growth Curve Instrument. Optical density was measured at absorbance at 600 nm every 2 h. B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with10μg/mL galectin-9 (Gal9) or inactivated galectin-9 (Gal9 HK, heat killed at 95 [for 5 min). C. CFU of Mtb H37Rv (Rv) on Middlebrook 7H10 solid medium with or without 10μg/mL Galectin-9 (Gal9). Cultures were grown at 37°C for 4-8 weeks. D. Growth profile of Mycobacterium smegmatis (MS) in Middlebrook 7H9 liquid medium with different concentrations of Galectin-9 (Gal9, 0, 0.01, 0.1, 1 μg/mL). E. Concentrations of galectin-9 in sera of healthy donors (n = 40) and active TB patients (n = 40). F. Confocal microscopy of M. bovis BCG-DsRed (BCG-DsRed, red) and Galectin[9 (Anti-Gal9, green) in THP-1 cells. Nuclei was stained with DAPI (blue). G. Percent of cells with galectin-9 positive (gal9 + ) BCG in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. H. Confocal microscopy of Mtb H37Rv-GFP (Rv-GFP, green) and Galectin[9 (Anti-Gal9, red) in THP-1 cells. Nuclei were stained with DAPI (blue). I. Percent of cells with galectin9 positive (gal9 + ) Mtb H37Rv in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Data are representative of three independent experiments with similar results (F and H). Two-tailed unpaired Student’s t test (A-D, G, and I) or Mann-Whitney U test (E). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Concentration Assay, Confocal Microscopy, Staining, Infection, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and lactose (1 μg/mL). B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and D-glucose (10 μg/mL). C. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and AG (1 μg/mL). D. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with 1μg/mL CRD1 or CRD2 of galectin-9. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Two-tailed unpaired Student’s t test (A-D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Morphologic characteristics for Mtb H37Rv strain grown in liquid culture with or without anti-AG mAbs (1 μg/mL) observed by ×[2 magnifier. B. Bacterial shape of Mtb H37Rv strain treated as in (A) observed by acid fast staining under a Leica DM2500 microscope using the 100× oil microscopy. EMB, Ethambutol. Scale bar, 20μm. C. Ultrastructural morphology of Mtb H37Rv treated as in (A) analyzed by transmission electron microscopy (TEM). The cell wall was labeled with red arrows. D. Cell wall thickness of bacteria in (C). E. Schematic presentation of Mtb growth arrest by Galectin-9 or anti-AG antibodies. Data are representative of three independent experiments with similar results (A, B and C). Data are means ± SD of 11 bacteria, representatives of three independent experiments (D). Two-tailed unpaired Student’s t test (D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Staining, Microscopy, Transmission Assay, Electron Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: Journal of Innate Immunity

Article Title: Intestinal Epithelium-Derived Galectin-9 Is Involved in the Immunomodulating Effects of Nondigestible Oligosaccharides

doi: 10.1159/000350515

Figure Lengend Snippet: Expression and secretion of galectin-9 in HT-29 cells is regulated by TLR9 ligation and scGOS/lcFOS. a-c Analysis of galectin (Gal) mRNA expression by HT-29 cells by quantitative PCR analysis. Evaluation of the expression profile of galectins by IEC (a). Apical TLR9 ligation of HT-29 cells, in the absence or presence of scGOS/lcFOS, specifically increases galectin-9 expression (b, c). Protein expression of galectin-4 and galectin-9 and its modulation upon TLR9 ligation and scGOS/lcFOS was confirmed by immunofluorescence microscopic staining of HT-29 monolayers (d). ELISA was performed in the basolateral supernatant of HT-29 monolayers (e). TLR9 ligation of HT-29 cells in the presence of scGOS/lcFOS enhanced galectin-9, but not galectin-4 secretion. n = 3 (a-d) or n = 6 (e) independent PBMC donors, means ± SEM, * p < 0.05, # p < 0.05, ** p < 0.01.

Article Snippet: IEC were fixed using 4% formalin in PBS for 10 min, permeabilized in 0.1% Triton X-100 (Sigma) and 1% BSA in PBS for 15 min and incubated with anti-human galectin-4 or galectin-9 antibodies or normal goat IgG as isotype control (all 0.75 μg/ml; R&D Systems) in 0.1% Triton X-100 and 1% BSA in PBS for 1 h. IEC were incubated with secondary Alexa Fluor 546 donkey anti-goat IgG (Invitrogen) in 0.1% Triton X-100 and 1% BSA for 30 min and embedded in Hoechst.

Techniques: Expressing, Ligation, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Intestinal Epithelium-Derived Galectin-9 Is Involved in the Immunomodulating Effects of Nondigestible Oligosaccharides

doi: 10.1159/000350515

Figure Lengend Snippet: Galectin-9 neutralization in IEC/PBMC cocultures abrogates IFN-γ and IL-10 secretion by PBMC. HT-29 (unpolarized) and T84 (polarized) IEC were cocultured with CD3/CD28-activated PBMC for 24 h and apically exposed to TLR9 ligand in the presence or absence of scGOS/lcFOS. Basolateral galectin-9 was neutralized using TIM-3-Fc fusion protein. Galectin-9 neutralization abrogated the induction of IFN-γ (a, e) and IL-10 (b, f) by PBMC, but did not modulate IL-17A secretion by PBMC (c, g). Furthermore, the immunomodulating effects on IL-13 secretion induced by exposure of HT-29 or T84 cells to TLR9 ligand in the absence or presence of scGOS/lcFOS was abolished upon neutralization of galectin-9 (d, h). Means ± SEM of 3 independent PBMC donors. * p < 0.05, ** p < 0.01, *** p < 0.001. GF = scGOS/lcFOS.

Article Snippet: IEC were fixed using 4% formalin in PBS for 10 min, permeabilized in 0.1% Triton X-100 (Sigma) and 1% BSA in PBS for 15 min and incubated with anti-human galectin-4 or galectin-9 antibodies or normal goat IgG as isotype control (all 0.75 μg/ml; R&D Systems) in 0.1% Triton X-100 and 1% BSA in PBS for 1 h. IEC were incubated with secondary Alexa Fluor 546 donkey anti-goat IgG (Invitrogen) in 0.1% Triton X-100 and 1% BSA for 30 min and embedded in Hoechst.

Techniques: Neutralization

Journal: Journal of Innate Immunity

Article Title: Intestinal Epithelium-Derived Galectin-9 Is Involved in the Immunomodulating Effects of Nondigestible Oligosaccharides

doi: 10.1159/000350515

Figure Lengend Snippet: Functional responses of galectin-9-stimulated naïve CD4+CD45RA+ T cells and moDC. To study whether galectin-9 acts on moDC, T cells or via modulation of the interaction between moDC and T cells, galectin-9 was added during the differentiation of monocytes to moDC or during coculture of moDC with naïve CD4+ T cells (a). Naïve CD4+CD45RA+ T cells, moDC or moDC-T-cell cocultures were exposed to recombinant human galectin-9. Mixed lymphocyte cultures were maintained for 5 days. For Treg-cell differentiation, exogenous TGF-β was added to the culture. For intracellular cytokines, T cells were restimulated as described in the Materials and Methods. Stimulation of naïve CD4+ T cells with galectin-9 did not induce CD25+Foxp3+ Treg cells (b, d), IL-10 (b, e) or IFN-γ expression by CD4+ T cells (b, f). However, moDC generated in the presence of galectin-9 (Gal-9 DC) or exogenous galectin-9 added to mixed lymphocyte cultures with unconditioned (control) moDC resulted in increased differentiation of CD25+Foxp3+ Treg cells (c, d) and IL-10-expressing CD4+ cells (c, e). Only Gal-9 DC had the capacity to induce IFN-γ expression by CD4+ T cells (c, f). n = 3 independent PBMC donors. * p < 0.05, ** p < 0.01 vs. control DC.

Article Snippet: IEC were fixed using 4% formalin in PBS for 10 min, permeabilized in 0.1% Triton X-100 (Sigma) and 1% BSA in PBS for 15 min and incubated with anti-human galectin-4 or galectin-9 antibodies or normal goat IgG as isotype control (all 0.75 μg/ml; R&D Systems) in 0.1% Triton X-100 and 1% BSA in PBS for 1 h. IEC were incubated with secondary Alexa Fluor 546 donkey anti-goat IgG (Invitrogen) in 0.1% Triton X-100 and 1% BSA for 30 min and embedded in Hoechst.

Techniques: Functional Assay, Recombinant, Cell Differentiation, Expressing, Generated

Journal: Infection and Immunity

Article Title: Intestinal Lamina Propria CD4 + T Cells Promote Bactericidal Activity of Macrophages via Galectin-9 and Tim-3 Interaction during Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.00769-17

Figure Lengend Snippet: The Tim-3/galectin pathway is involved in the cross talk between intestinal CD4+ T cells and macrophages. The expression levels of galectin-9 on intestinal macrophages (A and B) and Tim-3 on CD4+ T cells (A and C) were determined after S. Typhimurium infection. Then S. Typhimurium-infected intestinal macrophages were cultured alone or with CD4+ T cells in the presence of increasing amounts of α-lactose. The numbers of CFU in the macrophages were determined (D). Data are expressed as the means ± SDs from at least three separate experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (compared with the control group).

Article Snippet: The primary antibody solution (anti-F4/80 MAb, clone C-7 [Santa Cruz Biotechnology, CA]; rabbit anti-galectin-9 polyclonal antibody, bs-0604R [Bioss, China]; and fluorescein isothiocyanate [FITC]-labeled anti- Salmonella antibody, ab69253) was dropped to cover the tissue sections and incubated overnight at 4°C.

Techniques: Expressing, Infection, Cell Culture

Journal: Infection and Immunity

Article Title: Intestinal Lamina Propria CD4 + T Cells Promote Bactericidal Activity of Macrophages via Galectin-9 and Tim-3 Interaction during Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.00769-17

Figure Lengend Snippet: Tim-3/galectin-9 interaction promotes inflammasome activation, which causes caspase-1 cleavage and IL-1β secretion. The expression levels of NLRs, procaspase-1, caspase-1, pro-IL-1β, and mature IL-1β in macrophages were assessed at the indicated infection time points by Western blotting (A). S. Typhimurium-infected macrophages were cultured alone or with CD4+ T cells. One hour later, the double positivity for capase-1 fluorescent inhibitor probe (FAM-YVAD-FMK) and PI in macrophages was detected by FACS (B). S. Typhimurium-infected intestinal macrophages was cultured alone or with CD4+ T cells in the presence of increasing amounts of α-lactose. Macrophages were then isolated from the culture system by discarding CD4+ T cells using the EasySep Mouse CD4 Positive Selection kit. The expression levels of NLRC4, procaspase-1, and caspase-1 were detected by Western blotting (C). IL-1β expression levels in the cocultures were determined by ELISA (D) and intracellular IL-1β expression was detected by FACS (E). Data are representative of those from three independent experiments. Error bars indicate SDs from three replicate cultures. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (compared with the control group).

Article Snippet: The primary antibody solution (anti-F4/80 MAb, clone C-7 [Santa Cruz Biotechnology, CA]; rabbit anti-galectin-9 polyclonal antibody, bs-0604R [Bioss, China]; and fluorescein isothiocyanate [FITC]-labeled anti- Salmonella antibody, ab69253) was dropped to cover the tissue sections and incubated overnight at 4°C.

Techniques: Activation Assay, Expressing, Infection, Western Blot, Cell Culture, Isolation, Selection, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Intestinal Lamina Propria CD4 + T Cells Promote Bactericidal Activity of Macrophages via Galectin-9 and Tim-3 Interaction during Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.00769-17

Figure Lengend Snippet: IL-1β improves the bactericidal activity of intestinal macrophages and the expression of galectin-9. S. Typhimurium-infected intestinal macrophages were cultured alone or in the presence of increasing amounts of IL-1β for 12 h (A). S. Typhimurium-infected intestinal macrophages were cultured alone or with CD4+ T cells in the presence of anti-IL-1β antibodies or isotype (B). The numbers of CFU in the macrophages were determined (A and B). Intestinal macrophages were cultured alone or infected with S. Typhimurium and then treated with 10 ng/ml of IL-1β. Galectin-9 expression on intestinal macrophages was detected by FACS (C). Data are representative of those from three independent experiments. Error bars indicate SDs from three replicate cultures. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (compared with the control group).

Article Snippet: The primary antibody solution (anti-F4/80 MAb, clone C-7 [Santa Cruz Biotechnology, CA]; rabbit anti-galectin-9 polyclonal antibody, bs-0604R [Bioss, China]; and fluorescein isothiocyanate [FITC]-labeled anti- Salmonella antibody, ab69253) was dropped to cover the tissue sections and incubated overnight at 4°C.

Techniques: Activity Assay, Expressing, Infection, Cell Culture

Journal: Infection and Immunity

Article Title: Intestinal Lamina Propria CD4 + T Cells Promote Bactericidal Activity of Macrophages via Galectin-9 and Tim-3 Interaction during Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.00769-17

Figure Lengend Snippet: Proposed model for mechanisms of cross talk between intestinal macrophages and CD4+ T cells involved in the Tim-3/galectin-9 pathway. S. Typhimurium infection promotes the accumulation of macrophages and CD4+ T cells in the small intestinal LP. Meanwhile, the expressions of galectin-9 on intestinal macrophages and Tim-3 on CD4+ T cells are enhanced following infection. CD4+ T cells promote the activation and bactericidal activity of intestinal macrophages via Tim-3/galectin-9 interaction, which triggers the formation and activation of inflammasomes, leading to the cleavage of caspase-1 and IL-1β. The secretion of active IL-1β further improves the bactericidal activity of intestinal macrophages and expression of galectin-9 on macrophages.

Article Snippet: The primary antibody solution (anti-F4/80 MAb, clone C-7 [Santa Cruz Biotechnology, CA]; rabbit anti-galectin-9 polyclonal antibody, bs-0604R [Bioss, China]; and fluorescein isothiocyanate [FITC]-labeled anti- Salmonella antibody, ab69253) was dropped to cover the tissue sections and incubated overnight at 4°C.

Techniques: Infection, Activation Assay, Activity Assay, Expressing