Alarmin function of galectin-9 in murine respiratory tularemia.

Sepsis is a complex immune disorder that is characterized by systemic hyperinflammation. Alarmins, which are multifunctional endogenous factors, have been implicated in exacerbation of inflammation in many immune disorders including sepsis. Here we show that Galectin-9, a host endogenous β-galactoside binding lectin, functions as an alarmin capable of mediating inflammatory response during sepsis resulting from pulmonary infection with Francisella novicida, a Gram negative bacterial pathogen. Our results show that this galectin is upregulated and is likely released during tissue damage in the lungs of F. novicida infected septic mice. In vitro, purified recombinant galectin-9 exacerbated F. novicida-induced production of the inflammatory mediators by macrophages and neutrophils. Concomitantly, Galectin-9 deficient (Gal-9-/-) mice exhibited improved lung pathology, reduced cell death and reduced leukocyte infiltration, particularly neutrophils, in their lungs. This positively correlated with overall improved survival of F. novicida infected Gal-9-/- mice as compared to their wild-type counterparts. Collectively, these findings suggest that galectin-9 functions as a novel alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection

Gal-9^-/- mice show improved survival during pulmonary F.n. infection.

<p><b>(A)</b> Fifteen each C57Bl/6 WT and Gal-9^-/- mice in 3 separate experiments (5 mice per experiment) were inoculated intranasally with F.n and were monitored daily for 2 weeks. The improved survival of Gal-9^-/- mice compared to WT mice was statistically significant, as determined by Kaplan-Meier log-rank analysis (<i>P</i> value*** = 0.0003). <b>(B)</b> Bacterial burdens in lungs, blood, spleen and liver harvested from F.n. infected WT and Gal-9^-/- mice at indicated times post-infection. Lung, liver and spleen homogenates prepared as described in Materials and Methods and blood were serially diluted and plated on TSA plates to enumerate bacterial burdens. Each symbol on the plots represents one mouse and data is from 2–3 independent experiments.</p

Gal-9^-/- mice display reduced accumulation of neutrophils and reduced inflammatory mediators in lungs during F.n. infection.

<p><b>(A)</b> Frozen sections of lungs harvested at 3 dpi from F.n. infected WT or Gal-9^-/- mice were co-stained with antibodies against myeloid cell markers CD11b (red) and Ly6G (green). A high co-expression of both markers is depicted by yellow color and indicates neutrophils. Nuclei (blue) were stained with 4’6’ diamidino-2-phenylindol-dilactate (DAPI). Magnification X 200. Asterisks depict lesions in the lungs. <b>(B)</b> Flow cytometry analysis of neutrophils in mock control and F.n. infected WT and Gal-9^-/- (WT-Fn and Gal-9^-/--Fn) mice. Total lungs cells were isolated from mice by collagenase treatment at 3 dpi as described in Methods. The cells were stained with anti-Ly6G-APC and anti-CD11b-Pacific Blue antibodies as markers for neutrophils. The plots are representative of three mice per group in 3 independent experiments. <b>(C)</b> Lungs from mock infected and F.n. infected WT or Gal-9^-/- mice were harvested at 3dpi, homogenized with protease inhibitors in PBS and analyzed commercially for rodent multi-analyte profiles (Myriad Rules-Based Medicine, Austin, TX). Levels of inflammatory cytokines and neutrophil markers in lung homogenates are shown. Results shown are from 3–4 mice per group from 3 different experiments. MMP-9; matrix metalloproteinase 9, MPO; myeloperoxidase. * <i>p</i><0.05; ** <i>p</i><0.005; *** <i>p</i><0.001. Comparisons were made between infected WT and Gal-9^-/- groups.</p

Gal-9^-/- mice exhibit improved lung pathology and reduced TUNEL staining indicative of cell death upon pulmonary F.n. infection.

<p><b>(A)</b> The lungs from F.n. infected wild-type (WT) or Gal-9^-/- mice were harvested at indicated times post-infection, embedded in optimal-cutting-temperature (OCT) compound, and sectioned as described in Materials and Methods. The frozen sections were stained with Hematoxylin and Eosin (H&E). The images obtained are representatives of three experiments performed with 3 mice per group in each experiment. Magnification, ×200. <b>(B)</b> H&E sections were scored in blinded fashion according to the following scoring scale: 0, no inflammatory cells (macrophages or neutrophils) present in section; 1, <5% of section infiltrated by inflammatory cells; 2, 5–10% of section infiltrated; 3, 20% of section infiltrate; and 4, >20% of section infiltrated. <b>(C)</b> Frozen lung sections from F.n. infected WT or Gal-9^-/- mice were processed for in-situ TUNEL staining for detection of DNA fragmentation (red) in nuclei. Nuclei (blue) were stained with 4′,6′-diamidino-2-phenylindole dilactate. Bar graph shows Mean fluorescence intensity (MFI) quantified using Image J software. Magnification, ×100.</p

Galectin-9 is upregulated and released in lungs of mice during respiratory <i>F</i>. <i>novicida</i> (F.n.) infection.

<p><b>(A)</b> Total RNA was extracted by Trizol method from lungs harvested at the indicated times after infection with the F.n. strain U112. The mRNA levels of galectin-9 were analyzed by real-time PCR as described in Materials and Methods and are expressed as fold increase over the levels in mock control mice. Data shown are the averages of 3–4 mice per group. <b>(B)</b> In-situ IF staining of frozen lung sections from mock control and U112 infected mice harvested at 3 dpi. The sections were stained for galectin-9 (red) using a purified rat galectin-9 antibody followed by Alexa-546 conjugated chicken anti-rat antibody. Nuclei (blue) were stained with 4’6’ diamidino-2-phenylindol-dilactate (DAPI). Magnification X 200. Inset depicts possible extracellular galectin-9 in infected mouse lungs. The area indicated by green arrow has been enlarged and shown in <b>(C)</b>. Asterisks depict galectin-9 present in cell-free areas.</p

Galectin-9 regulates F.n. infection induced inflammatory response in vitro.

<p><b>(A)</b> Peritoneal neutrophils were isolated from mice 12–14h after injection with 4% thioglycollate and were stimulated with F.n. at an MOI 50 with or without pre-treatment with purified recombinant galectin-9 (15μg/ml). Stimulation with galectin-9 alone or phorbol myristate acetate (PMA, 10ng/ml) was used as a control. Production of reactive oxygen species (ROS) was measured one hour post-stimulation by flow-cytometry using Fc-OxyBURST dye following the manufacturer’s instructions. Numbers below the plots depict average percent of ROS positive cells from 3 independent experiments. Plots from a representative of these 3 independent experiments are shown. <b>(B)</b> Bone marrow derived macrophages (BMDMs) from C57Bl/6 wild-type mice were stimulated with F.n. Strain U112 at an MOI of 50 with or without pretreatment with 15μg/ml of purified recombinant galectin-9. UltraPure E.coli LPS (10ng/ml) and galectin-9 with and without heat-denaturation (boiled at 100°C for 45 min) as well as competitive inhibition with lactose (25mM) were used as controls to test the specificity of galectin-9 effect, as described in methods. Culture supernatants were collected 24h after infection and the amount of IL-6 was measured by ELISA. The data shown is average of three independent experiments. Statistical analysis between the data sets was performed by Two-way ANOVA with Tukey post-test where * <i>p</i><0.05; ** <i>p</i><0.005; *** <i>p</i><0.001.</p

Galectin-3 Functions as an Alarmin: Pathogenic Role for Sepsis Development in Murine Respiratory Tularemia

<div><p>Sepsis is a complex immune disorder with a mortality rate of 20–50% and currently has no therapeutic interventions. It is thus critical to identify and characterize molecules/factors responsible for its development. We have recently shown that pulmonary infection with <i>Francisella</i> results in sepsis development. As extensive cell death is a prominent feature of sepsis, we hypothesized that host endogenous molecules called alarmins released from dead or dying host cells cause a hyperinflammatory response culminating in sepsis development. In the current study we investigated the role of galectin-3, a mammalian β-galactoside binding lectin, as an alarmin in sepsis development during <i>F. novicida</i> infection. We observed an upregulated expression and extracellular release of galectin-3 in the lungs of mice undergoing lethal pulmonary infection with virulent strain of <i>F. novicida</i> but not in those infected with a non-lethal, attenuated strain of the bacteria. In comparison with their wild-type C57Bl/6 counterparts, <i>F. novicida</i> infected galectin-3 deficient (galectin-3^−/−) mice demonstrated significantly reduced leukocyte infiltration, particularly neutrophils in their lungs. They also exhibited a marked decrease in inflammatory cytokines, vascular injury markers, and neutrophil-associated inflammatory mediators. Concomitantly, in-vitro pre-treatment of primary neutrophils and macrophages with recombinant galectin-3 augmented <i>F. novicida</i>-induced activation of these cells. Correlating with the reduced inflammatory response, <i>F. novicida</i> infected galectin-3^−/− mice exhibited improved lung architecture with reduced cell death and improved survival over wild-type mice, despite similar bacterial burden. Collectively, these findings suggest that galectin-3 functions as an alarmin by augmenting the inflammatory response in sepsis development during pulmonary <i>F. novicida</i> infection.</p> </div

Galectin-3^−/− mice display reduced accumulation of neutrophils in lungs during F.n. infection.

<p>Frozen sections of lungs harvested at 3 d. p.i. from mock infected and <i>F. novicida</i> infected WT or galectin-3^−/− mice were co-stained with antibodies against myeloid cell markers CD11b (red) and Gr1 (green). A high co-expression of both markers is depicted by yellow color in infected WT lungs while cells infiltrating lungs of galectin-3^−/− mice exhibited expression of only CD11b. Nuclei (blue) were stained with 4′6′ diamidino-2-phenylindol-dilactate (DAPI). Magnification×200. Asterisks depict lesions in the lungs.</p

Galectin-3 deficiency leads to improved lung pathology, reduced leukocyte accumulation and reduced cell death upon pulmonary F.n. infection.

<p>(<b>A</b>) Lungs from mock infected and F.n. infected wild-type (WT) or galectin-3^−/− mice were harvested at the septic phase (3 d. p.i.), embedded in optimal-cutting-temperature (OCT) compound, and sectioned as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059616#s2" target="_blank">Materials and Methods</a>. The frozen sections were stained with Hematoxylin and Eosin. The images obtained are representatives of three experiments performed, and in each experiment each group contained three mice. Magnification, ×200. (<b>B</b>). Lungs from mock infected and F.n. infected WT or galectin-3^−/− mice were harvested 3 days after intranasal infection. Total immune cells infiltrating the lungs were isolated by collagenase treatment of lungs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059616#s2" target="_blank">Materials and Methods</a>. Total numbers of viable immune cells were counted by trypan blue exclusion staining (n = 5–6). Statistical analysis between the data sets was performed by Student’s t test where **<i>p</i><0.005. (<b>C</b>). Frozen lung sections from mock infected and Francisella infected WT or galectin-3^−/− mice were processed for in-situ TUNEL staining for detection of DNA fragmentation (red) in nuclei. Nuclei (blue) were stained with 4′,6′-diamidino-2-phenylindole dilactate. Magnification, ×100.</p

Upregulated expression and extracellular release of Galectin-3 in lungs during respiratory <i>F. novicida</i> infection.

<p>(<b>A</b>) Total RNA was extracted by Trizol method from lungs harvested at the indicated times after infection with the Wild-type bacteria (WT) or from mice vaccinated with an attenuated mutant strain followed by challenge with WT bacteria (Mut/WT mice). The mRNA levels of Galectin-3 were analyzed by real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059616#s2" target="_blank">Materials and Methods</a> and are expressed as fold changes over the levels in mock control mice. Data shown are the averages of 3–4 mice per group. Statistically significant differences are denoted by asterisks (**p<0.005). (<b>B</b>) In-situ IF staining of frozen lung sections from mock infected and WT U112 infected or Mut/WT mice harvested at 3 d. p.i Lung harvested 3 weeks after vaccination with the mutant alone (Mut-3 wk) served as controls for Mut/WT mice. The sections were stained for galectin-3 (red) using a purified rat anti-mouse galectin-3 antibody followed by Alexa-546 conjugated chicken anti-rat antibody. Nuclei (blue) were stained with 4′6′ diamidino-2-phenylindol-dilactate (DAPI). Magnification×200. Insets depict extracellular galectin-3 in WT <i>F. novicida</i> infected mouse lungs (B2’) and cytosolic galectin-3 in Mut/WT (B4’) mouse lungs.</p