Figure 1.
CAT2−/− mice develop larger pulmonary granulomas and fibrosis is increased.
WT C57BL/6 (N = 5) and CAT2−/− (N = 5) mice were sensitized i.p. with S. mansoni eggs and then challenged 2 weeks later i.v. with 5000 live eggs. Mice were sacrificed on days 4 and 7 post-challenge and granuloma volume (A) and fibrosis (B) was quantified from Giemsa (A) and Picrosirius Red (B) stained lung sections. Statistically significant differences are shown. The experiment was repeated a total of three times with similar results.
Figure 2.
CAT2−/− mice display increased granulomatous inflammation in the liver following infection with S. mansoni.
WT C57BL/6 (open squares) and CAT2−/− (filled circles) mice were infected with 30–35 S. mansoni cercariae and sacrificed on wks 8, 12 and 24 post-infection. The results shown are for individual mice pooled from 3 separate experiments. A. Liver granuloma volumes measured microscopically at 8, 12 and 24 wks post-infection. The * symbol denotes significant differences between WT and KO mice at that time point, p<0.05. B. Percentage of liver granuloma-associated eosinophils (% of total cells). C. Liver granuloma-associated mast cells (Scale 1–8). D. Representative granulomas from an infected WT C57BL/6 mouse (wk 8 post-infection). Arrows indicate the perimeter of an individual granuloma with the miracidium containing egg in the center. The bar in panel 2D = 200 microns. E. Representative granulomas from an infected CAT2−/− mouse. F. % Survival after infection with 100 cercariae. p<0.001.
Figure 3.
S. mansoni infected CAT2−/− mice develop exacerbated liver fibrosis.
WT C57BL/6 (open squares) and CAT2−/− (filled circles) mice were infected with 30–35 S. mansoni cercariae and sacrificed on wk 8, 12 and 24 post-infection. A. Liver Fibrosis adjusted per worm pair (µmol of hydroxyproline/worm pair). The * symbol denotes significant differences between WT and KO mice at that time point, p<0.05. B. Liver Fibrosis per 10,000 eggs (µmol of hydroxyproline/1×104 eggs). C. Representative liver granulomas stained with Masson's Trichrome (8 wk infected C57BL/6 mouse). D. Representative liver granulomas stained with Masson's Trichrome (8 wk infected CAT2−/− mouse). The bar in panel 3D = 200 microns.
Figure 4.
S. mansoni infected CAT2−/− mice develop marked hepatomegaly.
A. Serum aspartate transaminase (AST) values in WT C57BL/6 and CAT2−/− mice (wk 0, 8, 12 post-infection). The * symbol denotes significant differences between WT and KO mice at that time point, p<0.05. B. Serum alanine transaminase (ALT) values. C. Liver size as percentage of body weight at 6, 8, 12, and 24 wk post-S. mansoni infection.
Figure 5.
TH1/TH2 cytokine responses are reduced in infected CAT2−/− mice.
A. Leukocytes derived from the granulomatous livers of 8 wk S. mansoni infected mice were stimulated with PMA/Ionomycin/BFA for 3 hr and then stained for CD4, IFN-γ, IL-5 and IL-13. The data show the percentage of CD4+ lymphocytes producing a specific cytokine. Results for individual mice are shown and statistically significant differences are indicated in the figure. B. Frequency of non-CD4+ lymphocytes producing specific cytokines. C. Relative mRNA levels of ifn-γ, il-5, il-13, il-4, il-10 in S. mansoni infected liver tissue were determined by real-time Q-RT-PCR (wk 8,12 and 24), by normalizing mRNA levels to expressed, endogenous HPRT mRNA. Fold increases in relative mRNA levels were determined by the ratio of normalized infected mRNA to uninfected mRNAs. Results from individual mice are shown. The * symbol denotes significant differences between WT and KO mice at that time point, p<0.05.
Figure 6.
Cytokine production and proliferation of CD4+ lymphocytes is impaired in the granulomatous tissues of infected CAT2−/− mice.
A. Leukocytes derived from the granulomatous livers of 8 wk infected WT C57BL/6 and CAT2−/− mice were stained with CFSE and cultured for 72 hrs in the presence of media alone or Con A (1 µg/ml). Cells were subsequently stained for IL-13 (left panels) and IFN-γ (right panels). Representative FACS plots are shown. The experiment was repeated numerous times with similar results. B. Mesenteric lymph node cultures from 8 wk S. mansoni infected WT and CAT2−/− mice.
Figure 7.
CAT2−/− BMMφ display increased arginase activity but reduced NO responses.
A. Messenger RNA for CAT2 is induced in BMMφ following classical (CL) or alternative activation (AA). BMMφ were incubated with medium alone (MED), IFN-γ/LPS (100 U/ml/100 ng/ml, CL), or IL-13/IL-4 (20 ng/ml, AA) for 1–6 hrs. Total RNA was isolated from individual wells and CAT2 expression was analyzed by RT-PCR. The supernatants were also collected. B. NO expression by activated CAT2−/− BMMφ is impaired following classical (IFN- γ/LPS) or alternative activation (IL-13/IL-4). Supernatants were assayed for nitrate levels 24 and 48 hr post-stimulation. C. Urea production (a measure of arginase activity) was assessed in WT and CAT2−/− BMMφ's following stimulation with CL or AA stimuli. D. CAT2−/− BMMφ display increased arginase activity when exposed to multiple “alternative” activating stimuli. WT and CAT2−/− macrophages were stimulated with various combinations of IL-21, GM-CSF, IL-4 and IL-13 (all 20 ng/ml). E. Macrophages in granulomas at 8 and 12 wk post-infection with S. mansoni (% adjusted proportionally by granuloma size). Spots represent the mean of 30 granulomas analyzed per mouse.
Figure 8.
CAT2−/− primary fibroblasts display increased proliferative responses and spontaneous arginase activity.
A. WT C57BL/6 (open bars) and CAT2−/− (filled bars) primary lung fibroblasts were stimulated with medium alone, IFN-γ/LPS (100 U/ml/100 ng/ml, CL), or with IL-4/IL-13 (20 ng/ml, AA). After 24 hr, NO activity was quantified by measuring nitrate levels in the supernatants. B. WT and CAT2−/− fibroblasts were plated at 5 × 105 cells per well and stimulated with various type-2 cytokines that promote alternative activation. Arginase activity was quantified by measuring urea production (mg/dL). C. WT and CAT2−/− fibroblasts were plated at 1 × 105 cells/well, stimulated with medium alone or 50 ng/ml FGFb, and proliferation was measured by (H3) thymidine incorporation. D. WT and CAT2−/− fibroblasts were plated at 1 × 105 cells/well and cultured in the presence of medium alone or IL-4/IL-13 (20 ng/ml). After 24 hr incubation, the supernatants were assayed for IL-6. All assays were repeated with similar results. E. Fibroblasts in granulomas at 8 and 12 wk post-infection with S. mansoni (% adjusted proportionally by granuloma size). Spots represent the mean of 30 granulomas analyzed per mouse. F. WT and CAT2−/− were sensitized with 5000 live S. mansoni eggs i.p. and then challenged 2 wk later with 5000 eggs i.v. On day 4 and 7 post-challenge, lung tissue was harvested and Arg1 and Retlna mRNA expression was quantified by real-time PCR and graphed as fold-increase over naïve lung. The results for individual mice are shown. G. WT C57BL/6 (open squares) and CAT2−/− (filled circles) mice were infected with S. mansoni cercariae and serum was collected at various time points post-infection. Serum IL-13Rα2 levels were measured by ELISA in individual mice. The * symbol denotes significant differences between WT and KO mice at that time point, p<0.05.
Figure 9.
Arg-1 and α-SMA expression colocalized in CAT2−/− liver granulomas.
C57BL/6 and CAT2−/− mice were infected percutaneously with S. mansoni for 10 weeks. Portions of livers were removed and frozen. 8 µM liver sections from C57BL/6 and CAT2−/− mice were stained anti-F4/80-Alexa 488 (Green), anti-Arg1-Alexa 647(Red), and anti-Alpha Smooth Muscle Actin-Texas Red (Blue). Individual 20× images were taken for each channel and then combined to provide a composite image. Images are representative of 3 individual mice from independent experiments.
Figure 10.
Development of hepatic fibrosis in infected CAT2−/− mice is IL-13-independent.
WT C57BL/6 (squares) and CAT2−/− (circles) mice were infected with 30–35 cercariae. Separate groups were treated with either control antibody (C Ig, open symbols) or with a neutralizing mAb to IL-13 (α-IL-13, filled symbols) weekly for a total of 4 wk starting on wk 5 post-infection. The animals were sacrificed on wk 9 and fibrosis and granuloma formation were assessed. A. Liver hydroxyproline levels in individual mice, shown as µmol/liver, µmol/worm pair, and µmol/10K eggs. Significant differences are indicated in each figure. B. Granuloma size (volume, mm3 × 10−3), percentage of granuloma-associated eosinophils, and mast cell indices were determined microscopically.
Figure 11.
Development of Th1 immunity is compromised in CAT2−/− mice.
A. WT C57BL/6 (open squares), CAT2−/− (filled circles), NOS2−/− (filled inverted triangles) and IFN-γ−/− (filled triangles) mice were infected i.p. with 20 T. gondii cysts, and survival was assessed up to 50 days post-infection. (N = 5/group). B. Peritoneal exudate cells (PECs) were prepared from WT and CAT2−/− mice on day 7 post-infection. The percentage of infected cells was determined microscopically by evaluating a minimum of 700 cells per slide (3 mice/group). C. PECs were placed in culture for 24–48 hr and either left untreated or restimulated with STAG. Culture supernatants were collected and IFN-γ levels were determined by ELISA. D. NO activity was evaluated in the same culture supernatants by measuring nitrate production.