1. Volume 19, Issue 5, Pages 657-667 (November 2003)
WSX-1 Is Required for Resistance to Trypanosoma cruzi Infection by Regulation of Proinflammatory Cytokine Production 
Shinjiro Hamano, Kunisuke Himeno, Yoshiyuki Miyazaki, Kazunari Ishii, Atsushi Yamanaka, Atsunobu Takeda, Manxin Zhang, Hajime Hisaeda, Tak W. Mak, Akihiko Yoshimura, Hiroki Yoshida 
Immunity 
Volume 19, Issue 5, Pages (November 2003)
DOI: /S (03)00298-X

2. Figure 1
Sustained High Parasitemia, Increased Mortality, and Severe Liver Injury in WSX-1−/− Mice during T. cruzi Infection
(A) WSX-1−/− (closed circles) and WSX-1+/− (open circles) mice were infected intraperitoneally with 2 × 103 of blood trypomastigotes of the Tulahuén strain of T. cruzi. Parasitemia was monitored.
(B) WSX-1−/− (closed circles) and WSX-1+/− (open circles) mice were infected with 1 × 104 of T. cruzi. Mortality was daily monitored. Double dagger, the rest of WSX-1−/− mice finally recovered.
(C) Serum ALT and AST levels were measured after infection with T. cruzi (1 × 104). Shown are mean ± SD from five independent experiments with similar results (5 to 10 mice per group in one experiment). *; P < 0.05 by Student's t test.
(D) Hematoxylin and eosin staining of the liver at 14 days after infection (1 × 104). KO, WSX-1−/− and WT, WSX-1+/−.
Immunity  , DOI: ( /S (03)00298-X)

3. Figure 2
Enhanced Th2 Cytokine Production by WSX-1-Deficient Splenocytes during T. cruzi Infection
(A) Flow cytometric analysis of intracellular IFN-γ production at 7 and 14 days after T. cruzi infection. The splenocytes (1 × 106/ml) from WSX-1+/− (WT) and WSX-1−/− (KO) mice were cultured for 24 hr with or without 2.5 μg/ml of Con A. For the last 6 hr, GolgiStop and GolgiPlug were added to the culture. Then cells were stained for surface CD3 versus NK1.1 along with intracellular IFN-γ as described in Experimental Procedures. Numbers are the percentages of IFN-γ-positive splenocytes. Experiments were repeated three times with similar results.
(B) IFN-γ production by splenic CD4+ or CD8+ T cells from WSX-1+/− (WT) and WSX-1−/− (KO) mice on day 14 after T. cruzi infection. CD4+ or CD8+ T cells were cultured with irradiated naive APC (open columns) or infected APC (closed columns) for 66 hr. Infected APC were prepared as described in Experimental Procedures. IFN-γ concentration in the culture supernatants was measured by ELISA. Data shown are mean ± SD of triplicate samples from four mice per group and are representative of three independent experiments.
(C) RT-PCR analysis of cytokine mRNA expression in splenic CD4+, CD8+ T cells, and NK1.1+ cells isolated from WSX-1+/− (WT) and WSX-1−/− (KO) mice on day 14 after T. cruzi infection. Experiments were repeated three times with similar results.
(D) IFN-γ, IL-4 and IL-5 production by splenocytes from WSX-1+/− (WT; open columns) and WSX-1−/− (KO; closed columns) mice at 14 days after T. cruzi infection. The splenocytes were cultured for 66 hr. IFN-γ, IL-4, and IL-5 production was measured as in (B). Data shown are mean ± SD of triplicate samples from four mice per group and are representative of three independent experiments. *; P < 0.05 by Student's t test.
Immunity  , DOI: ( /S (03)00298-X)

4. Figure 3
Th2 Response Is Responsible for Sustained Parasitemia, but Not for High Mortality and Liver Injury in WSX-1−/− Mice
WSX-1−/− (closed symbols) and WSX-1+/− (open symbols) mice with (triangles) or without (circles) anti-IL-4 antibody treatment were infected intraperitoneally with 2 × 103 (A and C) or 1 × 104 (B and D) of T. cruzi. Parasitemia (A), mortality (B), and serum AST (C) levels were monitored as in Figure 1. Experiments were repeated five times with similar results and mean ± SD of representative experiment are shown. *; P < 0.05 by Student's t test compared with untreated mice. (D) Hematoxylin and eosin staining of the liver derived from anti-IL-4 antibody-treated WSX-1−/− mice at 14 days after infection.
Immunity  , DOI: ( /S (03)00298-X)

5. Figure 4
IFN-γ Is Responsible for the Necroinflammatory Liver Damages during T. cruzi Infection
IFN-γR−/− mice (squares), IL-12−/− mice (triangles), WSX-1−/− mice (closed circles), and WSX-1+/− mice (open circles) (5 to 10 mice per group) were infected with 2 × 103 (A and C) or 1 × 104 (B and D) of T. cruzi. Parasitemia (A), mortality (B), and serum AST activities (C) were monitored at indicated days after infection. Representative results of three independent experiments are shown. Double dagger, note that all the IFN-γR knockout mice died by day 25 of infection and all the IL-12 knockout mice died by day 30. (D) Hematoxylin and eosin staining of the liver from IFN-γR−/− and IL-12−/− mice at 14 days after infection. (E) Wild-type (WT) or WSX-1−/− (KO) mice were treated with anti-IFN-γ antibody as described in Experimental Procedures (closed circles) or control antibody (open circles) and infected with 1 × 104 of T. cruzi. Serum AST levels were monitored. *; P < 0.05 by Student's t test compared with control mice. (F) Hematoxylin and eosin staining of the liver from anti-IFN-γ antibody-treated WSX-1−/− mice at 14 days after infection.
Immunity  , DOI: ( /S (03)00298-X)

6. Figure 5
Overexpression of IFN-γ and Th2 Cytokines in WSX-1−/− Liver Mononuclear Cells after T. cruzi Infection
LMNC were purified from WSX-1+/− (WT) or WSX-1−/− (KO) mice at 4 days after T. cruzi infection. NK1.1+ cell population was sorted with magnetic beads. RT-PCR analysis of cytokine mRNA expression was performed as described in Experimental Procedures with RNA isolated from whole LMNC (Total) or NK1.1+ cells (NK1.1+). PCR products for β-actin were shown as internal control.
Immunity  , DOI: ( /S (03)00298-X)

7. Figure 6
Overproduction of Proinflammatory Cytokines in WSX-1−/− Mice after T. cruzi Infection
(A) LMNC were prepared from WSX-1+/− (open columns) or WSX-1−/− (closed columns) mice 11 days after infection. LMNC were cultured with infected APC for 66 hr and cytokine production in supernatants was measured.
(B) Serum cytokine levels were measured similarly on day 11 after infection. Data are mean ± SD from five mice per group and are representative of three independent experiments. *; P < 0.05 by Student's t test. N.D., not detected.
(C) LMNC prepared from WSX-1+/− (WT) or WSX-1−/− (KO) mice on day 7 of infection were stained for intracellular cytokines as described in Experimental Procedures. Numbers show percentages of cytokine production positive cells. Left lower panels, gated on NK cells. Experiments were repeated three times with similar results.
Immunity  , DOI: ( /S (03)00298-X)