Egan, C. E. et al.
Synergy Between Intraepithelial Lymphocytes and Lamina Propria T Cells
Drives Intestinal Inflammation During Infection
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. CCR2 deletion protects against T. gondii-induced ileal
inflammation. Wild type (A) and Ccr2-/- (B) mice were infected with 100 ME49 cysts and
intestines harvested 8 days later. Sections of ileum were stained with H&E and
examined for inflammatory lesions. The scale bar indicates 50 µm.
Supplementary Figure 2. Day 4 infected LP compartment is composed of a variety of
immune cells, and there are no detectable parasites in LP cells and IEL used for
adoptive transfer. LP cells from WT mice were subjected to flow cytometric analysis
using Ab to the indicated molecules (A). This experiment was performed twice with
essentially identical results. An anti-Toxoplasma Ab was used to detect intracellular
parasites in IEL (B) and LP (C) cells isolated 4 days after peroral infection. D, peritoneal
exudate cells obtained 24 hr after i. p. Toxoplasma infection were stained for intracellular
parasites using the same Ab. E, immunohistochemical anti-Toxoplasma staining of ileum
from Rag1-/- recipients of LP and IEL from infected mice. F, the same Ab was used to
stain ileal sections from WT mice after peroral infection. Parasites in F are visible as
extensive brown staining. Cell nuclei are counterstained with Hematoxylin (shown in
blue). The scale bar indicates 50 µm.
Supplementary Figure 3. Direct infection of Rag1-/- mice with Toxoplasma does not
induce intestinal lesions. Wild type and Rag1-/- mice were infected with 100 ME49 cysts
1
and intestines were harvested 8 days later. H&E staining of tissue sections revealed
characteristic necrotic lesions in ilea from C57BL/6 mice (A) whereas Rag1-/- animals on
the same genetic background displayed no evident small intestinal pathology (B).
Despite lack of lesions, immunohistochemical staining for Toxoplasma revealed large
numbers of parasites in Rag1-/- intestines at Day 8 post-infection (C).
Supplementary Figure 4. Linear plot of averaged gene expression relative to that of the
-actin gene. Each gene is represented by a point whose coordinates are determined by
relative expression under the indicated conditions. A, gene expression in ileum from
Rag1-/- mice adoptively transferred with lamina propria and intraepithelial lymphocytes
from day 4-infected wild-type mice (AT), compared to gene expression in ileum from
noninfected Rag1-/-. B, gene expression in ileum from day 4 infected wild-type mice (WT)
relative to gene expression in noninfected Rag1-/- ileum. C, gene expression in AT mice
compared to WT mice. The diagonal lines in each plot indicate the 5-fold boundaries for
changes in gene expression. In these analyses, n = 3 for noninfected Rag1-/- and WT,
and n = 4 for AT.
Supplementary Figure 5. Effectiveness of antibiotic treatment in eliminating intestinal
flora in Rag1-/- mice. Fecal samples, collected from the rectum at the time of necropsy,
were plated onto LB agar and cultured for 72 hours to allow growth of bacterial plaques.
No bacterial growth was observed in either of the antibiotic-treated groups (limit of
detection 50 cfu/g) whereas abundant numbers of bacteria were present in the nontreated control mice.
Supplementary Figure 6. Induction of immune-mediated ileitis by Toxoplasma. After
oral cyst administration, parasites provide a trigger signal for induction of pathogenic
2
immune mediators. This signal could be provided by foci of lysed infected cells resulting
in bacterial translocation, or possibly the trigger could result from suppressive effects of
the parasite on infected resident macrophages and dendritic cells. Subsequently, IEL
switch from homeostatic mediators to pathogenic effector cells. CCR2 and IFN-dependent TCR+ IEL recruit CCR2+TCR+CD4+ T cells from the lamina propria.
Acting alone, or possibly in conjunction with pathogenic IEL, lamina propria cells cause
necrosis of intestinal epithelial cells, resulting in damage to the epithelium, followed by
bacterial translocation and fulminant Crohn’s-like proinflammatory cytokine pathology.
3