Cardiac and gastrointestinal liabilities caused by deficiency in the immune modulatory enzyme indoleamine 2,3-dioxygenase

Abstract

Indoleamine 2,3-dioxygenase (IDO) modifies adaptive immunity, in part by determining the character of inflammatory responses in the tissue microenvironment. Small molecule inhibitors of IDO are being developed to treat cancer, chronic infections and other diseases, so the systemic effects of IDO disruption on inflammatory phenomena may influence the design and conduct of early phase clinical investigations of this new class of therapeutic agents. Here, we report cardiac and gastrointestinal phenotypes observed in IDO deficient mice that warrant consideration in planned assessments of the safety risks involved in clinical development of IDO inhibitors. Calcification of the cardiac endometrium proximal to the right ventricle was a sexually dimorphic strain-specific phenotype with ~30% penetrance in BALB/c mice lacking IDO. Administration of complete Freund's adjuvant containing Toll-like receptor ligands known to induce IDO caused acute pancreatitis in IDO deficient mice, with implications for the design of planned combination studies of IDO inhibitors with cancer vaccines. In an established model of hyperlipidemia, IDO deficiency caused a dramatic elevation in levels of serum triglycerides. In the large intestine, IDO loss only slightly increased sensitivity to induction of acute colitis, but it markedly elevated tumor incidence, multiplicity and staging during inflammatory colon carcinogenesis. Together, our findings suggest potential cardiac and gastrointestinal risks of IDO inhibitors that should be monitored in patients as this new class of drugs enter early clinical development.

Figure 1

Calcification of the cardiac endometrium in IDO deficient BALB/c mice. Representative illustrations of gross pathology (left panels) and representative histology (right panels) are shown of calcification of the cardiac endometrium proximal to the right ventricle which were observed in female animals with a ∼30% penetrance. RV, right ventricle.

Figure 2

Acute pancreatitis induced by complete Freund's adjuvant in IDO deficient mice. (A) Gross pathology. Freshly dissected pancreata are shown to illustrate the marked increase in organ size produced in mice 28 d after treatment with CFA + peptide (untreated WT n = 5, untreated IDO-/- n = 4, CFA + Peptide treated WT n = 5, CFA + Peptide treated IDO-/- n = 5) (see Material and Methods). (B) Pancreas histology. Representative samples are shown to illustrate pancreatitis that developed after treated with CFA or CFA + Peptide in WT and IDO-/- mice. Lower magnification images are presented in the upper row and boxed areas are magnified in the lower row. Areas indicated by arrows show normal acinar regions of pancreatic tissue. Scale bar = 100 µM. (CFA treated WT n = 5, CFA treated IDO-/- n = 5, CFA + Peptide treated WT n = 5, CFA + Peptide treated IDO-/- n = 5). (C) Insulin expression. Pancreatic tissue sections were stained with H&E for histology or processed for immunohistochemical expression of insulin in islet β-cells. Brown staining demonstrates the presence of insulin containing β-cells in these tissues. Scale bar = 100 µM.

Figure 3

IDO deficiency accentuates hyperlipidemia in a mouse model of atherosclerosis driven by high fat diet. Blood serum samples were analyzed from WT, LDLR-/- or IDO-/-LDLR-/- mice on the C57BL6/J strain background. A representative illustration of hyperlipidemia present in mice fed a high fat diet is shown. Quantification of serum lipids demonstrated a significant increase in serum triglycerides in IDO-/-LDLR-/- mice fed an atherogenic diet (p < 0.02). No significant differences in levels of serum cholesterol were observed. Total mice tested in each group was IDO-/- (n = 10), IDO-/-LDLR-/- (n = 10), LDLR-/- (n = 9).

Figure 4

IDO deficiency accentuates the effects of inflammatory insults that promote colon carcinogenenesis. (A) Colitis induction. Colons from WT and IDO-/- mice treated 3 d with 3% DSS in drinking water were fixed in paraffin and processed for H&E staining. Representative examples scored for no colitis, mild colitis and severe colitis are shown at original magnifications of 10× (middle) or 20× (bottom). (B) Colon carcinogenesis. WT and IDO-/- mice were treated once i.p. with 20 mg/kg DMH and then 7 d 2% DSS in drinking water followed by normal drinking water to an endpoint of 18 wks. Colons were harvested from euthanized mice, fixed in paraffin and processed for H&E staining. Representative images of gross pathology in WT or IDO-/- mice (top panels) illustrate observations of macroscopic tumors (arrows) or prolapsed rectums (arrowheads). Representative images of histopathology in IDO-/- mice (bottom panels) illustrate observations of (a) no tumor, (b) mucosal invasion, (c) submucosal invasion and (d) muscular invasion. Original magnifications are 4× (top), 10× (middle), 20× (bottom).