Non-hematopoietic expression of IDO is integrally required for inflammatory tumor promotion

Abstract

Indoleamine 2,3-dioxygenase (IDO) is generally considered to be immunosuppressive but recent findings suggest this characterization oversimplifies its role in disease pathogenesis. Recently, we showed that IDO is essential for tumor outgrowth in the classical two-stage model of inflammatory skin carcinogenesis. Here, we report that IDO loss did not exacerbate classical inflammatory responses. Rather, IDO induction could be elicited by environmental signals and tumor promoters as an integral component of the inflammatory tissue microenvironment even in the absence of cancer. IDO loss had limited impact on tumor outgrowth in carcinogenesis models that lacked an explicit inflammatory tumor promoter. In the context of inflammatory carcinogenesis where IDO was critical to tumor development, the most important source of IDO was radiation-resistant non-hematopoietic cells, consistent with evidence that loss of the IDO regulatory tumor suppressor gene Bin1 in transformed skin cells facilitates IDO-mediated immune escape by a cell autonomous mechanism. Taken together, our results identify IDO as an integral component of 'cancer-associated' inflammation that tilts the immune system toward tumor support. More generally, they promote the concept that mediators of immune escape and cancer-associated inflammation may be genetically synonymous.

Fig. 1

IDO is not anti-inflammatory. a Skin histology after TPA treatment alone. Similar epidermal thickening is observed in wild-type and Ido1 ^−/− animals at 24 and 48 h after a single application of TPA and remains evident through 5 weeks of repeated treatment. For comparison, the last panels illustrate histology of DMBA + TPA-treated skin at 22 weeks including papilloma formation in the wild-type animal. b Skin histology after abrasion wounding. Brackets indicate the nonabraded region 3 days after wounding with the abraded region noted beyond the asterisk. KO, Ido1 ^−/− mice. c Spleen weight after TPA treatment alone. Mice were exposed to TPA alone as noted in (b), killed on day 9, and wet weight of spleens dissected at necropsy determined. Standard error in the data presented is shown

Fig. 2

Tumor promoter TPA directly activates IDO expression and cooperates with pro-inflammatory regulators IFN-γ, LPS, and IL-1β to superactivate IDO expression. Human U937 cells were untreated or treated with 100 ng/ml TPA in the absence or presence of known immune and/or IDO-activating stimuli. Culture media were harvested 24 h later to quantitate relative levels of kynurenine by LC/MS as analyzed in triplicate. The stimuli included IFN-γ (100 ng/ml), TGF-β (50 ng/ml), IL-1β (5 ng/ml), TNF-α (10 ng/ml), alum (100 μg/ml), genomic E. coli DNA (9.5 μg/ml), imiquimod (3 μg/ml), lipopolysaccharide (LPS; 100 ng/ml), poly dI:dC (10 μg/ml), heat-killed S. aureus (1.5 × 10⁸ bacteria/ml), or HMGB1 (3 μg/ml). Additive effects are apparent between TPA (dot) and the stronger IDO inducers IFN-γ and LPS (carots) or the weaker inducer IL-1β (asterisk). The experiment was repeated once with similar results

Fig. 3

IDO is not critical for carcinogenesis in the absence of an explicit pro-inflammatory driver. a IDO is not critical for skin carcinogenesis in the absence of TPA-induced inflammation. Mice were enrolled on the standard protocol of complete carcinogenesis involving repetitive exposure to carcinogen DMBA alone and tumor incidence was determined. Standard error in the data presented is shown. b IDO is not critical for DMBA-induced mammary carcinogenesis. Mice were enrolled on a standard protocol of mammary carcinogenesis involving i.p. administration of DMBA and continuous exposure to the progesterone mimetic medroxyprogesterone acetate. WT, wild-type mice; KO, Ido1 ^−/− mice. (n = 10 all groups)

Fig. 4

IDO functions primarily in non-hematopoietic cells to support inflammatory skin carcinogenesis. a Experimental design. Four-week-old recipient mice were lethally irradiated and reconstituted with bone marrow from 6-week-old donor mice. All control and reciprocal transplant possibilities in wild-type and Ido1 ^−/− mice were performed. After allowing 6 weeks for the transplanted bone marrow to repopulate the hematopoietic system, host mice were subjected to two-stage inflammatory skin carcinogenesis. b Papilloma formation in bone marrow chimeric mice. Legend indicates host-to-donor configuration in each cohort (n = 10). Standard error is indicated for each of the incidence curves. KO, Ido1 ^−/− mice

Fig. 5

IDO can exert a tumor cell autonomous function to support inflammatory skin carcinogenesis. a Tumor cell autonomous role of IDO activity in tumor formation by transformed Bin1 ^−/− skin epithelial cells. Bin1 ^−/− keratinocytes transformed by the Myc and Hras1 oncogenes (MR KECs) were injected s.c. into syngeneic immunocompetent mice and tumor formation was monitored by caliper measurements as described [17]. Seven days after tumor cell engraftment the IDO inhibitor 1MT was administered b.i.d. by oral gavage at 400 mg/kg as described previously [47]. Standard error for the data is provided for each cohort (n = 5). b Bin1 ablation increases susceptibility to inflammatory skin cancer. Bin1 mosaic null mice have been described previously [20]. Mice were enrolled on the two-stage inflammatory skin carcinogenesis protocol and papilloma formation was monitored as before (n = 10). The results presented include data collected from two separate trials. Standard error is indicated for each of the incidence curves