Indoleamine 2,3-dioxygenase (IDO) induced by Leishmania infection of human dendritic cells

Abstract

Dendritic cells (DC) play a pivotal role in regulating immunity, establishing immunologically privileged tissue microenvironments and maintaining homoeostasis. It is becoming increasingly clear that one key mechanism that mediates many DC functions is production of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). For pathogens that cause chronic infection, exploitation of host DCs is a solution to establish and persist within a host. Leishmania parasites cause a range of clinical manifestations, all involving chronic infection, and are proficient at avoiding immune responses. We demonstrate here that infection of human myeloid-derived DC with L. major and L. donovani induces IDO expression using a mechanism that involves autocrine or paracrine stimulation with a DC-secreted factor. Leishmania-induced IDO suppresses allogeneic and tetanus toxoid-specific lymphocyte proliferation, an inhibition that is reversed with the IDO inhibitor, 1-methyl tryptophan (1-MT). Furthermore, IDO expression by human DC does not require live Leishmania infection, as parasite lysates also up-regulate IDO mRNA production. Our data suggest that one mechanism Leishmania parasites utilize to circumvent immune clearance may be to promote the induction of IDO among host DC within the infection microenvironment.

Figure 1. IDO is Induced in Human DC in Response to Leishmania Infection

A)IDO mRNA transcript expression as measured by qRT-PCR using equal concentrations of cDNA from six healthy adult human monocyte-derived DC and normalized to HPRT. Cells were left uninfected (UI) or infected in vitro with L. major (Lm) or L. donovani (Ld) or exposed to 1μg/mL of LPS (LPS) for 16 hrs. B) Western blot analysis of human monocyte-derived DC infected with L. major, L. donovani, or stimulated with LPS (1μg/mL) for 48 hrs. Blots were probed for IDO and GAPDH as a loading control. Data from one of six representative donors is presented. C) qRT-PCR of IDO mRNA from human monocyte-derived DC, infected in vitro with live L. major or stimulated with an equivalent amount of parasite lysate for 16 hrs. D) IDO mRNA detected from uninfected or L. major infected DC from the lower chamber of a transwell system. Infection in upper wells as indicated, n = nine donors. Data is expressed as mean ± standard error of th mean (SEM) fold induction over uninfected controls, *p ≤ 0.05 by Mann-Whitney Test.

Figure 2. Leishmania Infection Affects Differential Indoleamine and Interferon Transcript Expression Patterns over Time

qRT-PCR for A) IDO, B) IFNα, C) IFNβ,and D) IFNγ normalized to HPRT transcript expression levels using equal concentrations of cDNA from five healthy adult human monocyte-derived DC. Cells were either uninfected or infected in vitro with L. major and samples extracted after 2, 4, 8, and 24 hours post-infection. Data is expressed as mean transcript expression fold change over uninfected controls normalized to HPRT ± SEM. *p ≤ 0.05 compared with uninfected (UI) gene expression levels by one-way repeated measures ANOVA and Bonferroni multiple comparisons tests.

Figure 3. Leishmania-Infection Induced IDO is Active and Inhibits MLR

DC from nine healthy donors were either left uninfected (UI) or infected with either L. major (Lm) or L. donovani (Ld) and then mixed in auto- and allogeneic conditions at a ratio of 20 responders to 1 DC and incubated for 5 days. Proliferation was assessed by ³H-thymidine incorporation the last 18 hrs of culture. Data is expressed as β-particle counts per minute (CPM) with conditions listed with or without 1-MT. ^‡ p ≤ 0.05 compared to uninfected control; * p ≤ 0.05 compared to same infection lacking 1-MT as determined by Student’s T-Test. Data from three representative donors out of nine are shown.

Figure 4. Leishmania-Infection Induced IDO is Active and Inhibits Antigen-Specific Proliferation

Uninfected DC (UI) or DC infected with either L. major (Lm) or L. donovani (Ld), autologous responders, and 0.10 ng/mL tetanus toxoid were cultured for 5 days. Proliferation was assessed by ³H-thymidine incorporation the last 18 hrs of culture. Data is expressed as counts per minute (CPM) with conditions listed with or without 1-MT. ^‡ p ≤ 0.05 compared to uninfected control; * p ≤ 0.05 compared to same infection lacking 1-MT as determined by Student’s T Test.