Date Published: June 25, 2008
Publisher: Public Library of Science
Author(s): Nicholas A. Ettinger, Mary E. Wilson, Greg Matlashewski
Abstract: Visceral leishmaniasis is a potentially fatal infectious disease caused by the protozoan parasite Leishmania infantum/chagasi in the New World, or by L. donovani or L. infantum/chagasi in the Old World. Infection leads to a variety of outcomes ranging from asymptomatic infection to active disease, characterized by fevers, cachexia, hepatosplenomegaly and suppressed immune responses. We reasoned that events occurring during the initial few hours when the parasite encounters cells of the innate and adaptive immune systems are likely to influence the eventual immune response that develops. Therefore, we performed gene expression analysis using Affymetrix U133Plus2 microarray chips to investigate a model of early infection with human monocyte-derived macrophages (MDMs) challenged with wild-type L. chagasi parasites, with or without subsequent co-culture with Leishmania-naïve, autologous T-cells. Microarray data generated from total RNA were analyzed with software from the Bioconductor Project and functional clustering and pathway analysis were performed with DAVID and Gene Set Enrichment Analysis (GSEA), respectively. Many transcripts were down-regulated by infection in cultures containing macrophages alone, and the pattern indicated a lack of a classically activated phenotype. By contrast, the addition of autologous Leishmania-naïve T cells to infected macrophages resulted in a pattern of gene expression including many markers of type 1 immune cytokine activation (IFN-γ, IL-6, IL-1α, IL-1β). There was simultaneous up-regulation of a few markers of immune modulation (IL-10 cytokine accumulation; TGF-β Signaling Pathway). We suggest that the initial encounter between L. chagasi and cells of the innate and adaptive immune system stimulates primarily type 1 immune cytokine responses, despite a lack of classical macrophage activation. This local microenvironment at the site of parasite inoculation may determine the initial course of immune T-cell development.
Partial Text: Visceral leishmaniasis (VL) is a potentially fatal infectious disease caused by the protozoan parasites Leishmania chagasi/infantum in the New or in parts of the Old World, or by L. donovani in other regions of the Old World. Infection leads to a variety of outcomes ranging from asymptomatic infection to active disease, which is characterized by fevers, cachexia, hepatosplenomegaly and suppressed immune responses. Without treatment, most symptomatic patients die. Investigations into the mechanism underlying the immunosuppression during acute VL have demonstrated defective antigen-specific proliferation and IFN-γ responses to parasite antigen,– high expression of IL-10 in the spleen and serum of symptomatic VL patients– and high serum levels of IL-4, TGF-β and IL-2 receptor.–In vitro infection with Leishmania parasites suppresses macrophage microbicidal responses and IFN-γ pathway signaling,– suggesting that these suppressive changes begin at the earliest stages of infection. Whether this defect in macrophage responses to Leishmania infection is communicated to local adaptive immune cells is not known.
To test our hypothesis that the parasite induces unique changes in gene expression in both innate and adaptive cells of the immune system encountered early in infection, we examined gene expression in four parallel conditions: (a) Uninfected MDMs, (b) Infected MDMs, (c) Uninfected MDMs co-cultured with autologous T-cells and (d) Infected MDMs co-cultured with autologous T-cells. A box plot of the log2 ratios of the Uninfected expression values subtracted from the Infected expression values for all probe sets plotted separately for each donor demonstrated that although there was slightly more variation present in the MDM-only ratios for Donor 4, overall there did not appear to be substantial inter-donor variation (Figure 1). Most genes did not appear to change markedly from zero as evidenced by the narrow range of most of the inter-quartile boxes. Although the analyses presented below were generated employing all 4 donors’ data, the analyses were also tested leaving donor 4 out and did not differ substantially (data not shown). To generate the RNA samples, we only used infections where there was >70% infected macrophages at the 4 hour co-culture time point (MDM: 79.9±7.2% infection; MDM-T-cell: 76±7.7;see Methods). For subsequent experiments over longer time intervals, the percent of infected cells stayed roughly constant over the time intervals measured (see figure legends).
Our study was designed to test the hypothesis that a unique immune response to L. chagasi/infantum is initiated early during the initial interactions between the first immune system cells that encounter the parasite. These include macrophages and T-cells, elements of the innate and adaptive immune systems, respectively. To that end, we used microarrays to examine early gene expression patterns in purified Leishmania-naïve human T cells during their first encounter with infected human macrophages. The data suggested that macrophages exhibit a quiescent phenotype 24 hrs after infection with Leishmania, but Leishmania-naïve T cells respond to infected MDMs primarily with an inflammatory or a type 1 immune cytokine response. These in vitro data suggest that the initial microenvironment created at the site of Leishmania infection may be conducive to development of a type 1 adaptive immune response.