Orientia tsutsugamushi Stimulates an Original Gene Expression Program in Monocytes: Relationship with Gene Expression in Patients with Scrub Typhus

Orientia tsutsugamushi is the causal agent of scrub typhus, a public health problem in the Asia-Pacific region and a life-threatening disease. O. tsutsugamushi is an obligate intracellular bacterium that mainly infects endothelial cells. We demonstrated here that O. tsutsugamushi also replicated in monocytes isolated from healthy donors. In addition, O. tsutsugamushi altered the expression of more than 4,500 genes, as demonstrated by microarray analysis. The expression of type I interferon, interferon-stimulated genes and genes associated with the M1 polarization of macrophages was significantly upregulated. O. tsutsugamushi also induced the expression of apoptosis-related genes and promoted cell death in a small percentage of monocytes. Live organisms were indispensable to the type I interferon response and apoptosis and enhanced the expression of M1-associated cytokines. These data were related to the transcriptional changes detected in mononuclear cells isolated from patients with scrub typhus. Here, the microarray analyses revealed the upregulation of 613 genes, which included interferon-related genes, and some features of M1 polarization were observed in these patients, similar to what was observed in O. tsutsugamushi-stimulated monocytes in vitro. This is the first report demonstrating that monocytes are clearly polarized in vitro and ex vivo following exposure to O. tsutsugamushi. These results would improve our understanding of the pathogenesis of scrub typhus, during which interferon-mediated activation of monocytes and their subsequent polarization into an M1 phenotype appear critical. This study may give us a clue of new tools for the diagnosis of patients with scrub typhus

Activation of Indoleamine 2,3-Dioxygenase in Patients with Scrub Typhus and Its Role in Growth Restriction of <em>Orientia tsutsugamushi</em>

<div><h3>Background</h3><p>Our earlier genome-wide expression study revealed up-regulation of a tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO1), in patients with scrub typhus. This gene has been previously reported to have anti-microbial activity in a variety of infectious diseases; therefore, we aimed to prove whether it is also involved in host defense against <em>Orientia tsutsugamushi</em> (OT) infection.</p> <h3>Methodology/Principal Findings</h3><p>Using LC-MS, we observed an increased ratio of serum L-kynurenine to serum L-tryptophan in patients with scrub typhus, which suggests an active catalytic function of this enzyme upon the illness. To evaluate the effect of IDO1 activation on OT infection, a human macrophage-like cell line THP-1 was used as a study model. Although transcription of IDO1 was induced by OT infection, its functional activity was not significantly enhanced unless the cells were pretreated with IFN-γ, a potent inducer of IDO1. When the degree of infection was evaluated by quantitative real-time PCR, the relative number of OT 47 kDa gene per host genes, or infection index, was markedly reduced by IFN-γ treatment as compared to the untreated cultures at five days post-infection. Inhibition of IDO1 activity in IFN-γ treated cultures by 1-methyl-L-tryptophan, a competitive inhibitor of IDO1, resulted in partial restoration of infection index; while excessive supplementation of L-tryptophan in IFN-γ treated cultures raised the index to an even higher level than that of the untreated ones. Altogether, these data implied that IDO1 was partly involved in restriction of OT growth caused by IFN-γ through deprivation of tryptophan.</p> <h3>Conclusions/Significance</h3><p>Activation of IDO1 appeared to be a defensive mechanism downstream of IFN-γ that limited intracellular expansion of OT via tryptophan depletion. Our work provided not only the first link of in vivo activation of IDO1 and IFN-γ-mediated protection against OT infection but also highlighted the promise of this multifaceted gene in scrub typhus research.</p> </div

Kinetics of IDO1 expression in THP-1 cells.

<p>Before being infected with OT, THP-1 cells were treated with IFN-γ alone, IFN-γ combined with 1-MT, or neither. Mock-infected THP-1 cells were used as a control. Fold induction represents levels of IDO1 expression in each culture condition at indicated time points relative to that in mock-infected cells at 6 hours p.i.. For clearer illustration, data of OT-infected and mock-infected cultures are exclusively presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001731#pntd-0001731-g002" target="_blank">Figure 2A</a>, and those of all four culture conditions are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001731#pntd-0001731-g002" target="_blank">Figure 2B</a>. Means ± SEM from triplicate cultures are shown.</p

Clinical and laboratory characteristics of the patients with scrub typhus.

<p>IQR = interquantile range, DIC = disseminated intravascular coagulopathy, AST = aspartate aminotansferase, ALT = alanine aminotransferase.</p

Assessment of IDO1 activity in patients' sera.

<p>Serum concentrations of L-Trp (A) and L-Kyn (B) in patients with scrub typhus (n = 20) were compared with those in healthy donors (n = 10). L-Kyn/L-Trp ratios (C) were calculated to reflect IDO1 activity. Data were derived from triplicate measurement. Median and interquantile range are presented.</p

Assessment of OT growth in THP-1 cultures.

<p>Before being infected with OT, THP-1 cells were treated with IFN-γ alone, IFN-γ combined with 1-MT, or IFN-γ supplemented with L-Trp at 400 µg/ml (A) or 1 mg/ml (B). The infection index represents the degree of OT growth in each culture condition at indicated time points relative to that in mock-infected cells at 1 day p.i.. Means ± SEM from triplicate cultures are shown.</p

Assessment of IDO1 activity in OT-infected THP-1 cultures.

<p>Before being infected with OT, THP-1 cells were treated with IFN-γ alone, IFN-γ combined with 1-MT, or neither. Mock-infected THP-1 cells were used as a control. Levels of L-Trp (A) and L-Kyn (B, C) were measured in culture supernatant at 6, 24, 72, and 120 hours p.i.. IDO1 activity was reflected by the ratio of L-Kyn to L-Trp (D, E) at each indicated time point. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001731#pntd-0001731-g003" target="_blank">Figure 3B and 3D</a> represent the same dataset as <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001731#pntd-0001731-g003" target="_blank">Figure 3C and 3E</a> respectively, but those treated with IFN-γ combined with 1-MT were excluded for clearer illustration. Means ± SEM from triplicate cultures are shown. <u>Note</u>: L-Trp concentration fell below a detectable level at 24, 48 and 72 hours p.i.; therefore, the lower limit of detection of L-Trp, 0.05 ng/ml, was used in the calculation of L-Kyn/L-Trp ratio for these time points.</p