Phenotypic Characterization of Peripheral T Cells and Their Dynamics in Scrub Typhus Patients

<div><h3>Background</h3><p>Scrub typhus, caused by <em>Orientia tsutsugamushi</em> infection, is one of the main causes of febrile illness in the Asia-Pacific region. Although cell-mediated immunity plays an important role in protection, little is known about the phenotypic changes and dynamics of leukocytes in scrub typhus patients.</p> <h3>Methodology/Principal Findings</h3><p>To reveal the underlying mechanisms of immunological pathogenesis, we extensively analyzed peripheral blood leukocytes, especially T cells, during acute and convalescent phases of infection in human patients and compared with healthy volunteers. We observed neutrophilia and CD4⁺ T lymphopenia in the acute phase of infection, followed by proliferation of CD8⁺ T cells during the convalescent phase. Massive T cell apoptosis was detected in the acute phase and preferential increase of CD8⁺ T cells with activated phenotypes was observed in both acute and convalescent phases, which might be associated or correlated with elevated serum IL-7 and IL-15. Interestingly, peripheral Treg cells were significantly down-regulated throughout the disease course.</p> <h3>Conclusions/Significance</h3><p>The remarkable decrease of CD4⁺ T cells, including Treg cells, during the acute phase of infection may contribute to the loss of immunological memory that are often observed in vaccine studies and recurrent human infection.</p> </div

Apoptosis and proliferation of T cells during <i>Orientia</i> infection.

<p>A and B. PBMCs were stained with antibodies against CD4, CD8, Annexin V, or Ki-67 and then analyzed by flow cytometry. The frequencies of Annexin V- (<i>A</i>) or Ki-67- (<i>B</i>) positive cells in CD4⁺ and CD8⁺ T cells were compared among healthy controls (HC, n = 6, open circle) and scrub typhus patients at acute phase (AP, n = 13–15, gray circle) or convalescent phase (CP, n = 6, black circle). Red bars indicate the mean value. C. The CD8/CD4 ratio of apoptotic or proliferating cells were compared among the patients and healthy volunteers. D. The levels of IL-7 and IL-15 (pg/ml) in the sera were measured and compared. Error bars indicate standard error from the mean value (C <i>and</i> D). <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

Profiles of CD4⁺Foxp3⁺ or CD4⁺CD25⁺⁺ regulatory T cells in scrub typhus patients.

<p>A. PBMCs were stained with antibodies against CD4 and CD25 or Foxp3 and then analyzed on a flow cytometer. Representative dot plots show the identification of CD25⁺⁺ T cells (upper panels) or Foxp3⁺ T cells (lower panels) within CD4⁺ T cells. Numbers in the plots indicate the frequencies (%) of the gated cells in total PBMCs. B. The frequency of CD4⁺CD25⁺⁺ or CD4⁺Foxp3⁺ T cells were compared between healthy controls (HC, n = 7, open circle) and scrub typhus patients at acute phase (AP, n = 10, gray circle) or convalescent phase (CP, n = 12, black circle). C. PBMCs were stained with antibodies against CD4 and Fas or CCR4, followed by intracellular staining of Foxp3 and/or CTLA-4 after fixation and permeablization. Mean fluorescent Intensities (MFI) representing CTLA-4, Fas, or CCR4 expression in CD4⁺Foxp3⁺ regulatory cells from healthy controls (HC, n = 7–8, open circle) or the patients (AP, n = 7–10, gray circle and CP, n = 9–12, black circle) were compared. Red bars indicate the mean value and <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test or Wilcoxon signed-rank test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

Profiles of type 1 and type 2 T cells in peripheral blood of scrub typhus patients.

<p>PBMCs were stained with antibodies against CXCR3, CCR4, CD4, and CD8 and then analyzed on a flow cytometer. A. The frequencies of Th1 (CD4⁺CXCR3⁺) or Th2 (CD4⁺CCR4⁺) within CD4⁺ T cells were compared among healthy controls (HC, n = 6, open circle) and the patients in acute phase (AP, n = 12, gray circle) or convalescent phase (CP, n = 5, black circle). B. The frequencies of Tc1 (CD8⁺CXCR3⁺) and Tc2 (CD8⁺CCR4⁺) within CD8⁺ T cells were compared among healthy controls and the patients as in <i>A</i>. Red bars indicate the mean value and <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

Changes of effector and memory T cell subsets in scrub typhus patients.

<p>A. Representative contour plots show the frequencies of each subsets within CD4⁺ (upper panels) or CD8⁺ (lower panels) T cell populations. Numbers within the plots indicate the percentage of each subset. B. The frequencies of naïve (CCR7⁺CD45RA⁺), central memory (CM, CCR7⁺CD45RA⁻), and effector memory (EM, CCR7⁻CD45RA⁻), CD45RA⁺ EM (EM_CD45RA+, for CD8⁺ T cells), or CCR7⁻CD45RA⁺ (for CD4⁺ T cells) T-cell subsets in healthy controls (HC, n = 9) and scrub typhus patients at acute phase (AP, n = 15) or convalescent phase (CP, n = 17) were compared. Error bars indicate standard error of mean values. <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test or Wilcoxon signed-rank test. *, <i>p</i><0.05; **, <i>p</i><0.01; and ***, <i>p</i><0.001 (compared with that of healthy control); †, <i>p</i><0.05 and ††, <i>p</i><0.01 (compared with that of the patients at acute phase).</p

Profiles of peripheral blood leukocytes and T lymphocytes in scrub typhus patients.

<p>A. The frequencies (%) of neutrophils, lymphocytes, and monocytes in blood leukocytes from healthy controls (HC, n = 9, open circle) were determined using a hematology analyzer and compared to frequencies in scrub typhus patients during acute phase (AP, n = 17, gray circle) or convalescent phase (CP, n = 17, black circle) of infection. B. Peripheral blood mononuclear cells (PBMCs) from scrub typhus patients and healthy controls were stained with anti-CD3 and CD8 antibodies and analyzed by flow cytometry (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001789#pntd.0001789.s001" target="_blank">Figure S1</a>). The frequencies (% in lymphocytes) and absolute numbers (cells/mm²) of CD4⁺ or CD8⁺ T cells in PBMCs of the patients (AP, n = 15 and CP, n = 15) and healthy controls (HC, n = 9) were compared. The absolute number of each leukocyte subset was calculated based on the leukocyte differential counts and the frequency information obtained from flow cytometry. For example, CD4⁺ T cells count = (lymphocyte count) x (% of CD4+ T cells)/100. The data of each individual person are presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001789#pntd.0001789.s008" target="_blank">Table S3</a>. Red bars indicate the mean value and <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test or Wilcoxon signed-rank test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

Multiple <i>Orientia tsutsugamushi</i> Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α

<div><p>Background</p><p><i>Orientia tsutsugamushi</i>, the causative agent of scrub typhus, is an obligate intracellular bacterium. Previously, a large number of genes that encode proteins containing eukaryotic protein-protein interaction motifs such as ankyrin-repeat (Ank) domains were identified in the <i>O. tsutsugamushi</i> genome. However, little is known about the Ank protein function in <i>O. tsutsugamushi.</i></p><p>Methodology/Principal Findings</p><p>To characterize the function of Ank proteins, we investigated a group of Ank proteins containing an F-box–like domain in the C-terminus in addition to the Ank domains. All nine selected <i>ank</i> genes were expressed at the transcriptional level in host cells infected with <i>O. tsutsugamushi</i>, and specific antibody responses against three Ank proteins were detected in the serum from human patients, indicating an active expression of the bacterial Ank proteins post infection. When ectopically expressed in HeLa cells, the Ank proteins of <i>O. tsutsugamushi</i> were consistently found in the nucleus and/or cytoplasm. In GST pull-down assays, multiple Ank proteins specifically interacted with Cullin1 and Skp1, core components of the SCF1 ubiquitin ligase complex, as well as the eukaryotic elongation factor 1 α (EF1α). Moreover, one Ank protein co-localized with the identified host targets and induced downregulation of EF1α potentially via enhanced ubiquitination. The downregulation of EF1α was observed consistently in diverse host cell types infected with <i>O. tsutsugamushi</i>.</p><p>Conclusion/Significance</p><p>These results suggest that conserved targeting and subsequent degradation of EF1α by multiple <i>O. tsutsugamushi</i> Ank proteins could be a novel bacterial strategy for replication and/or pathogenesis during mammalian host infection.</p></div

Subcellular localization of Ank proteins in HeLa cells.

<p>Nine Ank proteins were ectopically expressed in HeLa cells as N-terminal Flag-tagged forms. Cells were transfected with each construct for 18 h and subsequently fixed, permeabilized, and stained with anti-Flag antibody. Host cell nuclei can be identified in the corresponding DIC image to the right of each immunofluorescent image.</p

Downregulation of EF1α in various types of host cell infected with <i>O. tsutsugamushi</i>.

<p>(A) The results of immunoblot analysis of the total cellular protein isolated from ECV304 cells infected with <i>O. tsutsugamushi</i> at the indicated time points are shown. Protein levels were standardized to GAPDH, and the level of TSA56, a type-specific antigen of <i>O. tsutsugamushi</i>, was monitored to confirm bacterial replication during the infection periods. EF1α levels during the infection were analyzed using anti-EF1α antibody. (B) EF1α levels were analyzed in lysates prepared from different types of cell lines at 2 d after the infection with <i>O. tsutsugamushi</i> and compared with that of uninfected cells. GAPDH and TSA56 were monitored simultaneously as protein loading and bacterial infection controls, respectively. UI, uninfected; I, infected. (C) EF1α levels were analyzed in HeLa cells treated with MG132 (10 µM) for 4 h at 2 d after infection with <i>O. tsutsugamushi</i>. GAPDH was used as protein loading control. (D) EF1α mRNA levels were examined by real-time RT-PCR and normalized to β-actin mRNAs in HeLa cells infected with <i>O. tsutsugamushi</i>. The data are presented as mean+SD of three independent experiments.</p

Identification of cellular proteins that interact with Ank proteins.

<p>(A) Glutathione-Sepharose beads containing GST or one of the nine Ank proteins fused with GST were mixed with ECV304 cell lysate. Cellular interacting proteins were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Arrows indicate Cullin1 and EF1α, which were identified by mass spectrometry. (B) Immunoblot analyses were performed using specific antibodies and the cellular protein precipitates obtained from GST pull-down assays. At the bottom of the image, GST and the recombinant Ank proteins used in the pull-down assays are visualized after Coomassie brilliant blue (CBB).</p