CD4⁺ T Cells Are as Protective as CD8⁺ T Cells against <i>Rickettsia typhi</i> Infection by Activating Macrophage Bactericidal Activity

<div><p><i>Rickettsia typhi</i> is an intracellular bacterium that causes endemic typhus, a febrile disease that can be fatal due to complications including pneumonia, hepatitis and meningoencephalitis, the latter being a regular outcome in T and B cell-deficient C57BL/6 RAG1^-/- mice upon <i>Rickettsia typhi</i> infection. Here, we show that CD4⁺ T_H1 cells that are generated in C57BL/6 mice upon <i>R</i>. <i>typhi</i> infection are as protective as cytotoxic CD8⁺ T cells. CD4⁺- as well as CD8⁺-deficient C57BL/6 survived the infection without showing symptoms of disease at any point in time. Moreover, adoptively transferred CD8⁺ and CD4⁺ immune T cells entered the CNS of C57BL/6 RAG1^-/- mice with advanced infection and both eradicated the bacteria. However, immune CD4⁺ T cells protected only approximately 60% of the animals from death. They induced the expression of iNOS in infiltrating macrophages as well as in resident microglia in the CNS which can contribute to bacterial killing but also accelerate pathology. <i>In vitro</i> immune CD4⁺ T cells inhibited bacterial growth in infected macrophages which was in part mediated by the release of IFNγ. Collectively, our data demonstrate that CD4⁺ T cells are as protective as CD8⁺ T cells against <i>R</i>. <i>typhi</i>, provided that CD4⁺ T_H1 effector cells are present in time to support bactericidal activity of phagocytes via the release of IFNγ and other factors. With regard to vaccination against TG <i>Rickettsiae</i>, our findings suggest that the induction of CD4⁺ T_H1 effector cells is sufficient for protection.</p></div

Immune CD4⁺ T cells induce NO release by <i>R</i>. <i>typhi</i>-infected macrophages <i>in vitro</i> and inhibit bacterial growth via IFNγ and TNFα.

<p>1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of 2×10⁶ purified CD4⁺ T cells from either naïve or immune BALB/c mice (day 7 post infection). IFNγ and TNFα were neutralized by the addition of 10 μg/ml anti-IFNγ and/or anti-TNFα as indicated on the x-axis. Cytokines were quantified in the supernatants 72h after T cell addition by LEGENDplex assay. IFNγ (left, y-axis), TNFα (middle, y-axis), IL-22 (right, y-axis) and IL-2 (below, left) are shown. Other cytokines were not detectable (<b>A</b>). In addition, NO was detected 72h after T cell addition (<b>B</b>). Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after T cell addition (<b>C</b>). 1×10⁶ bone-marrow-derived BALB/c macrophages were treated with recombinant IFNγ (1 U/ml) or TNFα (400 U/ml). NO was quantified in the cell culture supernatants after 72h (<b>D</b>). 1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of recombinant IFNγ (1 U/ml) or TNFα (400 U/ml). The cytokines were neutralized by simultaneous addition of either anti-TNFα or anti-IFNγ (10 μg/ml each) as indicated on the x-axis. Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after cytokine addition (<b>E</b>). Graphs show the mean±SEM of combined results from 2 independent experiments (n = 4 T cells from each group of mice (A-C) and n = 2 for the treatment with recombinant cytokines (D-E)). Statistical analysis was performed by One-way ANOVA (Kruskal-Wallis test followed by Dunn´s post test). Asterisks indicate significant differences (*<i>p</i><0.05, **<i>p</i><0.01).</p

Adoptive transfer of either CD8⁺ or CD4⁺ T cells protects CB17 SCID mice from severe disease and death.

<p>CD4⁺ and CD8⁺ T cells were isolated from naïve BALB/c mice. 1×10⁶ T cells were adoptively transferred into CB17 SCID mice 1 day prior to infection with 1×10⁶ sfu <i>R</i>. <i>typhi</i> or treatment with PBS (Control). Spleen and blood of the animals were analyzed for the presence of T cells on day 7 post infection. The dot plots show example stainings of spleen cells for CD4 and CD8 from a CB17 SCID control mouse, a CD4⁺ T cell recipient (middle) and a CD8⁺ T cell recipient (right). The graphs show the statistical analysis of the spleen (left) and blood (right). The percentage (y-axis) of CD4⁺ and CD8⁺ T cells (x-axis) was determined for control mice (n = 5; white bars), CD4⁺ T cell recipients (n = 5; gray bars) and CD8⁺ T cell recipients (n = 5; black bars). On average CD4⁺ T cell recipients contained 10.1±2.6% CD4⁺ T cells while CD8⁺ T cells were virtually absent (0.9±0.1%). 2.8±0.2% CD8⁺ T cells were detected in CD8⁺ T cells while CD4⁺ T cells were absent (0.6±0.1%). The percentage of T cells in the blood was lower (3.0±0.8% CD4⁺ T cells in CD4⁺ T cell recipient and 1.2±0.3% CD8⁺ T cells in CD8⁺ recipients) (<b>A</b>). Weight change (n = 5 for control animals and n = 6 for T cell recipient groups), clinical score (n = 5 for control animals and n = 6 for T cell recipient groups), survival (n = 11 for each group) and serum GPT levels (n = 3–5 for each group) were assessed (y-axis) at indicated points in time (x-axis). Differences in the weight change between CD4⁺ and CD8⁺ T cell recipients were compared by Mann-Whitney U test at indicated points in time. Statistical analysis of GPT levels was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post) test. Asterisks indicate significant differences compared to day 0 (*<i>p</i><0.05, **<i>p</i><0.01). The survival graph shows combined results from 2 independent experiments. Statistical analysis was performed with Log-rank (Mantel-Cox) test. Asterisks indicate significant differences compared to control animals (**<i>p</i><0.01, ***<i>p</i><0.001) (<b>B</b>).</p

Liver Necrosis and Lethal Systemic Inflammation in a Murine Model of <i>Rickettsia typhi</i> Infection: Role of Neutrophils, Macrophages and NK Cells

<div><p><i>Rickettsia</i> (<i>R</i>.) <i>typhi</i> is the causative agent of endemic typhus, an emerging febrile disease that is associated with complications such as pneumonia, encephalitis and liver dysfunction. To elucidate how innate immune mechanisms contribute to defense and pathology we here analyzed <i>R</i>. <i>typhi</i> infection of CB17 SCID mice that are congenic to BALB/c mice but lack adaptive immunity. CB17 SCID mice succumbed to <i>R</i>. <i>typhi</i> infection within 21 days and showed high bacterial load in spleen, brain, lung, and liver. Most evident pathological changes in <i>R</i>. <i>typhi</i>-infected CB17 SCID mice were massive liver necrosis and splenomegaly due to the disproportionate accumulation of neutrophils and macrophages (MΦ). Both neutrophils and MΦ infiltrated the liver and harbored <i>R</i>. <i>typhi</i>. Both cell populations expressed iNOS and produced reactive oxygen species (ROS) and, thus, exhibited an inflammatory and bactericidal phenotype. Surprisingly, depletion of neutrophils completely prevented liver necrosis but neither altered bacterial load nor protected CB17 SCID mice from death. Furthermore, the absence of neutrophils had no impact on the overwhelming systemic inflammatory response in these mice. This response was predominantly driven by activated MΦ and NK cells both of which expressed IFNγ and is considered as the reason of death. Finally, we observed that iNOS expression by MΦ and neutrophils did not correlate with <i>R</i>. <i>typhi</i> uptake <i>in vivo</i>. Moreover, we demonstrate that MΦ hardly respond to <i>R</i>. <i>typhi in vitro</i>. These findings indicate that <i>R</i>. <i>typhi</i> enters MΦ and also neutrophils unrecognized and that activation of these cells is mediated by other mechanisms in the context of tissue damage <i>in vivo</i>.</p></div

Enhanced protection by CD4⁺IFNγ^-/- T cells in the absence of TNFα.

<p>1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of 2×10⁶ purified CD4⁺ T cells from either naïve or immune BALB/c IFNγ^-/- mice (day 7 post infection). TNFα was neutralized by simultaneous addition of 10 μg/ml anti-TNFα. Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after T cell addition. Graphs show the mean and SEM of combined results from two independent experiments (n = 4 T cells from each group of mice) (<b>A</b>). CB17 SCID mice (n = 7 for each group) were infected with 1×10⁶ sfu <i>R</i>. <i>typhi</i>. 1×10⁶ purified CD4⁺ T cells from BALB/c IFNγ^-/- mice were adoptively transferred one day prior to the infection with <i>R</i>. <i>typhi</i>. Control groups of mice received PBS instead. TNFα was neutralized by intraperitoneal application of 500 μg anti-TNFα every three days beginning on day 3 post infection. Control animals received equal amounts of isotype antibody. The state of health of the mice was monitored by weight change (y-axis, upper left) and a clinical score (y-axis, upper right) and the survival rates (y-axis, below) were assessed. Dotted lines show the data for surviving animals of the isotype- and anti-TNFα-treated groups of CD4⁺IFNγ^-/- recipients. Statistical analysis of survival rates was performed with Log-rank (Mantel-Cox) test. Asterisks indicate significant differences compared to control animals (**<i>p</i><0.01) (<b>B</b>). Serum GPT levels (y-axis) were assessed from all groups of animals at the time of death and in surviving animals at the end of the experiment (day 34). Combined results are shown. Each dot represents a single mouse. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test) (*<i>p</i><0.05) (<b>C</b>). The bacterial content (y-axis) in the organs was quantified by <i>prsA</i>-specific qPCR from all animals that succumbed to the infection at the time of death and from surviving animals at the end of the experiment (day 34) as indicated on the x-axis (<b>D</b>). Statistical analysis for C and D was performed by One-way ANOVA (Kruskal-Wallis test followed by Dunn´s post test). Asterisks indicate statistically significant differences (*<i>p</i><0.05, **<i>p</i><0.01).</p

BALB/c mice generate cytotoxic CD8⁺ cells that are sporadically reactivated.

<p>BALB/c mice were infected with 1×10⁶ sfu <i>R</i>. <i>typhi</i>. Control mice received PBS instead and were used as "day 0" control. Spleen cells were isolated and stained for CD8, KLRG1 and CD11a or restimulated with PMA/Ionomycin for 4h and stained for CD8 and intracellular IFNγ and Granzyme B. The dot plots show example stainings from day 7 post infection. Mice were analyzed for cytokine and Granzyme B expression on day 0, 7 and 15 (n = 6) and day 35 (n = 4). 3–4 mice were analyzed for KLRG1 and CD11a expression. Graphs show the percentage of KLRG1⁺, CD11a⁺, Granzyme B⁺ and IFNγ⁺ T cells among CD8⁺ T cells (y-axis) at indicated days post infection (x-axis). Graphs show combined results from 2 independent experiments. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences compared to day 0 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Adoptively transferred CD4⁺ T cells produce IFNγ and TNFα and both CD4⁺ and CD8⁺ T cells provide long-term control of <i>R</i>. <i>typhi in vivo</i>.

<p>From the same mice as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005404#pntd.0005404.g003" target="_blank">Fig 3</a> cytokine levels (y-axis) were determined in plasma on day 7 post infection (n = 7 for each group as indicated on the x-axis). In addition, plasma from non-infected PBS-treated mice (ni; n = 6) was analyzed. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences (*<i>p</i><0.05, **<i>p</i><0.01) (<b>A</b>). Bacterial content (y-axis) in the organs indicated above was quantified by qPCR in each group (<i>R</i>. <i>typhi</i>-infected control mice: open circles, CD4⁺ T cell recipients: gray circles; CD8⁺ T cell recipients: black circles) on day 7 (n = 5 for each group) and when the experiment was terminated on day 175 (n = 5 for control mice and CD4⁺ T cell recipients; n = 4 for CD8⁺ T cell recipients) post infection (x-axis). Each symbol represents a single mouse. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences (*<i>p</i><0.05, **<i>p</i><0.01) (<b>B</b>). Bacterial content was quantified in <i>R</i>. <i>typhi</i>-infected control animals (n = 5) at the time of death in the indicated organs (x-axis) (<b>C</b>).</p

C57BL/6 mice mount a cytotoxic CD8⁺ T cell and CD4⁺ T_H1 response.

<p>CD8⁺ T cells in the spleen from non-infected control mice (d0) and <i>R</i>. <i>typhi</i>-infected C57BL/6 mice were analyzed for CD11a and KLRG1 expression at indicated points in time (x-axis). The percentage of CD11a⁺ and KLRG1⁺ as well as the absolute numbers of KLRG1-expressing CD8⁺ T cells (y-axis) are depicted (<b>A</b>). Spleen cells were restimulated <i>in vitro</i> with PMA and ionomycin. CD8⁺ T cells were analyzed for intracellular expression of IFNγ and granzyme B by flow cytometry. Representative dot plots of the stainings from a control mouse and a <i>R</i>. <i>typhi</i>-infected mouse at day 7 post infection are depicted. Graphs show the percentage of IFNγ⁺ and Granzyme B⁺ cells among CD8⁺ T cells (y-axis) at indicated points in time (x-axis) (<b>B</b>). PMA/Ionomycin-restimulated CD4⁺ T cells from the same cultures were analyzed for intracellular expression of IFNγ by flow cytometry. Representative dot plots of the stainings from a control mouse and a <i>R</i>. <i>typhi</i>-infected mouse at day 7 post infection are depicted. Graphs show the percentage of IFNγ⁺ cells among CD4⁺ T cells (y-axis) at indicated points in time (x-axis) (<b>C</b>). Each symbol represents a single mouse. Statistical analysis was performed with Kruskal-Wallis and Dunn´s post test. Asterisks indicate statistically significant differences compared to day 0 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

T cells enter the brain after adoptive transfer into <i>R</i>. <i>typhi</i>-infected C57BL/6 RAG1^-/- mice.

<p>CD4⁺ and CD8⁺ T cells were purified from C57BL/6 mice 21 days post infection to obtain immune T cells. T cells were adoptively transferred into C57BL/6 RAG1^-/- on day 63 post infection with <i>R</i>. <i>typhi</i>, which is approximately 20 days prior to the onset of disease. Control mice received PBS instead of T cells (<b>A</b>). Blood cells were analyzed for the presence of CD4⁺ and CD8⁺ T cells by flow cytometry on day 7 post T cell transfer by flow cytometry. Dot plots show representative stainings of the blood from a control mouse, a CD4⁺ and a CD8⁺ T cell recipient. Graphs show the percentage (y-axis) of CD4⁺ and CD8⁺ T cells as indicated on the x-axis among blood leukocytes in the groups of CD4⁺ and CD8⁺ recipient mice (<b>B</b>). Brain cells were analyzed for the presence of CD4⁺ and CD8⁺ T cells by flow cytometry gated on CD45^high cells. Representative stainings of a naïve C57BL/6 mouse that was used as an additional control, a <i>R</i>. <i>typhi</i>-infected C57BL/6 RAG1^-/- control mouse and a CD4⁺ and CD8⁺ T cell recipient are shown. Graphs show the percentage (y-axis) of CD4⁺ and CD8⁺ T cells as indicated on the x-axis among CD45^high cells in the brains of the groups of CD4⁺ and CD8⁺ recipient mice (<b>C</b>). Each symbol represents a single mouse.</p

Bacterial content in the organs of surviving CD8⁺Perforin^-/-, CD8⁺IFNγ^-/- and CD4⁺IFNγ^-/- T cell recipients.

<p>Bacterial content in the organs of surviving CD8⁺Perforin^-/-, CD8⁺IFNγ^-/- and CD4⁺IFNγ^-/- T cell recipients.</p