Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology

Abstract

Endemic typhus caused by Rickettsia (R.) typhi is an emerging febrile disease that can be fatal due to multiple organ pathology. Here we analyzed the requirements for protection against R. typhi by T cells in the CB17 SCID model of infection. BALB/c wild-type mice generate CD4+ TH1 and cytotoxic CD8+ T cells both of which are sporadically reactivated in persistent infection. Either adoptively transferred CD8+ or CD4+ T cells protected R. typhi-infected CB17 SCID mice from death and provided long-term control. CD8+ T cells lacking either IFNγ or Perforin were still protective, demonstrating that the cytotoxic function of CD8+ T cells is not essential for protection. Immune wild-type CD4+ T cells produced high amounts of IFNγ, induced the release of nitric oxide in R. typhi-infected macrophages and inhibited bacterial growth in vitro via IFNγ and TNFα. However, adoptive transfer of CD4+IFNγ-/- T cells still protected 30-90% of R. typhi-infected CB17 SCID mice. These cells acquired a TH17 phenotype, producing high amounts of IL-17A and IL-22 in addition to TNFα, and inhibited bacterial growth in vitro. Surprisingly, the neutralization of either TNFα or IL-17A in CD4+IFNγ-/- T cell recipient mice did not alter bacterial elimination by these cells in vivo, led to faster recovery and enhanced survival compared to isotype-treated animals. Thus, collectively these data show that although CD4+ TH1 cells are clearly efficient in protection against R. typhi, CD4+ TH17 cells are similarly protective if the harmful effects of combined production of TNFα and IL-17A can be inhibited.

Fig 1. BALB/c mice generate cytotoxic CD8⁺ cells that are sporadically reactivated.

BALB/c mice were infected with 1×10⁶ sfu R. typhi. 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 (*p<0.05, **p<0.01, ***p<0.001).

Fig 2. BALB/c mice generate CD4⁺ T_H1 cells that are sporadically reactivated.

Spleen cells from the same mice as described in Fig 1 were stained for CD4, CD11a and for intracellular IFNγ and Granzyme B after PMA/Ionomycin restimulation. The dot plots show example stainings from day 7 or day 15 post infection. Graphs show the percentage of CD11a⁺, IFNγ⁺ and Granzyme B⁺ T cells among CD4⁺ T cells (y-axis) at indicated days post infection (x-axis). 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 (*p<0.05, **p<0.01, ***p<0.001).

Fig 3. Adoptive transfer of either CD8⁺ or CD4⁺ T cells protects CB17 SCID mice from severe disease and death.

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 R. typhi 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) (A). 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 (*p<0.05, **p<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 (**p<0.01, ***p<0.001) (B).

Fig 4. Adoptively transferred CD4⁺ T cells produce IFNγ and TNFα and both CD4⁺ and CD8⁺ T cells provide long-term control of R. typhi in vivo.

From the same mice as described in Fig 3 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 (*p<0.05, **p<0.01) (A). Bacterial content (y-axis) in the organs indicated above was quantified by qPCR in each group (R. typhi-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 (*p<0.05, **p<0.01) (B). Bacterial content was quantified in R. typhi-infected control animals (n = 5) at the time of death in the indicated organs (x-axis) (C).

Fig 5. CD8⁺Perforin^-/-, CD8⁺ IFNγ^-/- and CD4⁺IFNγ^-/- T cells are still protective.

BALB/c wild-type mice (gray circles; n = 5), BALB/c Perforin^-/- (black circles; n = 5) and BALB/c IFNγ^-/- mice (black squares; n = 5) were infected with 1×10⁶ sfu R. typhi. Non-infected BALB/c Perforin^-/- mice (white circles; n = 5) were used as a control. Weight change (left), clinical score (middle) and survival (y-axis) was assessed at indicated points in time (x-axis) (A). CD8⁺ and CD4⁺ T cells were isolated from BALB/c Perforin^-/- and IFNγ^-/- mice. 1×10⁶ CD8⁺ IFNγ^-/- (black squares), CD8⁺ Perforin^-/- (black circles) and CD4⁺ IFNγ^-/- T cells (gray squares) were adoptively transferred into CB17 SCID mice 1 day prior to the infection with 1×10⁶ sfu R. typhi. Control animals were treated with PBS instead receiving T cells (white squares). Weight change (left; n = 5 for each group), clinical score (middle; n = 5 for each group) and survival (right; n = 5 for CD8⁺ Perforin^-/- recipients and n = 10–11 for CD8⁺ and CD4⁺ IFNγ^-/- recipients) was assessed (y-axis) at indicated points in time (x-axis). 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 (*p<0.05, **p<0.01) (B). All R. typhi-infected CB17 SCID control animals and two mice of the CD4⁺ T IFNγ^-/- cell recipient group died until day 20. At the time of death, the bacterial load (y-axis) was determined in the indicated organs (x-axis) in these animals (C). The bacterial load (y-axis) was also determined in the organs of surviving animals. Organs were taken at indicated points in time from CD8⁺Perforin^-/- (day 128 post infection; n = 4), CD8⁺ IFNγ^-/- (n = 6) and CD4⁺IFNγ^-/- T cell recipients (n = 8). For the transfer of CD8⁺ IFNγ^-/- and CD4⁺ IFNγ^-/- T cells the results from two independent experiments that were terminated on day 120 and day 168 post infection are shown (x-axis). The bacteria were not detectable at all in CD8⁺Perforin^-/- T cell recipients while low amounts of R. typhi were present in five animals of the CD8⁺ IFNγ^-/- and two mice of the CD4⁺IFNγ^-/- T cell recipient groups. The figure shows the bacterial content in the organs of all animals (mean±SEM) (D).

Fig 6. Immune CD4⁺ T cells induce NO release by R. typhi-infected macrophages in vitro and inhibit bacterial growth via IFNγ and TNFα.

1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of R. typhi 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 (A). In addition, NO was detected 72h after T cell addition (B). Bacterial content in the cultures (y-axis) was assessed by prsA-specific qPCR 72h after T cell addition (C). 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 (D). 1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of R. typhi 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 prsA-specific qPCR 72h after cytokine addition (E). 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 (*p<0.05, **p<0.01).

Fig 7. Enhanced protection by CD4⁺IFNγ^-/- T cells in the absence of TNFα.

1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of R. typhi 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 prsA-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) (A). CB17 SCID mice (n = 7 for each group) were infected with 1×10⁶ sfu R. typhi. 1×10⁶ purified CD4⁺ T cells from BALB/c IFNγ^-/- mice were adoptively transferred one day prior to the infection with R. typhi. 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 (**p<0.01) (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) (*p<0.05) (C). The bacterial content (y-axis) in the organs was quantified by prsA-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 (D). 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 (*p<0.05, **p<0.01).

Fig 8. CD4⁺IFNγ^-/- differentiate into T_H17 cells that produce large amounts of IL-17A and IL-22 upon R. typhi-specific restimulation.

1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of R. typhi per cell one day prior to the addition of 2×10⁶ purified CD4⁺ T cells from either naïve or immune wild-type BALB/c or BALB/c IFNγ^-/- mice (day 7 post infection). Cytokines were quantified in the cell culture supernatant by LEGENDplex assay 72h after T cell addition (n = 4 for each group). Statistical analysis of cytokine production by wild-type CD4⁺ and CD4⁺IFNγ^-/- T cells was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test) (*p<0.05).

Fig 9. Enhanced protection by CD4⁺IFNγ^-/- T cells in the absence of IL-17A.

1×10⁶ purified CD4⁺ T cells from BALB/c IFNγ^-/- mice were adoptively transferred into CB17 SCID mice one day prior to the infection with 1×10⁶ sfu R. typhi. IL-17A was neutralized by intraperitoneal application of 500 μg anti-IL-17A every two days beginning on day 2 post infection (n = 10). A second group of T cell recipients received isotype antibody (n = 10). R. typhi-infected control mice did not receive T cells and were treated with PBS instead of antibody (n = 5). The CD4⁺ T cell frequency among leukocytes in the blood (y-axis) was determined on day 8 post transfer (left). CD8⁺ T cells were not detectable. Plasma cytokines were detected by LEGENDplex assay. Plasma from naïve CB17 SCID mice (n = 10) was used as a control. Only IFNγ (y-axis) was detectable at significant amounts. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test) (*p<0.05, ***p<0.001) (A). The state of health of the mice was monitored by weight change (y-axis, left) and a clinical score (y-axis, middle) and the survival rates (y-axis, right) were assessed. Statistical analysis of survival rates was performed with Log-rank (Mantel-Cox) test. Differences in the survival of anti-IL-17A- and isotype-treated animals were not significant (B). The bacterial content (y-axis) in the organs was quantified by prsA-specific qPCR from all control animals that succumbed to the infection at the time of death and from all CD4⁺IFNγ^-/- T cell recipients including the mouse of the isotype-treated group that died on day 16 post infection. Samples from the surviving animals were taken at the end of the experiment (day 26). Statistical analysis was performed by One-way ANOVA (Kruskal-Wallis test followed by Dunn´s post test). Asterisks indicate statistically significant differences (**p<0.01, ***p<0.001) (C).