CD8⁺ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1β production

<div><p>Deregulated CD8+ T cell cytotoxicity plays a central role in enhancing disease severity in several conditions. However, we have little understanding of the mechanisms by which immunopathology develops as a consequence of cytotoxicity. Using murine models of inflammation induced by the protozoan parasite leishmania, and data obtained from patients with cutaneous leishmaniasis, we uncovered a previously unrecognized role for NLRP3 inflammasome activation and IL-1β release as a detrimental consequence of CD8+ T cell-mediated cytotoxicity, ultimately resulting in chronic inflammation. Critically, pharmacological blockade of NLRP3 or IL-1β significantly ameliorated the CD8+ T cell-driven immunopathology in leishmania-infected mice. Confirming the relevance of these findings to human leishmaniasis, blockade of the NLRP3 inflammasome in skin biopsies from leishmania-infected patients prevented IL-1β release. Thus, these studies link CD8+ T cell cytotoxicity with inflammasome activation and reveal novel avenues of treatment for cutaneous leishmaniasis, as well as other of diseases where CD8+ T cell-mediated cytotoxicity induces pathology.</p></div

Increased IL-1β in <i>L</i>. <i>braziliensis</i> lesions is dependent on CD8 T cell cytotoxicity.

<p>RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with CD8 T cells or did not receive cells and (a) the course of infection was monitored and representative images of lesions are shown. At 7 weeks post infection mice were euthanized and (b) mRNA levels for <i>IL1a</i> and <i>IL1b</i> were assessed. mRNA data is represented as a fold change (FC) over expression in naïve mice. At 7 weeks post infection, lesions were also digested and used for flow cytometric analysis. Depicted are (c) representative histogram and (d) bar graph of intracellular staining for IL-1β. (e) Ears were cultured for 48 hours and IL-1β release was measured in the supernatants by ELISA. RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with either WT or perforin-/- CD8 T cells or did not receive cells and (f) course of infection was monitored. At 7 weeks post infection mice were euthanized, lesions were digested and used for flow cytometric analysis. Depicted are (g) representative histogram and (h) bar graph of intracellular staining for IL-1β. Representative data from one of three or more independent experiments (n = 3 to 5 mice per group) with similar results are presented. <i>*p ≤ 0</i>.<i>05</i> or <i>***p ≤ 0</i>.<i>001;</i> ns, non-significant</p

Treatment of mice with NLRP3 inhibitors dampens the immunopathology caused by CD8 T cells.

<p>WT C57BL/6 mice were infected with <i>L</i>. <i>major</i> in the ear, and 2 weeks later mice were co-infected with 2×10⁵ PFU of LCMV Armstrong strain by i.p. injection. Ten days post LCMV infection mice were treated with MCC950, glyburide or vehicle; (a and b) ear thickness was assessed weekly. Five weeks post infection with <i>L</i>. <i>major</i>, mice were euthanized and the lesions were digested and the (c and d) frequency of neutrophils in the skin was determined directly ex vivo by flow cytometry. (e and f) Number of parasites in the skin was determined at 5 weeks post infection with <i>L</i>. <i>major</i>. Results in mice are data from one experiment with 5 mice per group. <i>*p</i> ≤ <i>0</i>.<i>05</i>, *<i>*p<0</i>.<i>01</i> or ***<i>p</i> ≤ <i>0</i>.<i>001</i></p

Eosinophils promote larval growth in an innate context.

<p>(A)–(C), C57BL/6, PHIL, Rag1^-/-, PHIL/Rag1^-/- and Rag2^-/-γc^-/- mice were injected IV with 25,000 NBL. (A) Body size (area) of larvae recovered from indicated strains 13 days post injection. (B) Body size (area) of larvae recovered from Rag1^-/- and Rag2^-/-γc^-/- mice, 13 days post injection. (C) PHIL/Rag1^-/- mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg⁺/IL-4^-/- mice every 48 h from 0–5 days post IV infection. Body size (area) of larvae, 13 days post injection. Each data set was collected from two experiments with similar results. Values represent mean ± SD; n = 3–4 mice. Significant differences were determined by Student’s <i>t</i> test or ANOVA and Tukey’s test. ***p < 0.0001.</p

IL-4/STAT6 signaling in eosinophils is required for larval growth.

<p>(A) Body size (area) of larvae, 17dpi. ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils every 48 h between 5 and 9 dpi or 11 and 15 dpi (oral infection). (B)–(C), ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg/STAT6^-/- mice every 48 h from 5–9 dpi. (B) Total body larval burdens in muscle, 28 dpi. (C) Body size (area) of larvae, 17 dpi. (D) ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg⁺/IL-4^-/- mice every 48 h from 5–9 dpi. Body size (area) of larvae, 17dpi. (E) ΔdblGATA mice received PBS or 5 × 10⁶ eosinophils from uninfected or infected IL-5Tg⁺ every 48 h from 5–9 dpi. Body size (area) of larvae, 17dpi. (F) Body size (area) of larvae recovered from C57BL/6 and IL-5^-/- mice injected 25,000 NBL IV, 13 days post injection. (G) IL-4^-/- mice received PBS or 5 × 10⁶ eosinophils from infected IL-5Tg⁺ or IL-5Tg⁺/IL-4^-/- mice every 48 h from 0–5 days post IV infection. Body size (area) of larvae, 13 days post injection. Each data set was collected from two experiments with similar results. Values represent mean ± SD; n = 4 mice. Significant differences were determined by ANOVA and Tukey’s test. *p < 0.05, **p < 0.001, ***p < 0.0001.</p

Immunopathology caused by CD8 T cells is IL-1β-dependent.

<p>RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with CD8 T cells or did not receive cells and at 2 weeks post infection mice were treated with (a) anti-IL-1R mAb, (b) anti-IL-1β mAb (c) anti-IL-1α mAb or (f) anakinra; ear thickness was assessed weekly. (d) 4 weeks or (g) 6 weeks post infection mice were euthanized and lesions were digested and used for flow cytometric analysis of intracellular IFN-γ and GzmB on CD8 T cells. (e and h) Parasite burden in the lesions. Graphs are data combined from 2 independent experiments (n = 3 to 5 mice per group in each experiment). C57BL/6 mice were infected with <i>L</i>. <i>major</i> in the ear, and 2 weeks later mice were co-infected with 2×10⁵ PFU of LCMV Armstrong strain by i.p. injection. Ten days post LCMV infection mice were treated with anakinra or were left untreated; (i) ear thickness was assessed weekly. Five weeks post infection with <i>L</i>. <i>major</i>, mice were euthanized and the (j) number of parasites in the skin was determined and lesions were digested and used for flow cytometric analysis of intracellular IFN-γ, and GzmB on (k) CD4 T cells or (l) CD8 T cells. Graphs are data from 2 independent experiments (n = 5 mice per group) with similar results are presented. <i>*p ≤ 0</i>.<i>05</i>, <i>**p ≤ 0</i>.<i>01</i> or <i>***p ≤ 0</i>.<i>001;</i> ns, non-significant</p

IL-4/STAT6 signaling pathway is essential for larval growth.

<p>Area of larvae recovered from (A)–(C), C57BL/6, STAT6^-/-, IL-4^-/- and IL-13^-/- mice injected 25,000 NBL IV, 13 days post injection. Total body larval burdens in muscles of (D) WT and STAT6^-/- and (E) WT and IL-4^-/-, 24 days post injection. (F) IL-10 detected in CLN cultures from WT and STAT6^-/- mice, 13 days post injection. (G) Number of CD4⁺IL-10⁺ cells per diaphragm of WT and IL-4^-/- mice, 13 days post injection. Each data set was collected from two experiments with similar results. Values represent mean ± SD; n = 4 mice. Significant differences were determined by Student’s <i>t</i> test. ***p < 0.0001.</p

The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to <i>Toxoplasma gondii</i>

<div><p>The protozoan parasite, <i>Toxoplasma</i>, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host–pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite <i>Toxoplasma</i> results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such “modifiers.”</p></div

STAT6 signaling in bone marrow-derived cells is essential for parasite growth.

<p>B6.SJL and STAT6^-/- mice were reconstituted with bone marrow cells from B6.SJL or STAT6^-/- mice for eight weeks, then infected by IV injection of 25,000 NBL. Areas of larvae were estimated, 13 days post injection. Each data set was collected from two experiments with similar results. n = 4 mice. Significant differences were determined by ANOVA and Tukey’s test. ***p < 0.0001.</p

GLUT4 and phosphorylation of Akt are enhanced in nurse cells.

<p>Detection of (A) Akt, phospho-Akt^Ser473 and (B) GLUT4 in tongues collected from Rag1^-/- mice infected by IV injection with NBL, 13 days post injection. Arrows indicate nurse cells. Solid arrowheads indicate muscle larvae. Opened arrowheads indicate uninfected muscle cells. Scale bar = 100μm. n = 3 mice.</p