Abstract
The innate immune system fights infection with inflammasomes and interferons. Facultative bacterial pathogens that inhabit the host cytosol avoid inflammasomes1,2,3,4,5,6 and are often insensitive to type I interferons (IFN-I), but are restricted by IFN-γ7,8,9,10,11. However, it remains unclear how obligate cytosolic bacterial pathogens, including Rickettsia species, interact with innate immunity. Here, we report that the human pathogen Rickettsia parkeri is sensitive to IFN-I and benefits from inflammasome-mediated host cell death that antagonizes IFN-I. R. parkeri-induced cell death requires the cytosolic lipopolysaccharide (LPS) receptor caspase-11 and antagonizes IFN-I production mediated by the DNA sensor cGAS. The restrictive effects of IFN-I require the interferon regulatory factor IRF5, which upregulates genes encoding guanylate-binding proteins (GBPs) and inducible nitric oxide synthase (iNOS), which we found to inhibit R. parkeri. Mice lacking both IFN-I and IFN-γ receptors succumb to R. parkeri, revealing critical and overlapping roles for these cytokines in vivo. The interactions of R. parkeri with inflammasomes and interferons are similar to those of viruses, which can exploit the inflammasome to avoid IFN-I12, are restricted by IFN-I via IRF513,14, and are controlled by IFN-I and IFN-γ in vivo15,16,17. Our results suggest that the innate immune response to an obligate cytosolic bacterial pathogen lies at the intersection of antibacterial and antiviral responses.
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Data availability
The RNA-seq datasets generated and analysed during this study are available in the Gene Expression Ombibus (GEO) repository, accession no. GSE128211. R. parkeri strains were authenticated by whole-genome sequencing and are available in the National Center for Biotechnology Information (NCBI) Trace and Short-Read Archive; Sequence Read Archive (SRA), accession no. SRX4401164. Source Data for Figs. 1–4 and Extended Data Figs. 3 and 5 are provided with the paper.
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Acknowledgements
We thank J. Coers (Duke University) for femurs from Gbpchr3−/− mice. We thank M. Diamond (Washington University, St. Louis) for femurs from Irf5−/−, Ifit1−/− and Ifit2−/− mice. We thank D. Rader (the University of Pennsylvania) for femurs from LipG−/− mice. We thank E. Harris (University of California, Berkeley) for AG129 mice (originally obtained from M. Aguet, Swiss Institute for Experimental Research). We thank G. Barton (University of California, Berkeley) for advice and for Irf3−/−Irf7−/− mice and Tnfrsf1a−/−Tnfrsf1b−/− mice. We thank D. Raulet (University of California, Berkeley) and C. Nicolai for Rag2−/− mice. We thank N. Fischer for the critical reading of this manuscript. P.E. was supported by postdoctoral fellowships from the Foundation Olle Engkvist Byggmästare, the Swedish Society of Medical Research (SSMF) and the Sweden–America Foundation. M.D.W. was supported by NIH/NIAID grant nos. R01 AI109044, R21 AI109270 and R21 AI138550. J.A.F. was supported by NIH/National Institute of General Medical Sciences (NIGMS) grant no. 2T34GM008612-24. R.E.V. is a Howard Hughes Medical Institute (HHMI) investigator and is supported by NIH/NIAID grant nos. AI075039 and AI063302.
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T.P.B. performed and analysed in vitro and in vivo experiments. P.E. provided reagents and contributed to in vivo experiments. R.A.C. contributed to the breeding of the mice. J.A.F. collected microscopy images and contributed to image analysis. T.P.B. wrote the original draft of this manuscript with guidance and edits from M.D.W. Critical reading and further edits were also provided by P.E. and R.E.V. Supervision was provided by T.P.B., R.E.V. and M.D.W.
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Extended data
Extended Data Fig. 1 Inflammasome activation benefits R. parkeri by antagonizing the IFN-I response in mouse macrophages.
a, Measurement of Listeria monocytogenes (Lm) CFU in BMDMs, MOI of 1. 3,000 U of IFN-β added at t = 0. n = 3 independent experiments. b, Measurement of IFN-I in supernatants of WT BMDMs infected with R. parkeri (24 hpi) or L. monocytogenes (8 hpi), MOI of 1. Supernatants were used to stimulate a luciferase-expressing cell line and relative light units (RLU) were measured and compared between each sample and uninfected cells. n = 7 and 7 biological replicates. c, Time course of LDH release (blue) and IFN-I abundance as measured by RLU production (pink), in WT BMDMs infected with R. parkeri, MOI of 1. n = 3 independent experiments. d, Time course of LDH release (blue) and IFN-I abundance (pink), in Casp1/11-/- BMDMs infected with R. parkeri, MOI of 1. n = 3 independent experiments. e, Images of BMDMs infected with R. parkeri, MOI of 1, at 72 hpi. Scale bar = 100 μm. Experiments were repeated 3 times with similar results. f, Measurement of R. parkeri abundance in BMDMs, MOI of 0.2. ‘Supe’ indicates 200 μl of conditioned supernatant collected at 24 hpi from Casp1/11-/- BMDMs infected at an MOI of 1. Antibody was added at t = 0. The indicated statistical differences (*) are between WT and WT+supernatant. No statistical differences were observed between the samples treated with supernatant. g, Measurement of R. parkeri abundance in BMDMs, MOI of 1. Antibodies were added at t = 0. n = 3 independent experiments. h, Host cell death during R. parkeri infection of BMDMs. LDH release was measured at 24 hpi upon R. parkeri infection of the indicated BMDMs, MOI of 1. n = 6, 4, 4, and 4 biological replicates. Statistical comparisons in panel h were made between each sample and WT. Statistical analyses in panels a, b, f, and g used a two-tailed Student’s T-test. Statistical analyses in panel h used a one-way ANOVA with multiple comparisons and Tukey post-hoc test. For all panels: data are expressed as means and error bars represent the SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.
Extended Data Fig. 2 IRF5-regulated genes, including Gbp2 and Nos2, are antirickettsial ISGs.
a, R. parkeri abundance in BMDMs. “supe” indicates 200 μl conditioned supernatant from infected Casp1/11-/- BMDMs. n = 3 independent experiments. b, qPCR of ISGs, normalized to actin. WT and mutant BMDMs were infected with R. parkeri and treated with IFN-β and RNA was analyzed at 12 hpi. Data are fold upregulation as compared to infected cells not treated with IFN-β. n = 3 separate experiments. For statistics, values were compared to the WT value for each primer set. c, R. parkeri abundance in BMDMs. “supe” indicates 200 ul conditioned supernatant from infected Casp1/11-/- BMDMs. n = 3 independent experiments. Statistical differences (*) are shown between WT and WT + supernatant. No statistical differences (ns) were observed between WT+ supernatant and mutant cells + supernatant. d, R. parkeri abundance in BMDMs. “supe” indicates 200 μl conditioned supernatant from infected Casp1/11-/- BMDMs. The L-NIL final concentration was 1 mM, added at t = 0. n = 3 independent experiments. e, Quantification of GBP2 colocalization with R. parkeri using immunofluorescence microscopy, in BMDMs, MOI of 1. Each data point is an independent experiment and includes quantification from more than 5 images totaling at least 150 bacteria. n = 3 independent experiments. Lines connect means for each time point. f, Quantification of GBP2 colocalization with R. parkeri using immunofluorescence microscopy, in BMDMs, MOI of 1 at 3 hpi. Each data point is an independent experiment and includes quantification from more than 5 images totaling at least 150 bacteria. n = 7, 7, 3, and 3 independent experiments. For experiments with exogenous IFN-I, 100 U of rIFN-β was added overnight prior to infection. Statistical analyses in panels a, b, c, d, and e used a two-tailed Student’s T-test; statistical analyses in panel f used a one-way ANOVA with multiple comparisons and Tukey post-hoc test; For all panels, data are expressed as means and error bars represent the SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.
Extended Data Fig. 3 IFN-I and IFN-γ play overlapping roles in protecting against R. parkeri in vivo.
a, R. parkeri abundance in mouse organs, infected i.v. with 107 bacteria, at 72 hpi. Bars denote medians. n = 4 (control) and 5 (α-IFN-γ) individual mice, for each organ. Data are the combination of two independent experiments. Each individual data point represents an individual mouse. Statistics used a two-tailed Mann Whitney U test. *p < 0.05. b, Mouse weight after i.v. infection with 107R. parkeri. Data are normalized to the weight at t = 0. Each line represents an individual mouse. n = 5 (Ifnar-/-), 7 (Ifngr-/-), and 7 (Ifnar-/-Ifngr-/-). c, Mouse body temperature after i.v. infection with 107R. parkeri. Each line represents an individual mouse. n = 5 (Ifnar-/-), 7 (Ifngr-/-), and 7 (Ifnar-/-Ifngr-/-). d, Survival of AG129 genotype mice (lacking IFN-I and IFN-γ receptors) after i.v. infection. n = 5 (107), 7 (106), and 5 (105). Data for each group are the combination of 2 independent experiments.
Extended Data Fig. 4 Tissue necrosis, leukocyte infiltration, and vascular damage is increased in spleens and livers of infected Ifnar-/-Ifngr-/- mice.
Organs were harvested from mice intravenously infected with 107R. parkeri at 72 hpi. Samples were fixed, sliced, and stained with hematoxylin and eosin (H&E) and commercially analyzed by a pathologist for inflammation and vascular damage. Inflammation observed was infiltration of mononuclear cells including macrophages, plasma cells, and lymphocytes in both organs, and also granulocytes in the liver. Vascular changes include fibrinoid vascular wall degeneration, hypertrophy of the endothelium, perivascular fibrinous material, and fibrin thrombi in medium caliber vessels. Double-headed arrows indicate aberrations at the vasculature and single-headed arrows indicate regions of necrosis and/or regions of mononuclear infiltrates. Scale bars in the liver are 100 μm (20×), 500 μm (4×) and 1 mm (2×); scale bars in the spleen are 100 μm (20×), 200 μm (10×), and 500 μm (4×); asterisks indicate defined splenic follicles in uninfected mice, which are lost in infected Ifnar-/-Ifngr-/- mice; results were similar in 3 independent experiments.
Extended Data Fig. 5 NK and CD8+ T cells do not play a critical role in protecting against intravenous R. parkeri infection in mice.
a, R. parkeri abundance in mouse organs, infected i.v. with the indicated amounts of bacteria, at 72 hpi. Bars denote medians. From left to right, n = 4, 5, 5, and 5 individual mice, for each organ. Statistics used a two-tailed Mann Whitney U test, where each condition was compared to the WT+IgG control for each organ. *p < 0.05, **p < 0.01, and ns=not significant. b, Survival of mice after i.v. infection. n = 4 (Rag2-/-) and 5 (Ifnar-/-Ifngr-/-) individual mice. Data for each group are combined from 2 independent experiments.
Supplementary Information
Supplementary Information
Supplementary Fig. 1.
Supplementary Dataset 1
RNA-seq analysis of infected WT, Irf1-/-, Irf5-/-, and Irf3/7-/- BMDMs infected with R. parkeri.
Supplementary Table 1
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Burke, T.P., Engström, P., Chavez, R.A. et al. Inflammasome-mediated antagonism of type I interferon enhances Rickettsia pathogenesis. Nat Microbiol 5, 688–696 (2020). https://doi.org/10.1038/s41564-020-0673-5
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