1. Microbiology and Infectious Disease
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Interferon receptor-deficient mice are susceptible to eschar-associated rickettsiosis

  1. Thomas P Burke  Is a corresponding author
  2. Patrik Engström
  3. Cuong J Tran
  4. Ingeborg M Langohr
  5. Dustin R Glasner
  6. Diego A Espinosa
  7. Eva Harris
  8. Matthew D Welch  Is a corresponding author
  1. Molecular and Cell Biology, University of California, Berkeley, United States
  2. Division of Infectious Disease and Vaccinology, School of Public Health, University of California, Berkeley, United States
  3. Department of Pathobiological Sciences, Louisiana State University, United States
Research Article
Cite this article as: eLife 2021;10:e67029 doi: 10.7554/eLife.67029
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
I.d. infection of Ifnar1-/-;Ifngr1-/- mice with R. parkeri elicits skin lesions that are grossly similar to human eschars.

(a) Representative images of WT, Ifnar1-/-, Ifngr1-/-, and Tlr4-/- single mutant mice, infected intradermally with 107 WT R. parkeri PFU, at 8 d.p.i. WT mice were injected with PBS. White arrows indicate the infection site on the right flank of the mouse. Scale bar, 1 cm. Data are representative of three independent experiments. (b) Representative images of a DKO Ifnar1-/-;Ifngr1-/- mouse after i.d. inoculation with 107 R. parkeri PFU. Data are representative of three independent experiments. The white arrow indicates the injection site on the right flank of the mouse. Scale bar, 1 cm. (c) Gross pathology of a human R. parkeri infection, reproduced from Figure 2a of Paddock et al., 2008, with permission from Oxford Academic. It is not covered by the CC-BY 4.0 license and further reproduction of this panel would require permission from the copyright holder. (d) Representative images of Ifnar1-/-;Ifngr1-/- mice infected intradermally with the indicated amounts of WT R. parkeri at 9 d.p.i. Scale bar, 1 cm. Data are representative of two independent experiments.

© 2008, Oxford Academic Permissions. Gross pathology of a human R. parkeri infection, reproduced from Figure 2a of Paddock et al., 2008, with permission from Oxford Academic. It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.

Figure 1—figure supplement 1
Ifnar1-/-;Ifngr1-/- mice develop disseminated disease upon intradermal R. parkeri infection.

(a) Representative image of the right flank of CD-1 mice intradermally infected with 107 PFU of R. parkeri. Scale bar, 1 cm. Data are representative from two independent experiments. (b) Representative images of tails of Ifnar1-/-;Ifngr1-/- and Ifngr1-/- mice, infected via the i.v. or i.d. route (as indicated), with 107 PFU of WT R. parkeri. Some Ifnar1-/-;Ifngr1-/- and Ifngr1-/- mice had no gross pathological manifestations in the tail, whereas some mice exhibited inflamed, necrotic tails at various times post-infection. Scale bar, 1 cm. Data are representative from three independent experiments.

Figure 2 with 1 supplement
I.d. infection of Ifnar1-/-;Ifngr1-/- mice by R. parkeri elicits disseminated, lethal disease.

(a) Weight changes over time in mice infected i.d. with R. parkeri. Data are shown as a percentage change to initial weight. In the left panel, all mice were infected with 107 PFU of R. parkeri: n = 7 (WT), n = 11 (Ifnar1-/-), n = 7 (Ifngr1-/-), n = 9 (DKO Ifnar1-/-;Ifngr1-/-), and n = 4 (Tlr4-/-) individual mice. In the right panel, DKO Ifnar1-/-;Ifngr1-/- mice were infected with the indicated R. parkeri PFU: n = 7 (105), n = 7 (104), n = 8 (103), n = 7 (102), n = 7 (101) individual mice. WT data is the same in both panels. Data for each genotype are combined from two or three independent experiments. (b) Temperature changes over time in mice intradermally infected with 107 R. parkeri PFU. Each line is an individual mouse. Mice were euthanized if their temperature fell below 90°F, as indicated by the dotted line. Data are the combination of three independent experiments with n = 7 (WT) and n = 9 (Ifnar1-/-;Ifngr1-/-) individual mice. (c) Analysis of gross skin pathology after i.d. infection. Ifnar1-/-;Ifngr1-/- mice were infected with the indicated PFU of R. parkeri and monitored over time. WT mice were infected with 107 R. parkeri PFU. Data are the combination of three independent experiments for WT and the 107 dose in Ifnar1-/-Ifngr1-/- mice; data for all other doses are the combination of two independent experiments. n = 9 (107), n = 5 (105), n = 5 (104), n = 8 (103), n = 7 (102), n = 7 (101), and n = 7 (WT) individual mice. (d) Mouse survival after i.d. infection with R. parkeri. In the left panel, all mice were infected with 107 R. parkeri PFU: n = 7 (WT), n = 11 (Ifnar1-/-), n = 7 (Ifngr1-/-), n = 4 Tlr4-/-, and n = 12 (DKO Ifnar1-/-;Ifngr1-/-) individual mice. Data are the combination of three separate experiments for WT, Ifnar1, and DKO Ifnar1-/-;Ifngr1-/- and two separate experiments for Ifngr1-/- and Tlr4-/-. In the right panel, DKO Ifnar1-/-;Ifngr1-/- mice were infected with the indicated amounts of R. parkeri. Data are the combination of two independent experiments: n = 7 (105), n = 7 (104), n = 8 (103), n = 7 (102), and n = 7 (101) individual mice. (e) Bacterial burdens in organs of intradermally infected WT and Ifnar1-/-;Ifngr1-/-mice. Mice were intradermally inoculated with 107 R. parkeri, and spleens and livers were harvested and plated for p.f.u. at 72 h.p.i. Dotted lines indicate the limit of detection. Data are the combination of two independent experiments. n = 4 (WT) and 7 (Ifnar1-/-;Ifngr1-/-) individual mice. Data in (a), (c) are the mean ± SEM. Statistical analyses in (a) used a two-way ANOVA where each group was compared to WT at t = 20 d.p.i. Statistical analyses in (c) used a two-way ANOVA at t = 20 d.p.i. Statistical analyses in (d) used a log-rank (Mantel−Cox) test to compare Ifnar1-/- to DKO Ifnar1-/-;Ifngr1-/- at each dose. Statistical analysis in (e) used a two-tailed Mann–Whitney U test. NS, not significant; **p<<0.01; ***p<0.001; ****p<0.0001.

Figure 2—source data 1

I.d. infection of Ifnar1-/-;Ifngr1-/- mice by R. parkeri elicits disseminated, lethal disease.

https://cdn.elifesciences.org/articles/67029/elife-67029-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Ifnar1-/- or Ifngr1-/- mice develop limited disease upon intradermal infection, and Ifnar1-/-;Ifngr1-/- develop lesions of dose-dependent severity.

(a) Weight changes over time in mice intradermally infected with 107 PFU of WT R. parkeri. Data are the combination of two independent experiments for WT and three for Ifnar1-/-;Ifngr1-/-; n = 7 (WT) and n = 9 (Ifnar1-/-;Ifngr1-/-) individual mice. Each line is an individual mouse. (b) Gross pathological analysis of the skin infection site after i.d. infection. Ifnar1-/-;Ifngr1-/- mice were infected with the indicated number of R. parkeri and monitored over time. Data are the combination of three independent experiments for the 107 dose and two independent experiments for all other doses. n = 7 (WT), n = 9 (107), n = 5 (105), n = 5 (104), n = 8 (103), n = 7 (102), and n = 7 (101) individual mice. Data are the same as in Figure 2c but are extended to 40 d.p.i. Dara are represented as means and error bars indicate SEM. (c) Temperature changes over time in mice infected i.d. with the indicated amounts of WT R. parkeri. Data are the combination of two independent experiments; n = 7 (WT), n = 7 (105), n = 7 (104), n = 8 (103), n = 7 (102), and n = 7 (101) individual mice. Each bar represents an individual mouse. Mice were euthanized if their body temperature fell below 90°F, as indicated by the dotted line.

Pathology of infected Ifnar1-/-;Ifngr1-/- mice reveals necrosis, inflammation, and rickettsial staining of macrophage and neutrophils.

(a–d) Representative images of the skin from WT and Ifnar1-/-;Ifngr1-/- mice, infected i.d. with 103 R. parkeri. Top row panels are skin stained with hematoxylin and eosin. Bottom row panels are a nearby Z plane, stained with an anti-Rickettsia antibody (brown). (e) Representative image of lungs from Ifnar1-/-;Ifngr1-/- mice, infected i.d. with 103 R. parkeri at 7 d.p.i. (f) Representative image of brains (choroid plexus) from Ifnar1-/-;Ifngr1-/- mice, infected i.d. with 106 R. parkeri at 7 d.p.i. (g, h) Representative images of livers and spleens from WT and Ifnar1-/-;Ifngr1-/- mice, infected i.v. with 107 R. parkeri at 7 d.p.i. The arrow in (g) for WT mice indicates rickettsial staining within a Kupffer cell. The asterisk for Ifnar1-/-;Ifngr1-/- indicates a granuloma. Scale bars are indicated in each panel. Data are representative of two independent replicates.

Figure 4 with 3 supplements
R. parkeri Sca2 contributes to dissemination from skin to spleens and livers.

(a) Survival of Ifnar1-/-;Ifngr1-/- mice upon i.v. infection with 5 × 106 PFU of R. parkeri. n = 7 (WT), 10 (sca2::Tn), and 7 (MC1_RS08740::Tn R. parkeri) individual mice. Data are the combination of two independent experiments. (b) Survival of Ifnar1-/-;Ifngr1-/- mice upon i.v. infection with 107 R. parkeri. n = 7 (WT) and 10 (sca2::Tn) individual mice. Data are the combination of two independent experiments. (c) Survival of Ifnar1-/-;Ifngr1-/- mice upon i.d. infection with 107 PFU of R. parkeri. n = 6 (WT) and 8 (sca2::Tn) individual mice. Data are the combination of two independent experiments. (d) Weight changes of Ifnar1-/-;Ifngr1-/- mice upon i.d. infection with 107 PFU of R. parkeri. n = 6 (WT) and 8 (sca2::Tn) individual mice. Data are the combination of two independent experiments. (e) Analysis of gross skin pathology after i.d. infection. Ifnar1-/-;Ifngr1-/- mice were infected with 107 PFU of the indicated strains of R. parkeri and monitored over time. n = 9 (WT) and 8 (sca2::Tn) individual mice. Data are the combination of two independent experiments. (f) Representative images of fluorescence in mouse skin after i.d. infection with 106 R. parkeri and delivery of a fluorescent dextran, at 5 d.p.i. Scale bars, 1 cm. The larger black dashed circle represents the area that was measured for fluorescence for each sample, as indicated in (g) (~80,000 pixels). The smaller, black-dashed circle represents of the injection site area that was measured for fluorescence for each sample, as indicated in Figure 4—figure supplement 1 (~7800 pixels). (g) Quantification of fluorescence in mouse skin after i.d. infection. Mice were infected with 107 R. parkeri, and 150 µl fluorescent dextran was intravenously delivered at 5 d.p.i. Skin was harvested 2 hr later, and fluorescence was measured using a fluorescence imager. Data indicate measurements of larger areas of skin, as indicated in (f) by the larger black circle. n = 6 (WT R. parkeri) and n = 6 (sca2::Tn R. parkeri) individual Ifnar1-/-;Ifngr1-/- mice; n = 6 (WT R. parkeri) individual WT mice. For each experiment, the average of uninfected samples was normalized to 100; each sample was divided by the average for uninfected mice and multiplied by 100; the dotted horizontal line indicates 100 arbitrary units, corresponding to uninfected (unin.) mice. Data are the combination of two independent experiments. (h) Quantification of Listeria monocytogenes abundance in organs of WT C57BL/6J mice upon i.v. infection with 104 bacteria, at 72 h.p.i. Data are the combination of two independent experiments. n = 6 (WT), n = 7 (ΔactA) individual mice. (i) Quantification of R. parkeri abundance in spleens and livers of WT C57BL/6J mice upon i.v. infection, at 72 h.p.i. Data are the combination of two independent experiments. n = 10 (WT) and 10 (Sca2::Tn) individual mice. (j) Quantification of R. parkeri abundance in organs upon i.d. infection with 107 PFU of R. parkeri. n = 7 (WT) and 7 (sca2::Tn) individual mice. Data are the combination of two independent experiments. Data for WT R. parkeri in Ifnar1-/-;Ifngr1-/- mice are the same as in Figure 2e. (k) Quantification of R. parkeri abundance in organs upon i.d. infection with 105 PFU of R. parkeri. n = 7 (WT) and 6 (sca2::Tn). Data are the combination of two independent experiments. Solid horizontal bars in (g) indicate means; solid horizontal bars in (h–k) indicate medians; error bars indicate SEM. Statistical analyses for survival in (a–c) used a log-rank (Mantel–Cox) test. Statistical analysis in (d) used a two-way ANOVA at t = 20. Statistical analysis in (e) used a two-way ANOVA from 0 to 10 d.p.i. Statistical analyses in (g) used a two-tailed Student’s T test. Statistical analyses in (h–k) used a two-tailed Mann–Whitney U test. The fold change in (hk) indicates differences of medians. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 4—source data 1

R. parkeri Sca2 contributes to dissemination from skin to spleens and livers.

https://cdn.elifesciences.org/articles/67029/elife-67029-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Intradermal infection of Ifnar1-/-;Ifngr1-/- mice with sca2::Tn R. parkeri causes less severe temperature loss as compared to WT bacteria.

Temperature changes over time in mice infected i.d. with 107 R. parkeri. Data are the combination of two independent experiments; n = 5 (WT), n = 8 (sca2::Tn) individual mice. Each line represents an individual mouse. Mice were euthanized if their body temperature fell below 90°F, as indicated by the dotted line.

Figure 4—figure supplement 2
WT and sca2::Tn R. parkeri elicit similar amounts of vascular damage in skin upon i.d. infection of Ifnar1-/-;Ifngr1-/- mice.

(a) Quantification of fluorescence in mouse skin after i.d. infection. Mice were infected i.d. with 107 R. parkeri and fluorescent dextran was intravenously delivered at 5 d.p.i. Skin was harvested 2 hr after delivery of dextran and analyzed with a fluorescence imager. n = 6 (WT R. parkeri) and n = 6 (sca2::Tn R. parkeri) individual Ifnar1-/-;Ifngr1-/- mice; n = 6 (WT R. parkeri) individual WT mice. Data in the ‘extended area’ are the same as those reported in Figure 4e. (b) Quantification of fluorescence in mouse skin after i.d. infection. Mice were infected i.d. with 106 R. parkeri, and fluorescent dextran was intravenously delivered at 5 d.p.i. Skin was harvested 2 hr after delivery of dextran and analyzed with a fluorescence imager. n = 8 (WT R. parkeri) and n = 8 (sca2::Tn R. parkeri) individual Ifnar1-/-;Ifngr1-/- mice. (c) Quantification of fluorescence in mouse skin after i.d. infection. Mice were infected i.d. with 105 R. parkeri and fluorescent dextran was intravenously delivered at 5 d.p.i. Skin was harvested 2 hr after delivery of dextran and analyzed with a fluorescence imager. n = 6 (WT R. parkeri) and n = 5 (sca2::Tn R. parkeri) individual Ifnar1-/-;Ifngr1-/- mice. For each experiment, the average of uninfected samples was normalized to 100, and each sample was divided by the average for uninfected mice and multiplied by 100; the dotted horizontal line indicates 100 arbitrary units, corresponding to uninfected (unin.) mice. Representative sizes for the larger ‘extended areas’ of skin and the smaller ‘injection site’ areas of skin are indicated in Figure 4d. Data are each the combination of two independent experiments. Solid horizontal bars indicate means; error bars indicate SEM. For statistical analyses, a two-tailed Student’s T test was used to compare the indicated samples.

Figure 4—figure supplement 3
Sca2 does not significantly enhance R. parkeri avoidance of antibacterial innate immune responses in vitro.

(a) R. parkeri abundance in HMEC-1s, multiplicity of infection (MOI) of 0.2. Data are the combination of three independent experiments, each with two biological replicates. For statistics, a two-tailed Student’s T test was used to compare WT to sca2::Tn at 48, 72, and 96 h.p.i. No statistically significant differences were observed at any time. (b) R. parkeri abundance in bone-marrow-derived macrophage (BMDMs), MOI of 1. Data are the combination of three independent experiments, each with two biological replicates. Data were normalized by multiplying fold difference between WT and Sca2::Tn at 4 h.p.i. to Sca2::Tn and Sca2::Tn + IFN I data at all time points. (c) Host cell death upon R. parkeri infection of BMDMs, as measured by lactate dehydrogenase (LDH) release assay, MOI of 1. From left to right, n = 6 and 3 biological replicates and are the combination of two independent experiments. (d) IFN-I abundance in supernatants of infected BMDMs (24 h.p.i.; MOI of 1), measured using a luciferase reporter assay. The data show the fold change over uninfected cells. n = 7 and 7 biological replicates and are the combination of two independent experiments. (e) A representative image using x100 confocal immunofluorescence microscopy of WT BMDMs infected with sca2::Tn R. parkeri in the presence of 100 U recombinant IFN-β (3 h.p.i.; MOI of 1). Green, α-GBP2; red, α-Rickettsia (Rp). The dotted square indicates the image that is expanded in the other images, separated into two individual and one merged channel. Scale bars, 2.5 µm. White arrows indicate a bacterium that colocalizes with GBP2. Data are representative of three independent experiments. (f) Quantification of GBP2 colocalization with R. parkeri in BMDMs at 24 h.p.i. Each data point is an average of at least five separate images totaling >150 bacteria. Data are the combination of three independent experiments. Statistical analyses used a two-tailed Student’s T test. NS, not significant. Data in (a, b) are means; bars in (c, d, and f) are means; error bars indicate SD.

ompB mutant R. parkeri elicit no lethality and reduced skin lesion formation in Ifnar1-/-;Ifngr1-/-mice.

(a) Survival of Ifnar1-/-;Ifngr1-/- mice upon i.d. infection with 107 PFU of R. parkeri. n = 6 (WT) and 8 (ompB::TnSTOP) individual mice. Data are the combination of two independent experiments. Data for WT are the same as in Figure 4c. (b) Weight changes of Ifnar1-/-;Ifngr1-/- mice upon i.d. infection with 107 PFU of R. parkeri. n = 6 (WT) and 8 (ompB::TnSTOP) individual mice. Data are the combination of two independent experiments. Data for WT are the same as in Figure 4d. (c) Analysis of gross skin pathology after i.d. infection. Ifnar1-/-;Ifngr1-/- mice were infected with 107 of the indicated strains of R. parkeri and monitored over time. n = 9 (WT) and 8 (ompB::TnSTOP) individual mice. Data are the combination of two independent experiments. Data for WT are the same as in Figure 4e. Error bars indicate SEM. Statistical analyses in (a) used a log-rank (Mantel–Cox) test. Statistical analysis in (b) used a two-way ANOVA from 0 to 20 d.p.i. Statistical analysis in (c) used a two-way ANOVA from 0 to 10 and 20 d.p.i.; **p<0.01; ***p<0.001.

Figure 5—source data 1

ompB mutant R. parkeri elicit no lethality and reduced skin lesion formation in Ifnar1-/-;Ifngr1-/-mice.

https://cdn.elifesciences.org/articles/67029/elife-67029-fig5-data1-v2.xlsx
ompB and sca2 mutant R. parkeri elicit immunity in Ifnar1-/-Ifngr1-/- mice.

(a) Survival of immunized and naïve Ifnar1-/-;Ifngr1-/- mice upon i.v. R. parkeri infection. Immunized mice were first infected with 5 × 106 PFU of sca2::Tn or 107 PFU of ompB:TnSTOP R. parkeri and were re-challenged 40 d later with 107 WT R. parkeri. n = 6 (naïve); n = 5 (sca2::Tn immunized); n = 5 (ompB::TnSTOP immunized) individual mice. Data are the combination of two independent experiments. (b) Weight changes over time in mice infected i.d. with 107 R. parkeri. Data are representative of two independent experiments. n = 3 (naïve); n = 3 (sca2::Tn immunized); n = 3 (ompB::Tnstop immunized) individual mice. Each line represents an individual mouse. (c) Temperature changes over time in mice infected i.d. with 107 R. parkeri. Data are representative from two independent experiments. n = 3 (naïve); n = 3 (sca2::Tn immunized); n = 3 (ompB::TnSTOP immunized) individual mice. Each line represents an individual mouse. (d) Survival of naïve or immunized Ifnar1-/-;Ifngr1-/- mice that were infected i.d. with 105 R. parkeri. (e) Skin lesion pathology of naïve or immunized Ifnar1-/-;Ifngr1-/- mice that were infected i.d. with 105 R. parkeri; n = 7 (naïve); n = 8 (sca2::Tn i.d. immunized); n = 5 (sca2::Tn i.v. immunized); n = 8 (ompB::TnSTOP) individual mice. Statistical analyses in (a, d) used a log-rank (Mantel–Cox) test to compare each group of immunized mice to naïve mice; **p<0.01. Statistical analysis in (e) used a two-way ANOVA from 0 to 10 and 20 d.p.i.; **p<0.01; ***p<0.001.

Figure 6—source data 1

ompB and sca2 mutant R. parkeri elicit immunity in Ifnar1-/-Ifngr1-/- mice.

https://cdn.elifesciences.org/articles/67029/elife-67029-fig6-data1-v2.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Biological sample (Mus musculus)WT C57Bl/6JJackson labsStock #: 000664
Biological sample (Mus musculus)DKO Ifnar1-/-;Ifngr1-/-Jackson labsStock #: 029098
Biological sample (Mus musculus)B6.129S2-Ifnar1tm1Agt/Mmjax; aka Ifnar1-/-Jackson labsStock #: 32045-JAXRRID:MMRRC_032045-JAX
Biological sample (Mus musculus)B6.129S7-Ifngr1tm1Agt/J; akaIfngr1-/-Jackson labsStock #: 003288
AntibodyI7205 anti-Rickettsia (Rabbit polyclonal)Dr. Ted HackstadtIHC (1:1000);IF (1:1000)
Biological sample (Rickettsia parkeri)WT R. parkeri strain PortsmouthOriginally from Dr. Chris Paddock (CDC)
Biological sample (Rickettsia parkeri)Sca2::Tn R. parkeriWelch lab (UC Berkeley)PMID:24361066
Biological sample (Rickettsia parkeri)OmpB::TnSTOPR. parkeriWelch lab (UC Berkeley)PMID:31611642

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