Caspase-1-Independent Interleukin-1β Is Required for Clearance of <i>Bordetella pertussis</i> Infections and Whole-Cell Vaccine-Mediated Immunity

<div><p>Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1) response, which is required for the clearance of <i>Bordetella pertussis</i>. We show that IL-1β but not IL-1α is required for mediating the clearance of <i>B. pertussis</i> from the lungs of mice. We further found that IL-1β and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during <i>B. pertussis</i> infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a <i>B. pertussis</i> challenge following vaccination with heat-killed whole cell <i>B. pertussis,</i> despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.</p></div

Bacterial clearance is mediated by IL-1β and IL-1R but not IL-1α.

<p>Groups of 5–8 mice were inoculated with <i>B. pertussis</i> (5×10⁵ CFU) suspended in a 50 µL droplet of PBS and euthanized at indicated time points to determine bacterial CFUs in lung homogenates. The mean ± standard error was graphed for each group at the specified time-point and is representative of three independent experiments. Significance was calculated using one-way ANOVA with Dunnet’s multiple comparison post-hoc analysis. <i>*p<0.05, **p<0.01</i>; WT vs IL-1β^−/− (*), vs IL-1α^−/− (†), vs IL-1R^−/− (‡).</p

Adaptive immune responses are IL-1 dependent but Caspase-1 independent.

<p>Groups of four mice were either sham vaccinated with PBS or vaccinated with heat-killed <i>B. pertussis</i> (wP) by administering 200 µL <i>i.p.</i> at 28 and 14 days prior to challenge inoculation with 5×10⁵ CFU. Lungs were excised at seven days post-challenge and to determine bacterial CFUs. Bar graphs represent the mean CFU per lung ± standard deviation. Significance was determined by one-way ANOVA and Tukey’s multiple comparison post-hoc test with *p<0.05, ***p<0.001. Data is representative of two independent experiments.</p

IL-1 signaling limits lung inflammation during <i>B. pertussis</i> infection.

<p>Groups of four mice were either (A–D) uninfected or inoculated with <i>B. pertussis</i> and euthanized at (E–H) 7 days or (I–L) 28 days post-inoculation. Automated H&E staining was performed on 5 µm paraffin-embedded sections. Images were taken and are representative of 10–12 fields per group. Lung homogenate cytokine measurements (M–N) were measured by ELISA from uninfected mice or on days 3, 7, 14, or 28 post-inoculation (n = 4–8). ELISA significance was determined by one-way ANOVA with Dunnet’s post-hoc test. *p<0.05 represents a significant difference from WT mice.</p

IL-1β production is caspase-1 dependent in vitro but independent in vivo.

<p>To determine if <i>in vitro</i> IL-1β processing is dependent upon caspase-1, bone marrow-derived macrophages were (A) primed 3 h prior to inoculation and were washed and treated with fresh media, ATP (5 mM), or live <i>B. pertussis</i> (MOI 10) or (B) seeded at 10⁶ cells/well and primed with LPS or washed and treated with media, ATP (5 mM), or live <i>B. pertussis</i> (MOI 100) for 6 hours in FBS-free DMEM, and supernatant was collected. The requirement for caspase-1 during infection and the ability to produce IL-1β in response to live <i>B. pertussis</i> was assessed by (C) inoculating groups of four mice per time point with <i>B. pertussis</i> (5×10⁵ CFU) and lungs were dissected for determining bacterial CFUs ± standard error. (D) To identify whether cleaved IL-1β was produced in caspase-1 deficient mice, lung homogenates from day 3 post-inoculation were run on 12% gels and probed with anti-IL-1β. Data is representative of at least two independent experiments.</p

Antigen-specific CD8⁺ IFN-γ and IL-17A responses during and after LVS-OVA infection.

<p>OT-I cells were adoptively transferred into recipient mice 24 hours prior to infection with 1000 CFU of LVS-OVA. Intracellular cytokine staining was performed on single cell suspensions incubated with BrefeldinA, +/- PMA and calcium ionophore. n = 3 per group, means +/- SEM are plotted. * p<0.05 according to two-tailed, unpaired T test.</p

Antigen-specific proliferation of CD8⁺ OT-I cells in response to LVS-OVA.

<p>Thy1.1^-/Thy1.2⁺ OT-I cells were transferred in to Thy1.1⁺/Thy1.2⁺ recipient mice, 24 hours prior to infection with 1000 CFU of LVS-OVA or LVS-pKK214 (LVS with empty vector). A) representative scatterplots of CD8⁺ cells from the lungs of animals infected with LVS-OVA. B) CFSE dilution of CD8⁺ OT-I cells isolated from animals infected with LVS-OVA. C) The frequency of CD8⁺ OT-I cells in animals infected with LVS-OVA or LVS-pKK214. n = 3 per group, means +/- SD are plotted; * p<0.05 according to two-tailed, unpaired T test.</p

Kinetics of the CD8⁺ T cell response to intranasal LVS infection.

<p>OT-I cells were adoptively transferred into recipient mice 24 hours prior to infection with 1000 CFU of LVS-OVA. A) Bacterial burden of infected animals over time. B-D) number of CD8⁺ OT-I cells in infected animals over time. Day 0 data are for uninfected mice 24h after adoptive transfer. n = 3 per group, means +/- SEM are plotted. Statistical significance (p<0.05) for one-way ANOVA with Tukey’s post test, for the indicated column compared to: # day 0, * day 7.</p

Antigen-specific lung CD8⁺ T cells with resident memory markers following prime-boost vaccination strategies.

<p>A) relative frequency, and B) number of CD69⁺CD103⁺ OT-I cells. n = 3 per group, means +/- SD are plotted. No statistically significant differences according to two-tailed, unpaired t-tests.</p

Generation and stability of LVS-OVA.

<p>A) Map of the pKK214-vgrG-OVA plasmid. B) Immunoblot demonstrating expression of the VgrG-OVA construct by cells harboring plasmid (pKK214-vgrG-OVA). C) Stability of the pKK214-vgrG-OVA plasmid during an <i>in vivo</i> infection. C57BL/6J mice (n = 3 per group) were infected with 1000 CFU of wild-type <i>F</i>. <i>tularensis</i> LVS, LVS harboring empty vector (LVS-pKK214), or LVS harboring pKK214-vgrG-OVA (LVS-OVA). Serial dilutions of lung homogenates were plated with or without tetracycline as indicated.</p