Caspase-11 Activation in Response to Bacterial Secretion Systems That Access the Host Cytosol

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense

TLR4 sensing of IsdB of Staphylococcus aureus induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade

The iron-regulated surface determinant protein B (IsdB) of Staphylococcus aureus is involved in the acquisition of iron from hemoglobin. Moreover, IsdB elicits an adaptive immune response in mice and humans. Here, we show that IsdB also has impact on innate immunity. IsdB induces the release of proinflammatory cytokines, including IL-6 and IL-1β, in innate immune cells of humans and mice. In silico analysis and thermophoresis show that IsdB directly binds to TLR4 with high affinity. TLR4 sensing was essential for the IsdB-mediated production of IL-6, IL-1β, and other cytokines as it was abolished by blocking of TLR4-MyD88-IRAK1/4-NF-κB signaling. The release of IL-1β additionally required activation of the NLRP3 inflammasome. In human monocytes infected with live S. aureus, IsdB was necessary for maximal IL-1β release. Our studies identify S. aureus IsdB as a novel pathogen-associated molecular pattern that triggers innate immune defense mechanisms. IMPORTANCE The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.</p

IL-1α and IL-1β control bacterial burden and neutrophil recruitment <i>in vivo</i>.

<p>(<b>A</b>) 8–12 week old B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA L. pneumophila</i> intranasally (IN). Lungs were plated to quantify CFU per gram. Graph shows the mean ± SEM of three or four infected mice per group. Dashed line represents the limit of detection. (<b>B</b> and <b>C</b>) B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA</i> Lp IN. 24 hours post-infection, bronchoalveolar lavage fluid (BALF) was collected and the percentage of neutrophils in the BALF was quantified by flow cytometry. Percentages are reported as the frequency of live cells in the BALF. (B) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (C) Graph showing the percentage of neutrophils. Each point represents an individual mouse and lines indicate the mean of 4 mice per group. (<b>D, E</b>, and <b>F</b>) B6 mice were injected intraperitoneally (IP) with either PBS, 100 µg isotype control antibody (iso), 100 µg anti-IL-1α antibody, 100 µg anti-IL-1β antibody, or 100 µg each of anti-IL-1α and anti-IL-1β (anti-IL-1α/β) 16 hours before infection. The mice were then intranasally infected with either 1×10⁶ Δ<i>flaA</i> Lp or mock infected with PBS. (D and E) 24 hours post-infection, BALF was collected and flow cytometry was performed to quantify the percentage of neutrophils. (D) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (E) Graph showing the percentage of neutrophils. Each point represents an individual mouse, lines indicate the mean of 8 mice per group, and error bars represent SEM. Shown are the pooled results of two independent experiments. (F) 72 hours post-infection, the lungs were plated to quantify CFU per gram. Each point represents an individual mouse. Line indicates the mean of 4 infected mice per group with error bars representing SEM. *** is p<0.001 by one-way ANOVA with Tukey post-test or unpaired t-test (C). **is p<0.01 and *is p<0.05 by unpaired t-test. NS is not significant.</p

Caspase-11 mediates both NLRP3-dependent and NLRP3-independent immune responses.

<p>(<b>A</b>) B6, <i>Casp1^−/−Casp11^−/−</i>, <i>Asc</i>^−/−, <i>Nlrc4</i>^−/−, or <i>Asc^−/−Nlrc4^−/−</i> BMDMs were primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT <i>L. pneumophila</i> (WT Lp), Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) or treated with 2.5 mm ATP for 4 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA and cell death (% cytotoxicity) was measured by LDH release into the supernatants relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. (<b>B</b> and <b>C</b>) B6 or <i>Nlrp3^−/−</i> BMDMs were primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infected) or treated with 2.5 mm ATP for 1 hour (C) or 4 hours (B). (B) Levels of IL-1α and IL-1β in the supernatants were measured by ELISA and cell death (% cytotoxicity) was measured by LDH release into the supernatants relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. (C) Levels of processed caspase-1 (casp-1 p10) in the supernatants and pro-caspase-1 in the cell lysates were determined by immunoblot analysis. Data are representative of two (A,C) or three (B) independent experiments. *** is p<0.001 by one-way ANOVA with Tukey post-test. NS is not significant.</p

Non-canonical inflammasome responses to <i>L.</i><i>pneumophila</i> occur independently of TRIF and IFNAR.

<p>(<b>A</b>) Unprimed B6, <i>Ifnar^−/−</i>, or <i>Trif^−/−</i> BMDMs were infected with WT <i>L. pneumophila</i> (WT Lp), Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, <i>E. coli</i>, or PBS (mock infection) for 16 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (<b>B</b>) Unprimed B6, <i>Ifnar^−/−</i>, or <i>Trif^−/−</i> BMDMs were infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) for 16 hours. Cell death (% cytotoxicity) was measured by LDH release into the supernatants relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. (<b>C</b>) B6, <i>Ifnar^−/−</i>, or <i>Trif^−/−</i> BMDMs were primed with 0.4 µg/mL Pam3CSK4 for 4 hours and infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS for 16 hours. Levels of full-length caspase-11 (pro-casp-11) and processed caspase-11 (casp11 p26) in the supernatants and pro-casp-11 and β-actin (loading control) in the cell lysates were determined by immunoblot analysis. Data are representative of two independent experiments.</p

Caspase-11 mediates inflammasome activation in response to a functional <i>Yersinia</i> type III secretion system.

<p>BMDMs from B6, <i>Casp1^−/−Casp11^−/−</i>, <i>Casp1^−/−</i>, or <i>Casp11^−/−</i> mice were primed with 0.05 µg/mL LPS for 4 hours and infected with type III secretion system-deficient <i>Y. pseudotuberculosis</i> (Δ<i>yopB</i> Yp), effectorless <i>Y. pseudotuberculosis</i> ΔHOJMEK (Δ6 Yp), or PBS (mock infection) or treated with 2.5 mm ATP for 4 hours. (<b>A</b>) Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (<b>B</b>) Cell death (% cytotoxicity) was measured by lactate dehydrogenase (LDH) release relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. Data are representative of two independent experiments. *** is p<0.001 and ** is p<0.01 by two-way ANOVA with Bonferroni post-test. NS is not significant.</p

Caspase-11 controls multiple pathways of inflammasome activation in response to bacterial secretion systems that access the host cytosol.

<p>Three distinct inflammasome pathways are induced upon interaction of virulent bacteria with host cells. Translocation of flagellin into the host cytosol by specialized secretion systems triggers a NAIP5/NLRC4/caspase-1 inflammasome that leads to cell death, IL-1α, and IL-1β release. Virulent bacteria induce two separate pathways of caspase-11-dependent inflammasome activation through a two-signal model. First, TLR stimulation by PAMPs (signal one) leads to upregulation of pro-IL-1α, pro-IL-1β, NLRP3, and pro-caspase-11. Next, cytosolic detection of virulence activity, namely type III or type IV secretion (signal two), leads to caspase-11 processing and activation. Active caspase-11 contributes to NLRP3-mediated inflammasome activation and caspase-1-dependent IL-1β secretion. Caspase-11 also mediates caspase-1-independent cell death and IL-1α release through a pathway that is independent of the NLRP3/ASC and NAIP5/NLRC4 inflammasomes and involves an unknown host sensor.</p