Skin Commensal Antigens: Taking the Road Less Traveled

Although our knowledge of host-commensal interactions has increased exponentially, the mechanisms linking a specific commensal, its detection by the immune system, and its impact on tissue function are still often poorly understood. In a recent study in Cell, Linehan et al. dissect one of these interactions in the context of the skin, and demonstrate that Staphylococcus epidermidis antigens, presented through a non-classical pathway, drive the accumulation of CD8+ T cells that promote wound healing

Host stress and immune responses during aerosol challenge of Brown Norway rats with Yersinia pestis

Inhalation exposure models are becoming the preferred method for the comparative study of respiratory infectious diseases due to their resemblance to the natural route of infection. To enable precise delivery of pathogen to the lower respiratory tract in a manner that imposes minimal biosafety risk, nose-only exposure systems have been developed. Early inhalation exposure technology for infectious disease research grew out of technology used in asthma research where predominantly the Collison nebulizer is used to generate an aerosol by beating a liquid sample against glass. Although infectious aerosol droplets of 1-5µm in size can be generated, the Collison often causes loss of viability. In this work, we evaluate a gentler method for aerosolization of living cells and describe the use of the Sparging Liquid Aerosol Generator (SLAG) in a rat pneumonic plague model. The SLAG creates aerosols by continuous dripping of liquid sample on a porous metal disc. We show the generation of 0.5 to 1µm Y. pestis aerosol particles using the SLAG with spray factors typically ranging from 10-7 to 10-8 with no detectable loss of bacterial viability. Delivery of these infectious particles via nose-only exposure led to the rapid development of lethal pneumonic plague. Further, we evaluated the effect of restraint-stress imposed by the nose-only exposure chamber on early inflammatory responses and bacterial deposition. Elevated serum corticosterone which peaked at 2 hrs post-procedure indicated the animals experienced stress as a result of restraint in the nose-only chamber. However, we observed no correlation between elevated corticosterone and the amount of bacterial deposition or inflammation in the lungs. Together these data demonstrate the utility of the SLAG and the nose-only chamber for aerosol challenge of rodents by Y. pestis

Early Apoptosis of Macrophages Modulated by Injection of <i>Yersinia pestis</i> YopK Promotes Progression of Primary Pneumonic Plague

<div><p><i>Yersinia pestis</i> causes pneumonic plague, a disease characterized by inflammation, necrosis and rapid bacterial growth which together cause acute lung congestion and lethality. The bacterial type III secretion system (T3SS) injects 7 effector proteins into host cells and their combined activities are necessary to establish infection. <i>Y. pestis</i> infection of the lungs proceeds as a biphasic inflammatory response believed to be regulated through the control of apoptosis and pyroptosis by a single, well-conserved T3SS effector protein YopJ. Recently, YopJ-mediated pyroptosis, which proceeds via the NLRP3-inflammasome, was shown to be regulated by a second T3SS effector protein YopK in the related strain <i>Y. pseudotuberculosis</i>. In this work, we show that for <i>Y. pestis</i>, YopK appears to regulate YopJ-mediated apoptosis, rather than pyroptosis, of macrophages. Inhibition of caspase-8 blocked YopK-dependent apoptosis, suggesting the involvement of the extrinsic pathway, and appeared cell-type specific. However, in contrast to <i>yopJ</i>, deletion of <i>yopK</i> caused a large decrease in virulence in a mouse pneumonic plague model. YopK-dependent modulation of macrophage apoptosis was observed at 6 and 24 hours post-infection (HPI). When YopK was absent, decreased populations of macrophages and dendritic cells were seen in the lungs at 24 HPI and correlated with resolution rather than progression of inflammation. Together the data suggest that <i>Y. pestis</i> YopK may coordinate the inflammatory response during pneumonic plague through the regulation of apoptosis of immune cells.</p></div

YopK contributes to caspase-3 cleavage through the extrinsic pathway.

<p>A) RAW 264.7 macrophages were infected with KIM5, KIM5 <i>yopJ</i>C172A, or KIM6⁻ at an MOI of 20 and assayed for caspase-3 activity after 3.5 hours of infection. Data shown are the mean of four biological replicates, collected in two independent trials, and are shown as a percentage of caspase-3 activity from WT-infected cells. B–D) RAW 264.7 macrophages were left untreated (white bars) or treated with inhibitors (grey bars) for (B) caspase-9 (Z-LEHD-FMK), (C) caspase-8 (IETD), or (D) JNK (SP600125) and infected with either <i>Y. pestis</i> KIM D27 or KIM D27 <i>yopK</i> at an MOI of 20. After 3.5 hours of infection, cell pellets were analyzed for cleaved caspase-3. Data shown are the mean of four replicates, collected in two independent trials and are shown as percent of untreated for each strain. Statistical significance by one-way ANOVA followed by Tukey post-hoc test, *<i>p</i><0.05, ns not significant.</p

The presence of YopK leads to increased populations of dendritic cells and macrophages in the lungs.

<p>BALB/c mice were challenged by intranasal infection with 1×10⁴ CFU <i>Y. pestis</i> CO92 or 6×10⁵ CFU CO92 <i>yopK.</i> At 24 HPI, infected lungs were isolated and homogenized for analysis by flow cytometry. A–B) Gating of events based on CD11c-PE and F4/80-FITC in naive (A), CO92- (B), and <i>yopK</i>- (C) infected lungs. D) Graphical representation of average numbers of CD11c⁺F4/80⁻, CD11c⁻F4/80⁺, and CD11c⁺F4/80⁺ cells in the lungs of CO92- or CO92 <i>yopK</i>-infected mice, *<i>p</i><0.05, evaluated by one-way ANOVA followed by Tukey post-hoc test. n = 3–4 mice per group.</p

Similar early inflammation following pulmonary challenge with WT and <i>yopK Y. pestis</i> is resolved in the absence of YopK.

<p>BALB/c mice were challenged by intranasal infection with 1×10⁴ CFU of WT <i>Y. pestis</i> CO92 or 1×10⁶ CFU of CO92 <i>yopK</i>. At 6, 24 and 72 HPI, mice were euthanized and lungs, livers, and spleens were divided with one-half tissue analyzed for bacterial titers (A) including lungs (open), liver (grey) and spleen (black) and the second half formalin fixed, sectioned and stained for histochemistry (B–E). H&E stains of WT-infected lungs (B–C) and <i>yopK</i>-infected lungs (D–E) from 24 (B,D) and 72 (C,E) HPI. Data shown were collected in two independent experiments, n = 6 mice per time point per strain. Statistical significance of differences in bacterial titer of WT- and <i>yopK</i>- infected lungs was analyzed by unpaired Student's t-test, *<i>p</i><0.05. (B–E) Boxes show 4× magnified section of indicated area.</p

CCR2 contributes to host defense from primary pneumonic plague following intranasal challenge with <i>Y. pestis</i> CO92 <i>yopK</i>.

<p>A) WT C57BL/6 and <i>Ccr</i>2^−/− mice were challenged by intranasal infection with 5×10⁶ CFU of <i>Y. pestis</i> CO92 <i>yopK</i> and monitored for development of disease for 14 days. Data shown are representative; n = 10 mice per group, data were collected in two independent experiments. *<i>p</i><0.05, evaluated by Log rank test. B) Bacterial titers collected from lungs of C57BL/6 (white circles) and C57BL/6 <i>Ccr</i>2^−/− mice (grey circles) infected intranasally with 5×10⁶ CFU CO92 <i>yopK</i> at 72 hpi, data shown from two independent experiments, n = 9–12 mice per group. (C–D) H&E stains of formalin fixed lungs from mice challenged by intranasal infection with 5×10⁶ CFU of <i>Y. pestis</i> CO92 <i>yopK</i> at 72HPI: (C) C57BL/6, (D) C57BL/6 <i>Ccr2^−/−</i>. Images are representative; boxes show 4× magnified section of indicated area. Data were collected in two independent experiments (n = 9–12 mice per group).</p

A model for <i>Yersinia pestis</i> induced apoptosis of macrophages.

<p>Infection of RAW macrophages induces caspase-3-apoptosis through the extrinsic pathway by manipulating host signaling through type III secretion system effectors YopJ and YopK. YopK is located at the plasma membrane where it may control injection of effector Yops but may also be required to induce formation of the DISC death receptor complex, composed of pro-caspase-8 and -10, FADD and cFLIP, which is necessary for activation of caspase-8. In the absence of DISC complex formation, small amounts of active caspase-8 can cleave Bid to tBid in the absence of anti-apoptotic factors whose expression is repressed through the action of YopJ (green X). Formation of the DISC complex is regulated by YopJ. The small molecule SP600125 inhibits JNK phosphorylation, thereby causing reduced production of anti-apoptotic genes. Yellow background denotes the signaling pathway that may be specific to RAW cells and perhaps other immune cells.</p

YopK modulates caspase-3 activation <i>in vitro</i> in <i>Yersinia pestis</i> infected macrophages.

<p>A) Intravenous infection of C57BL/6 mice with the indicated doses of KIM D27 <i>yopK</i> for determination of 50% lethal dose (data shown are representative of two trials; n = 8–10 mice per group total). RAW 264.7 cells were infected with <i>Y. pestis</i> KIM D27, <i>yopK</i>, pYopK and CO92 pCD1⁻, (B) harvested after 3.5 hours and assayed for caspase-3 activation (shown are the mean values from four trials, each sample run in duplicate) or (C) harvested after 4 hours for release of lactate dehydrogenase (LDH) (shown are the mean values from nine trials, each sample run in duplicate). (D–E) MH-S cells, an alveolar macrophage cell line, were infected with KIM D27, <i>yopK</i>, pYopK, and KIM6- (pCD1⁻) and assayed for caspase-3 activation (D) and LDH release (E) at 2, 3.5, and 5 HPI, shown are the mean values from two (5 HPI), four (2 HPI) or six (3.5 HPI) trials, each sample run in duplicate. (F) RAW 264.7 macrophages were infected at an MOI of 20 for 3.5 hours with <i>Y. pestis</i> KIM D27<i>yopK</i> complemented with wild type <i>yopK</i> or the T45Y or D46K mutants; data shown are the mean values collected in 2–5 independent trials, with each sample run in duplicate. Caspase data are reported as a percentage of caspase activation by WT <i>Y. pestis</i> KIM D27; LDH data are reported as a percent of total LDH released following lysis of macrophages that were not infected. Data from each trial were evaluated for statistical significance using one-way ANOVA followed by the Tukey post-hoc test, *<i>p</i><0.05, ns not significant.</p