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Title: Early Host Cell Targets of Yersinia pestis during Primary Pneumonic Plague}
Article:
Abstract:
Inhalation of Yersinia pestis causes primary pneumonic plague, a highly lethal syndrome with mortality rates approaching 100%. Pneumonic plague progression is biphasic, with an initial pre-inflammatory phase facilitating bacterial growth in the absence of host inflammation, followed by a pro-inflammatory phase marked by extensive neutrophil influx, an inflammatory cytokine storm, and severe tissue destruction. Using a FRET-based probe to quantitate injection of effector proteins by the Y. pestis type III secretion system, we show that these bacteria target alveolar macrophages early during infection of mice, followed by a switch in host cell preference to neutrophils. We also demonstrate that neutrophil influx is unable to limit bacterial growth in the lung and is ultimately responsible for the severe inflammation during the lethal pro-inflammatory phase.
Introduction:
The historical impact of Yersinia pestis on humanity cannot be understated, as three major pandemics including the “Black Death” of the Middle Ages have been attributed to Y. pestis[1],[2]. The bacterium can be transmitted person-to-person via respiratory droplets resulting in primary pneumonic plague, which is the deadliest manifestation of Y. pestis infection[1],[2],[3]. In the event of respiratory exposure in humans, mortality rates are nearly 100% with a time to death of typically between four and seven days[1]. Its extreme lethality and history of weaponization have led to the assignment of Y. pestis as a Tier 1 Select Agent and compound fears of its intentional release as a weapon of bioterrorism[1]. Using a murine intranasal infection model, our laboratory demonstrated that the primary pneumonic plague syndrome progresses in two distinct phases[4]: an initial “pre-inflammatory phase” characterized by rapid bacterial replication in the lung in the absence of host innate immune responses, followed by a “pro-inflammatory” phase marked by extensive neutrophil influx, a massive pro-inflammatory cytokine storm, and considerable tissue destruction within the lung. In mice and humans, progression into this pro-inflammatory phase invariably results in death without immediate treatment[5],[6]. Recently, our laboratory showed that Y. pestis creates a unique protective environment in the lungs of mice that allows for the growth of typically avirulent organisms[7], suggesting that Y. pestis suppresses host innate immune responses in the lung early during infection. The mechanism and host cell types involved in this phenomenon remain unknown. Y. pestis utilizes a plasmid-encoded type III secretion system( T3SS) to deliver Yersinia effector proteins( Yops) directly to the cytosol of target cells. Injection of Yop effectors is essential for Y. pestis virulence and is known to have anti-inflammatory and anti-phagocytic effects on mammalian cells[2],[8],[9]. The pulmonary cells targeted by Y. pestis during primary pneumonic plague have yet to be identified. In the work presented here, we use fully virulent Y. pestis CO92 expressing a YopE-TEM β-lactamase hybrid protein to identify the host cells targeted in the lung, and to evaluate the effect of depletion of these cell types on the progression of pneumonic plague. We show that the Y. pestis T3SS primarily targets macrophages and neutrophils early during the pre-inflammatory phase of disease. We also monitor the host cell dynamics in the lung in response to Y. pestis challenge and show that neutrophils are ultimately responsible for the severe necrotizing pneumonia during the pro-inflammatory disease phase. This work is the first identification/evaluation of the host cells targeted by fully virulent Y. pestis in the lung, and the results give insight into the dynamic events occurring shortly after pulmonary exposure to the highly lethal pathogen Y. pestis.
Discussion:
The work presented here is the first to identify the initial host cell targets of fully virulent Y. pestis during pulmonary infection. Using flow cytometry to monitor injection of a YopE-TEM fusion protein by the T3SS, we showed that Y. pestis initially targets CD11chigh alveolar macrophages and neutrophils in the lung. Previously, Bosio et al. demonstrated that the attenuated( pgm- pCD1-) Y. pestis strain A1122 was taken up by a distinct CD11c+ CD11b− host population in the lungs following intratracheal inoculation, confirming that Y. pestis initially encounters alveolar macrophages early during infection[14]. By 12 hpi there is a dramatic shift in host target cell preference from macrophages to neutrophils that continues to increase by 24 hpi. This corresponds with a massive influx of neutrophils into the lung that is detected as early as 6 hpi. In addition to neutrophils, flow cytometry analysis of infected lungs revealed a 10-fold increase in numbers of CD11bhigh DCs by 24 hpi. CD11bhigh DCs are known to accumulate in airways adjacent to alveolar spaces after allergen challenge, and have been implicated in the events that define asthma as well as contributing to CD4+ T-cell priming[19],[20]. Infected lungs also saw an overall increase in levels of F4/80+CD11b+CD11c+ cells. These cells exhibited positivity for both interstitial( CD11b+) and alveolar( CD11c+) macrophage markers and were not targeted by clodrosome in uninfected mice. This population likely consists of a subset of dendritic cells expressing F4/80, activated alveolar macrophages expressing CD11b, and macrophages that have entered the lung in response to bacterial challenge and are induced to express CD11c[21]. Taken together, these results outline a highly dynamic lung environment early during infection where different host innate immune populations are expanding in the lung in an attempt to control Y. pestis infection, albeit without much success. Despite the expansion of these cell types, macrophages and neutrophils remained the primary targets of YopE injection. The Y. pestis specificity for injection of macrophages and neutrophils cannot be explained by abundance alone, as the mammalian respiratory tract is comprised of dozens of host cell types with direct access to the airways. We observed minimal injection of CD45−CD3− epithelial/endothelial populations at each of the time points examined, despite the fact that this includes alveolar epithelial type I cells which are known to cover roughly 95% of the surface area of the alveoli[22]. Depletion of approximately 92% of alveolar macrophages hastened the shift in target cells seen during normal infection but resulted in only slight increases in levels of injection for other cell types, even though the abundance of these populations was roughly equivalent to that of neutrophils. Conversely, the depletion of roughly 99% of neutrophils in the lung prior to infection resulted in a roughly 10-fold decrease in total injection events. This is similar to what was seen for the enteric pathogen and recent Y. pestis ancestor Y. pseudotuberculosis, where depletion of neutrophils resulted in a significant overall decrease in total translocation events in Peyer's patches of orogastrically inoculated mice[10]. After treatment with α-Ly-6G antibody, neutrophils remained the primary host cell target. It is known that neutrophils can regenerate rapidly, particularly in the bone marrow, which may partly explain the failed emergence of an additional preferred cell target after depletion of lung neutrophils[23]. The targeting of professional phagocytes, specifically neutrophils, has been seen in other bacterial species and may represent a common virulence mechanism for pathogens harboring a functional T3SS[10],[24],[25]. P. aeruginosa has been shown to target macrophages and neutrophils in the lung[24], and Salmonella enterica has been shown to target neutrophils in the spleen via type III secretion to promote intracellular survival[25]. This phenomenon also occurs in other organs of the body during Yersinia infection. In the first example of in vivo detection of Yop translocation via a Yop-β-lactamase fusion, Marketon et al. showed that the attenuated Y. pestis pgm− strain KIM D27 targeted CD11b+, CD11c+, and Gr-1+ cells in the spleen when delivered intravenously, suggesting targeting of macrophages, dendritic cells, and neutrophils[12]. It will be intriguing to compare the injected host cell repertoire of Y. pestis delivered via additional routes of infection, including subcutaneous and intradermal, and in other tissues including lymph nodes and in the skin. As alveolar macrophages and neutrophils are the primary targets of Y. pestis type III secretion, we sought to further evaluate their roles during pulmonary infection. Depletion of roughly 92% of alveolar macrophages had little or no effect on bacterial burden in the lung. This indicates that either macrophages are not involved in limiting Yersinia survival in the lung, or that Y. pestis is able to neutralize the anti-bacterial effects of sentinel alveolar macrophages, presumably as a result of antiphagocytic/anti-inflammatory factors including the Yops, F1 capsular protein, and pH 6 antigen[2]. Also, depletion of alveolar macrophages did not significantly alter progression into the pro-inflammatory phase of disease. Infected animals demonstrated typical disease symptoms as well as the formation of inflammatory foci in infected lungs. Thus, the absence of greater than 90% of alveolar macrophages did not inhibit early host signaling that leads to the massive influx of neutrophils into the lung during pulmonary Y. pestis infection. This indicates that either the remaining number of alveolar macrophages is sufficient, or an additional cell type is responsible for this early host response. Neutrophils have been shown to play a fundamental role in protecting the host during pulmonary infection with a number of organisms, including Streptococcus pneumoniae, Klebsiella pneumoniae, and Legionella pneumophila[26],[27],[28],[29]. We hypothesized that deleting these key mediators of innate immunity would significantly exacerbate disease progression. Surprisingly, depletion of neutrophils had little to no effect on bacterial burden in the lung. This is in contrast to what has been reported previously, as Laws et al. reported a modest increase in bacterial titers in lungs of infected mice early after neutrophil depletion[30]. Differences in mouse lines and Y. pestis strains may have contributed to this disparity, as this group utilized a strain of Y. pestis that has poor fitness in the lung, as demonstrated by a decrease in bacterial burden by 24 hpi[30]. The unaltered pattern of bacterial proliferation in the lungs of neutrophil-depleted mice indicates that fully virulent Y. pestis is highly resistant to the antimicrobial effects of neutrophils that are recruited in response to infection. In support of this, the presence of the Y. pestis T3SS has been shown to be important for resisting neutrophil-mediated killing by inhibiting phagocytosis and repressing reactive oxygen species production[31]. Also, gene expression profiling in neutrophils exposed to Y. pestis indicated that the T3SS suppresses the activation of a number of genes responsible for pro-inflammatory cytokine output and prolonged cell survival[32]. Surprisingly, infected animals pre-treated with neutrophil-depleting antibody did not demonstrate outward signs of disease at 48 hpi despite significant bacterial burdens, indicating a failure to fully progress into the pro-inflammatory phase of pneumonia. Upon histological examination, the small sporadic foci that were detected in the lungs of these animals were primarily made up of alveolar-residing bacteria and a relatively small number of mononuclear cells and neutrophils. These animals eventually succumbed, not from the typical lung inflammation and pathology of pneumonic plague, but as a result of the unhindered spread of Y. pestis to other tissues of the body. The information gathered in this study supports a model of the pre-inflammatory phase of pneumonic plague where Y. pestis initially targets resident alveolar macrophages and then neutrophils to facilitate proliferation in the lung. As neutrophils are unable to sufficiently control Y. pestis, their continued chemotaxis results in the formation of massive inflammatory foci and causes severe damage to the alveolar architecture of the lung. Work evaluating the fate of injected macrophages and neutrophils will provide insight into the downstream signaling events that lead to progression into the pro-inflammatory phase of disease. In summary, the work presented here is the first to identify target host cells and evaluate population dynamics in the lung during primary pneumonic plague. Though much of the previous literature in the field highlights interactions between Y. pestis and macrophages, we show that Y. pestis primarily targets neutrophils early after inoculation in the lung, presumably to limit host innate immune mechanisms aimed at bacterial killing and clearance. To our surprise, depletion of neutrophils, a key innate immune mechanism for controlling infection during pneumonia, attenuated much of the intense inflammation seen in the lung during the pro-inflammatory phase of disease. Thus, neutrophils appear to be key players in the manifestation of what is ultimately a host-mediated necrotizing pneumonia. Understanding the early delay in innate immune response is crucial to understanding the events that ultimately lead to the progression of this highly lethal syndrome. Further, therapy targeting both bacterial growth as well as the destructive capacity of neutrophils in the lung may be key to expanding the time window during which treatment can be administered during pneumonic plague.
Inhalation of the bacterium Yersinia pestis results in primary pneumonic plague , a severe necrotizing pneumonia with mortality rates approaching 100% in the absence of timely antibiotic administration . Despite the notoriety of Y . pestis as a potential biological weapon and its well-established pandemic potential , very little is known regarding early host-pathogen interactions that lead to the progression of pulmonary infection . Y . pestis harbors a type III secretion system ( T3SS ) for delivery of Yersinia outer protein ( Yop ) effectors into host cells , an early and essential step in pathogenesis . In the work presented here , we identify the host cell targets of Y . pestis Yop secretion in the lung . We show that Y . pestis initially targets alveolar macrophages , followed by a shift in host cell preference to neutrophils . Through cellular depletion studies , we demonstrate that Y . pestis is highly resistant to macrophage- and neutrophil-mediated clearance , and that the accumulation of neutrophils in the lung is responsible for the severe necrotizing pneumonia that develops during the pro-inflammatory phase of pneumonic plague .