Community-acquired pneumonia is a common condition that leads to prolonged hospitalization, impaired clinical outcome, and increased mortality of patients, especially those with compromised immunity, such as children, the elderly, and those with immune suppression as a result of disease or injury.[1, 2] The gram-positive bacterium Streptococcus pneumoniae is a major human pathogen, and the most common cause of bacterial community-acquired pneumonia.
Infection also contributes to the development of cardio- and cerebrovascular diseases and is associated with poor outcomes after heart disease and stroke.[4-8] S. pneumoniae infection could contribute to stroke or cardioembolism by triggering the rupture of an atherosclerotic plaque in humans,[7-10] and molecular signatures of common bacteria, including Streptococcus species, have been identified in atherosclerotic lesions.[11, 12] Vaccination against S. pneumoniae reduces atherosclerosis; however, the effect of S. pneumoniae infection on atherogenesis has not been demonstrated experimentally.
Infection is a key risk factor for stroke in young patients, a cohort without any atherosclerotic burden, suggesting that infection could contribute to an ischemic event independently of inducing plaque rupture. Clinical data indicate that S. pneumoniae, together with other common infections such as Chlamydia pneumoniae and Haemophilus influenzae, also contributes to impaired outcome, prolonged hospitalization, and death after stroke. Experimental stroke in mice propagates pneumonia.[13, 14] However, it is not known if sustained bacterial infections preceding an acute cerebrovascular event could trigger stroke or contribute to increased stroke pathophysiology. Therefore, the main hypothesis tested in the study was whether preceding infection by a clinically highly relevant bacterial strain, S. pneumoniae, induces systemic vascular inflammation and worsens stroke outcome and whether this occurs via inflammatory mechanisms that could be blocked therapeutically.
Here we demonstrate that S. pneumoniae infection promotes atherogenesis, and exacerbates inflammatory responses to cerebral ischemia, leading to increased brain injury via platelets and interleukin (IL) 1. Thus, we suggest that complications after acute vascular events is preceded by a rapid systemic inflammatory response and excessive coagulation induced by pre-existing infection that profoundly impairs outcome. This could be prevented by treatment with IL-1 receptor antagonist (IL-1Ra).
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We present evidence that sustained pulmonary S. pneumoniae infection, the most common cause of bacterial community-acquired pneumonia, generates an IL-1–mediated systemic inflammatory response involving granulocytosis and platelet activation that accelerates atherogenesis and leads to cerebrovascular inflammation. S. pneumoniae–induced systemic inflammation preceding an acute cerebrovascular event profoundly exacerbates inflammation in the brain via IL-1– and platelet-dependent mechanisms that augment microglial IL-1α production and lead to increased neuronal injury.
Clinical data indicate that infection contributes to the development of vascular disease, is a trigger for an acute vascular event, and/or contributes to outcome once an acute vascular event has occurred. In patients, infection could mediate all of these effects in both heart disease and stroke,[5, 8, 10] although the mechanisms are not known, and current experimental evidence is insufficient. Chlamydia pneumoniae infection has been shown to induce an unstable atherosclerotic plaque phenotype in low-density lipoprotein receptor, ApoE double-knockout mice. There is clinical evidence to show that S. pneumoniae contributes to atherogenesis,[11, 12] although the efficacy of antibiotic treatment on heart disease is controversial. However, no experimental studies have addressed the role of sustained S. pneumoniae infection in plaque growth or plaque rupture to date. We show that systemic effects of infection induce inflammation in distant vascular beds, and lead to vascular pathologies in both the aorta and the brain, although the extent of the response in individual mice was different. Our study was not designed to specifically investigate plaque rupture, hence it is not surprising that no acute cardio- or cerebrovascular events were observed in infected, atherosclerotic rodents. In contrast, our data clearly indicate the acceleration of atherogenesis by S. pneumoniae infection in atherosclerosis-prone rodent models, arguing for a contribution of S. pneumoniae to vascular disease.
To examine the effects of sustained and localized pulmonary S. pneumoniae infection on systemic inflammatory changes and stroke outcome, we infected mice intranasally with S. pneumoniae ATCC 49619 serotype 19F (Danish), a clinically relevant, human isolate. Because major virulence factors such as pneumolysin are conserved across all S. pneumoniae strains, we used mock infection in control mice to account for any unexpected effects caused by the inoculum in the lungs. In general, serotype 19F isolates are associated with colonization of mucosa, and are less commonly associated with invasive disease (serotype 19F is included in the 7-valent conjugated pneumococcal polysaccharide vaccine PCV-7). Lack of invasiveness of ATCC 49619 was also confirmed in our experimental model. The infectious challenge was titrated over 5 days to maximize the stimulation of the immune system while minimizing the incidence of invasive disease in the acute phase. Although in this model the infection is localized to the lung, we found marked systemic inflammatory changes in several organs, dominated by the key proinflammatory cytokine, IL-1. It has been shown that serious pulmonary infection can cause multiorgan dysfunction and a high level of mortality. We developed our inoculation protocol to result in sustained infection over a course of several days using intermittent sublethal challenges, without causing major weight loss, fever, behavioral alterations, or death.
In patients, infections preceding a stroke are associated with increased stroke risk and result in impaired outcome, similarly to infections that develop poststroke.[5, 8, 35, 36] Our data clearly show that although pneumonia was associated with reduced pO2 levels (with O2 saturation unchanged) prior to stroke that could contribute to poor outcome, intervention against both IL-1 and platelet GPIbα reversed infection-induced cerebrovascular inflammation, neuronal injury, and impaired functional outcome, arguing for a major role of inflammation in brain pathologies caused by systemic effects of pulmonary S. pneumoniae infection. It is also possible that local and/or systemic inflammation could compromise perfusion and oxygen uptake/release in various organs including the lung or the brain. However, mild hypoxia due to lung inflammation was found to have no direct effect on infarct size. Nevertheless, our results strongly suggest that patients with stroke and pneumonia could benefit substantially from anti-inflammatory therapies, such as IL-1Ra. We investigated central and systemic inflammatory mechanisms in detail to explain how IL-1 can mediate injury after infection. As in our rodent model, infection-induced neutrophil and platelet activation has been documented in patients with pneumococcus-induced lung infection.[26, 38] Granulocyte levels were increased after infection and cerebral ischemia in the brain, and were marginally reduced by GPIbα blockade, but not IL-1Ra, indicating that granulocyte responses might not fully explain the effect of infection on brain injury. Granulocyte numbers were not increased in infected mice independently of infarct size either. Although after IL-1–mediated cerebrovascular transmigration granulocytes acquire a neurotoxic phenotype, and IL-1 actions can worsen injury in the brain via granulocytes in vivo,[30, 39] increased numbers of parenchymal granulocytes in the current experimental model might be indirectly associated with infection status and thus bigger infarcts. Nevertheless, systemic or perivascular granulocyte responses might mediate brain injury independently of the cells in the brain parenchyma, although this was not tested experimentally in the present study. In contrast, both platelet intervention and IL-1Ra uniformly reversed microglial IL-1α after cerebral ischemia in infected mice, suggesting that altered microglial activation (which was evident even prior to infection) could contribute to infection-induced inflammatory responses. Similarly, increased BBB injury was evident in infected mice with even smaller infarcts, supporting our earlier observations that systemic inflammatory mechanisms could mediate inflammation and injury in the brain independently of changes in infarct size.
Vascular activation after infection, and increased platelet aggregation in infected mice after cerebral ischemia, suggested that inflammatory signals in the brain might be mediated by activated platelets. According to a recent study, S. pneumoniae can induce platelet aggregation in a strain-specific manner via toll-like receptor 2, which is dependent on GPIIb/IIIa, but is not affected by aspirin. In addition, our previous data indicated that a chronic infection resulting in a systemic, Th1-polarized immune response leads to the accumulation of platelets in brain microvessels after cerebral ischemia, although the functional role of platelets in the brain has not been investigated earlier after infection. GPIbα blockade did not reduce microvascular platelet aggregates in the ischemic brain after S. pneumoniae infection, arguing for an inflammatory role for platelet GPIbα-mediated interactions in augmenting cerebrovascular pathologies. We reported recently that platelet-derived IL-1α can induce cerebrovascular inflammation. Activated platelets release IL-1 and/or IL-1–containing microparticles upon interacting with the endothelium.[42, 43]
We have demonstrated previously that peripheral IL-1 administration exacerbates brain injury, and inflammatory mechanisms reportedly contribute to cerebral ischemia even in the absence of systemic inflammation.[28, 29] Both GPIbα blockade and IL-1Ra have been shown to be protective in uninfected animals.[17, 18] However, due to the small cerebral injury (that mostly corresponded to the core of the infarct) seen in uninfected animals, we did not expect these interventions to significantly reduce brain injury in control animals in the current experimental model. Importantly, our present results indicate that IL-1–dependent systemic inflammation and pathologies in the central nervous system can develop in response to sustained bacterial infection in vivo, and this disease mechanism is central to the pathologies caused by a clinically important gram-positive bacterium, S. pneumoniae. It is likely that such mechanisms could be important in other vascular, noncommunicable diseases as well, such as myocardial infarction, and liver or kidney ischemia.
In conclusion, our data identify IL-1 as a key mediator of infection-induced brain injury and indicate that selective targeting of IL-1– and platelet-mediated mechanisms could be therapeutically useful to prevent infection-induced thromboinflammatory mechanisms, which predispose to acute vascular events and lead to profound impairment in outcome after stroke.
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A.D., N.J.R., and S.M.A. designed research. A.D., J.M.P., C.D., A.S., P.W., K.N.M., B.R., J.C., H.C., and B.M. performed research. P.W., D.H.D., S.F., and B.N. contributed new reagents/analytic tools. A.D., J.M.P., C.D., A.S., P.W., K.N.M., B.R., J.C., H.C., and B.M. analyzed data. A.D., N.J.R., and S.M.A. wrote the article. A.D. and J.M.P. contributed equally.