Impact of Key Nicotinic AChR Subunits on Post-Stroke Pneumococcal Pneumonia

Pneumonia is the most frequent severe medical complication after stroke. An overactivation of the cholinergic signaling after stroke contributes to immunosuppression and the development of spontaneous pneumonia caused by Gram-negative pathogens. The α7 nicotinic acetylcholine receptor (α7nAChR) has already been identified as an important mediator of the anti-inflammatory pathway after stroke. However, whether the α2, α5 and α9/10 nAChR expressed in the lung also play a role in suppression of pulmonary innate immunity after stroke is unknown. In the present study, we investigate the impact of various nAChRs on aspiration-induced pneumonia after stroke. Therefore, α2, α5, α7 and α9/10 nAChR knockout (KO) mice and wild type (WT) littermates were infected with Streptococcus pneumoniae (S. pneumoniae) three days after middle cerebral artery occlusion (MCAo). One day after infection pathogen clearance, cellularity in lung and spleen, cytokine secretion in bronchoalveolar lavage (BAL) and alveolar-capillary barrier were investigated. Here, we found that deficiency of various nAChRs does not contribute to an enhanced clearance of a Gram-positive pathogen causing post-stroke pneumonia in mice. In conclusion, these findings suggest that a single nAChR is not sufficient to mediate the impaired pulmonary defense against S. pneumoniae after experimental stroke

Paralyse der pulmonalen Immunität durch neurohumorale Mechanismen nach Schlaganfall

Bacterial pneumonias belong to the most serious complications after stroke whose underlying pathophysiological mechanisms are poorly understood. Overactivation of the autonomic, especially the cholinergic system, after central nervous system (CNS) injury contributes to a rapid onset of temporary immunosuppression and thus to the development of stroke-associated pneumonia (SAP). The α7 nicotinic acetylcholine receptor (nAChR) was identified in the experimental stroke model as an important mediator of the impaired immune response against spontaneous SAP. Since in vitro experiments indicated that non-α7nAChRs contribute to pulmonary immunosuppression, the first study investigated the role of α2, α5, α7 and α9/10nAChRs, expressed in the lung, in a mouse model of an aspiration-induced pneumococcal pneumonia after stroke. The absence of different nAChRs did not affect leukocyte recruitment to the lung and spleen, as well as pro- and anti-inflammatory cytokine responses in the lung and ultimately did not improve pathogen clearance, suggesting that nAChRs do not mediate the impaired immune response to pneumococci after stroke. The cholinergic system can be influenced at the level of acetylcholine synthesis, the transport, the release, the cognate receptors, but also by the regulation of the expression of underlying genes using small RNAs. In the second study, small RNAs in the blood of stroke patients were analyzed and demonstrated a decrease of microRNAs (miRs) and an increase of transfer RNA fragments (tRFs) two days after stroke. The stroke-induced tRFs contained complementary motifs to active transcription factors in monocytes regulating, among others, cholinergic genes and inflammation, suggesting that tRFs play a fundamental role in the regulation of the immune response after stroke. Besides cholinergic overactivation, the sympathetic nervous system is also activated after stroke, resulting, among others, in an IFN-γ-deficit and thus to an impaired bacterial defense. In the third study, intratracheal IFN-γ treatment of SAP was tested in a mouse model. Pulmonary IFN-γ administration had no negative effect on infarct maturation and can therefore be considered safe with respect to possible negative pro-inflammatory effects in the CNS. Despite an enhanced lung cell functionality in the IFN-γ treated stroke mice, both spontaneous infections and aspiration-induced pneumococcal pneumonias were not prevented. Although this work failed in an attempt to prevent SAP in the experimental stroke model by cholinergic blockade or by pulmonary cytokine therapy, the data provide basis for new therapeutic approaches. Combination therapy with different immunostimulators or manipulation of small RNAs to treat SAP would be conceivable here.Bakterielle Pneumonien gehören zu den schwerwiegendsten Komplikationen nach Schlaganfall, deren zugrundeliegenden pathophysiologischen Mechanismen nur unzureichend verstanden sind. Eine Überaktivierung des autonomen und insbesondere auch des cholinergen Systems nach Schädigung des zentralen Nervensystems (ZNS) trägt maßgeblich zu einer schnell einsetzenden, temporären Immunsuppression und damit zur Entwicklung von Schlaganfall-assoziierten Pneumonien (SAP) bei. Der α7 nikotinerge Acetylcholinrezeptor (nAChR) wurde im experimentellen Schlaganfall als wichtiger Mediator der beeinträchtigten Immunantwort in einer spontanen SAP identifiziert. Da in vitro Versuche auf eine Beteiligung von nicht-α7nAChR an einer pulmonalen Immunsuppression hinwiesen, wurde in der ersten Studie die Rolle von den in der Lunge exprimierten α2, α5, α7 und α9/10nAChR in einem Mausmodell der Aspirations-induzierten Pneumokokken-Pneumonie nach Schlaganfall untersucht. Die Abwesenheit verschiedener nAChR hatte keinen Einfluss auf die Rekrutierung von Leukozyten in Lunge und Milz sowie die pulmonale pro- und anti-inflammatorische Zytokinantwort und führte letztlich nicht zur verbesserten Beseitigung des Erregers, was dafürspricht, dass nAChR die gestörte Immunantwort gegen Pneumokokken nach Schlaganfall nicht vermitteln. Das cholinerge System kann auf der Ebene der Acetylcholinsynthese, des Transportes, der Freisetzung, der kognaten Rezeptoren, aber auch durch die Regulation der Expression der zugrundeliegenden Gene mittels kleiner RNAs beeinflusst werden. In der zweiten Studie wurden daher kleine RNAs im Blut von Schlaganfallpatienten untersucht und eine Verringerung der Mikro-RNAs (miRs) und einen Anstieg der Transfer-RNA-Fragmente (tRFs) zwei Tage nach Schlaganfall nachgewiesen. Die Schlaganfall-induzierten tRFs enthielten komplementäre Motive zu aktiven Transkriptionsfaktoren in Monozyten, die unter anderem cholinerge Gene und Inflammation regulieren. Diese Befunde sprechen dafür, dass tRFs eine fundamentale Rolle in der Regulation der Immunantwort nach Schlaganfall spielen. Neben der cholinergen Überaktivierung kommt es auch zu einer Stimulation des sympathischen Nervensystems, was unter anderem zu einem IFN-γ-Defizit und damit zu einer gestörten bakteriellen Abwehr führt. In der dritten Studie wurde die intratracheale IFN-γ Administration bei SAP im Mausmodell getestet. Die pulmonale IFN-γ Behandlung hatte keinen negativen Einfluss auf die Infarktreifung und kann daher in Bezug auf mögliche negative pro-inflammatorische Effekte im ZNS als sicher betrachtet werden. Trotz einer Verbesserung der Lungenzell-Funktionalität in den IFN-γ behandelten Schlaganfall-Mäusen konnten spontane Pneumonien sowie Aspirations-induzierte Pneumokokken-Pneumonien nicht verhindert werden. Obwohl diese Arbeit im experimentellen Schlaganfall-Modell keinen Nachweis dafür erbringen konnte, dass durch eine cholinerge Blockade oder durch gezielte pulmonale Zytokin-Therapie die SAP verhindert werden kann, bieten die Ergebnisse eine Grundlage für neue Therapieansätze. Basierend auf den hier vorgelegten Daten könnten Kombinationstherapien mit verschiedenen Immunstimulatoren oder die Manipulation kleiner RNAs zur Behandlung der SAP sinnvolle Optionen darstellen

Puumala Virus in Bank Voles, Lithuania

Little is known about the presence of human pathogenic Puumala virus (PUUV) in Lithuania. We detected this virus in bank voles (Myodes glareolus) in a region of this country in which previously PUUV-seropositive humans were identified. Our results are consistent with heterogeneous distributions of PUUV in other countries in Europe

Spatial and Temporal Evolutionary Patterns in Puumala Orthohantavirus (PUUV) S Segment

The S segment of bank vole (Clethrionomys glareolus)-associated Puumala orthohantavirus (PUUV) contains two overlapping open reading frames coding for the nucleocapsid (N) and a non-structural (NSs) protein. To identify the influence of bank vole population dynamics on PUUV S segment sequence evolution and test for spillover infections in sympatric rodent species, during 2010–2014, 883 bank voles, 357 yellow-necked mice (Apodemus flavicollis), 62 wood mice (A. sylvaticus), 149 common voles (Microtus arvalis) and 8 field voles (M. agrestis) were collected in Baden-Wuerttemberg and North Rhine-Westphalia, Germany. In total, 27.9% and 22.3% of bank voles were positive for PUUV-reactive antibodies and PUUV-specific RNA, respectively. One of eight field voles was PUUV RNA-positive, indicating a spillover infection, but none of the other species showed evidence of PUUV infection. Phylogenetic and isolation-by-distance analyses demonstrated a spatial clustering of PUUV S segment sequences. In the hantavirus outbreak years 2010 and 2012, PUUV RNA prevalence was higher in our study regions compared to non-outbreak years 2011, 2013 and 2014. NSs amino acid and nucleotide sequence types showed temporal and/or local variation, whereas the N protein was highly conserved in the NSs overlapping region and, to a lower rate, in the N alone coding part

Transfer RNA fragments replace microRNA regulators of the cholinergic post-stroke immune blockade

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced two days after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by RT-qPCR validated the top 6 upregulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes upregulated the top 6 stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using an ssRNA mimic induced downregulation of immune regulator Z-DNA binding protein 1 (Zbp1). In summary, we identified a “changing of the guards” between RNA types that may systemically affect homeostasis in post-stroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein- and RNA-level. Significance Statement Ischemic stroke triggers peripheral immunosuppression, increasing the susceptibility to post-stroke pneumonia that is linked with poor survival. The post-stroke brain initiates intensive communication with the immune system, and acetylcholine contributes to these messages; but the responsible molecules are yet unknown. We discovered a “changing of the guards,” where microRNA levels decreased but small transfer RNA fragments (tRFs) increased in post-stroke blood. This molecular switch may re-balance acetylcholine signaling in CD14+ monocytes by regulating their gene expression and modulating post-stroke immunity. Our observations point out to tRFs as molecular regulators of post-stroke immune responses that may be potential therapeutic targets

Transfer RNA fragments replace microRNA regulators of the cholinergic poststroke immune blockade

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a “changing of the guards” between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level