Responses of the innate immune system to the human pathogen Streptococcus pyogenes

Streptococcus pyogenes, auch bekannt als Gruppe A Streptokokkus (GAS), ist ein bedeutendes humanes Pathogen, das für eine Reihe von Krankheiten verantwortlich gemacht wird. Diese Krankheiten können relativ harmlos sein, wie zum Beispiel Mandelentzündung, aber auch extrem gefährlich für den Patienten, wie zum Beispiel invasive Infektionen der Haut und der darunterliegenden Gewebe, die zu einer nekrotisierenden Fasziitis, Sepsis oder zu einem streptokokkalen toxischen Schock-Syndrom führen können. Bakterielle Pathogene werden von Zellen des angeborenen Immunsystems durch spezielle Rezeptoren, die auf die Erkennung von bestimmten bakteriellen Mustern von Bakterien spezialisiert sind, erkannt. Die Toll-like Rezeptoren (TLRs) sind die wohl am besten charakterisierte Familie unter diesen Rezeptoren und jedes Pathogen wird von mindestens einem dieser Rezeptoren erkannt. Jedoch sind die Moleküle, die für die Aktivierung einer Immunantwort gegen S. pyogenes verantwortlich sind, noch nicht bekannt. Wir konnten zeigen, dass S. pyogenes zur Aktivierung einer inflammatorischen Antwort in aus dem Knochenmark von Mäusen gewonnenen Makrophagen führt, die durch die Produktion von Zytokinen wie TNF-alpha und IL-6 gekennzeichnet ist. Diese Aktivierung ist im Fall von S. pyogenes abhängig von dem Adapter-Molekül MyD88, jedoch unabhängig von den wichtigsten bakteriellen Rezeptoren TLR2, TLR4 und TLR9. Zusätzlich konnten wir zeigen, dass die Infektion von Zellen des angeborenen Immunsystems mit S. pyogenes zu einer Produktion von Typ I Interferonen (IFNs) und einer anschließenden Aktivierung des Jak/Stat Signaltransduktionsweges führt. Experimente mit Makrophagen, denen bestimmte Komponenten des IFN- Signaltransduktionsweges fehlen (IRF3, IRF1, STAT1, IFNAR1) und mittels siRNA-vermittelter Inaktivierung einer anderen Komponente dieses Signalwegs (TBK1) führten zu dem Schluss, dass die IFN-Produktion unterhalb eines noch nicht bekannten Rezeptors abhängig von IRF3, TBK1 und IFNAR1 aber unabhängig von bekannten TLRs geschieht, obwohl eine partielle Abhängigkeit von MyD88 beobachtet wurde. Ferner deuteten mit Enzymen verdaute streptokokkale Extrakte darauf hin, dass streptokokkale DNA für die Induktion der IFN- Produktion wichtig ist. Allerdings sind auch in diesem Fall die Moleküle die für diese Induktion noch nicht bekannt. Zusammengefasst wurde in dieser Studie gezeigt, dass Streptococcus pyogenes in der Lage ist, gleichzeitig mehrere Signaltransduktionswege im Wirt zu aktivieren. Dies könnte zu der bekannten Eigenschaft des Bakteriums beitragen, viele schwere inflammatorische Krankheiten hervorzurufen.Streptococcus pyogenes, also known as group A streptococcus (GAS), is an important human pathogen responsible for a variety of diseases ranging from mild (e.g. tonsillitis) to more severe infections (e.g. invasive infections of skin and soft tissues that can develop into necrotizing fasciitis, sepsis or lethal toxic shock syndrome). Bacterial pathogens are recognized by cells of the innate immune system through pattern recognition receptors (PRRs). Among them, the family of Toll-like receptors (TLRs) is the best characterized class of PRRs and virtually all pathogenic bacteria are recognized by at least one of them. However, the molecule(s) responsible for the activation of an immune response against S. pyogenes are not known yet. We were able to demonstrate that activation of an inflammatory response in murine bone-marrow derived macrophages (BMDMs) against S. pyogenes, involving the production of cytokines such as TNF-alpha and IL-6, depends on the adaptor molecule MyD88, but not on the prototype bacterial receptors TLR 2, 4 and 9. Moreover, we could show that infection of innate immune cells with S. pyogenes causes the production of type I interferons (IFNs) and subsequent activation of the Jak/Stat signaling pathway. Experiments using BMDMs from mice deficient in components of the interferon pathway (IRF3, IRF1, STAT1, IFNAR1) and siRNA-mediated knock down of another important gene (TBK1) from this pathway revealed that IFN-production occurs downstream of an unknown PRR via IRF3, TBK1 and IFNAR1, but without the involvement of any described TLRs, although a partial dependence on MyD88 was observed. Furthermore, digested streptococcal extracts suggest that streptococcal DNA is required for the induction of IFN-beta production. However, the receptor molecule which promotes the induction of the interferon response stays elusive. An experimental mouse model of skin infection using IFNAR1-deficient mice emphasized the important role of type I IFNs during the host immune response against GAS. Taken together, our work demonstrates that Streptococcus pyogenes has the ability to induce multiple signalling pathways within the host, which may contribute the known high capability to cause severe inflammatory diseases

Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection

Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs

Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

International audienceInnate immune recognition of the major human-specific Gram-positive pathogen Strepto-coccus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2-and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon in-ternalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 −/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms

Machine learning algorithms performed no better than regression models for prognostication in traumatic brain injury

Objective: We aimed to explore the added value of common machine learning (ML) algorithms for prediction of outcome for moderate and severe traumatic brain injury. Study Design and Setting: We performed logistic regression (LR), lasso regression, and ridge regression with key baseline predictors in the IMPACT-II database (15 studies, n = 11,022). ML algorithms included support vector machines, random forests, gradient boosting machines, and artificial neural networks and were trained using the same predictors. To assess generalizability of predictions, we performed internal, internal-external, and external validation on the recent CENTER-TBI study (patients with Glasgow Coma Scale <13, n = 1,554). Both calibration (calibration slope/intercept) and discrimination (area under the curve) was quantified. Results: In the IMPACT-II database, 3,332/11,022 (30%) died and 5,233(48%) had unfavorable outcome (Glasgow Outcome Scale less than 4). In the CENTER-TBI study, 348/1,554(29%) died and 651(54%) had unfavorable outcome. Discrimination and calibration varied widely between the studies and less so between the studied algorithms. The mean area under the curve was 0.82 for mortality and 0.77 for unfavorable outcomes in the CENTER-TBI study. Conclusion: ML algorithms may not outperform traditional regression approaches in a low-dimensional setting for outcome prediction after moderate or severe traumatic brain injury. Similar to regression-based prediction models, ML algorithms should be rigorously validated to ensure applicability to new populations

How do 66 European institutional review boards approve one protocol for an international prospective observational study on traumatic brain injury? Experiences from the CENTER-TBI study

Abstract: Background: The European Union (EU) aims to optimize patient protection and efficiency of health-care research by harmonizing procedures across Member States. Nonetheless, further improvements are required to increase multicenter research efficiency. We investigated IRB procedures in a large prospective European multicenter study on traumatic brain injury (TBI), aiming to inform and stimulate initiatives to improve efficiency. Methods: We reviewed relevant documents regarding IRB submission and IRB approval from European neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI). Documents included detailed information on IRB procedures and the duration from IRB submission until approval(s). They were translated and analyzed to determine the level of harmonization of IRB procedures within Europe. Results: From 18 countries, 66 centers provided the requested documents. The primary IRB review was conducted centrally (N = 11, 61%) or locally (N = 7, 39%) and primary IRB approval was obtained after one (N = 8, 44%), two (N = 6, 33%) or three (N = 4, 23%) review rounds with a median duration of respectively 50 and 98 days until primary IRB approval. Additional IRB approval was required in 55% of countries and could increase duration to 535 days. Total duration from submission until required IRB approval was obtained was 114 days (IQR 75–224) and appeared to be shorter after submission to local IRBs compared to central IRBs (50 vs. 138 days, p = 0.0074). Conclusion: We found variation in IRB procedures between and within European countries. There were differences in submission and approval requirements, number of review rounds and total duration. Research collaborations could benefit from the implementation of more uniform legislation and regulation while acknowledging local cultural habits and moral values between countries

Primary versus early secondary referral to a specialized neurotrauma center in patients with moderate/severe traumatic brain injury: a CENTER TBI study

Funder: ZNS - Hannelore Kohl Stiftung; doi: http://dx.doi.org/10.13039/501100007731Funder: Integra LifeSciences CorporationFunder: OneMindAbstract: Background: Prehospital care for patients with traumatic brain injury (TBI) varies with some emergency medical systems recommending direct transport of patients with moderate to severe TBI to hospitals with specialist neurotrauma care (SNCs). The aim of this study is to assess variation in levels of early secondary referral within European SNCs and to compare the outcomes of directly admitted and secondarily transferred patients. Methods: Patients with moderate and severe TBI (Glasgow Coma Scale < 13) from the prospective European CENTER-TBI study were included in this study. All participating hospitals were specialist neuroscience centers. First, adjusted between-country differences were analysed using random effects logistic regression where early secondary referral was the dependent variable, and a random intercept for country was included. Second, the adjusted effect of early secondary referral on survival to hospital discharge and functional outcome [6 months Glasgow Outcome Scale Extended (GOSE)] was estimated using logistic and ordinal mixed effects models, respectively. Results: A total of 1347 moderate/severe TBI patients from 53 SNCs in 18 European countries were included. Of these 1347 patients, 195 (14.5%) were admitted after early secondary referral. Secondarily referred moderate/severe TBI patients presented more often with a CT abnormality: mass lesion (52% vs. 34%), midline shift (54% vs. 36%) and acute subdural hematoma (77% vs. 65%). After adjusting for case-mix, there was a large European variation in early secondary referral, with a median OR of 1.69 between countries. Early secondary referral was not associated with functional outcome (adjusted OR 1.07, 95% CI 0.78–1.69), nor with survival at discharge (1.05, 0.58–1.90). Conclusions: Across Europe, substantial practice variation exists in the proportion of secondarily referred TBI patients at SNCs that is not explained by case mix. Within SNCs early secondary referral does not seem to impact functional outcome and survival after stabilisation in a non-specialised hospital. Future research should identify which patients with TBI truly benefit from direct transportation

Occurrence and timing of withdrawal of life-sustaining measures in traumatic brain injury patients: a CENTER-TBI study

Funder: National Institute for Health Research (UK)Abstract: Background: In patients with severe brain injury, withdrawal of life-sustaining measures (WLSM) is common in intensive care units (ICU). WLSM constitutes a dilemma: instituting WLSM too early could result in death despite the possibility of an acceptable functional outcome, whereas delaying WLSM could unnecessarily burden patients, families, clinicians, and hospital resources. We aimed to describe the occurrence and timing of WLSM, and factors associated with timing of WLSM in European ICUs in patients with traumatic brain injury (TBI). Methods: The CENTER-TBI Study is a prospective multi-center cohort study. For the current study, patients with traumatic brain injury (TBI) admitted to the ICU and aged 16 or older were included. Occurrence and timing of WLSM were documented. For the analyses, we dichotomized timing of WLSM in early (< 72 h after injury) versus later (≥ 72 h after injury) based on recent guideline recommendations. We assessed factors associated with initiating WLSM early versus later, including geographic region, center, patient, injury, and treatment characteristics with univariable and multivariable (mixed effects) logistic regression. Results: A total of 2022 patients aged 16 or older were admitted to the ICU. ICU mortality was 13% (n = 267). Of these, 229 (86%) patients died after WLSM, and were included in the analyses. The occurrence of WLSM varied between regions ranging from 0% in Eastern Europe to 96% in Northern Europe. In 51% of the patients, WLSM was early. Patients in the early WLSM group had a lower maximum therapy intensity level (TIL) score than patients in the later WLSM group (median of 5 versus 10) The strongest independent variables associated with early WLSM were one unreactive pupil (odds ratio (OR) 4.0, 95% confidence interval (CI) 1.3–12.4) or two unreactive pupils (OR 5.8, CI 2.6–13.1) compared to two reactive pupils, and an Injury Severity Score (ISS) if over 41 (OR per point above 41 = 1.1, CI 1.0–1.1). Timing of WLSM was not significantly associated with region or center. Conclusion: WLSM occurs early in half of the patients, mostly in patients with severe TBI affecting brainstem reflexes who were severely injured. We found no regional or center influences in timing of WLSM. Whether WLSM is always appropriate or may contribute to a self-fulfilling prophecy requires further research and argues for reluctance to institute WLSM early in case of any doubt on prognosis

Frequency of fatigue and its changes in the first 6 months after traumatic brain injury: results from the CENTER-TBI study

Background: Fatigue is one of the most commonly reported subjective symptoms following traumatic brain injury (TBI). The aims were to assess frequency of fatigue over the first 6 months after TBI, and examine whether fatigue changes could be predicted by demographic characteristics, injury severity and comorbidities. Methods: Patients with acute TBI admitted to 65 trauma centers were enrolled in the study Collaborative European NeuroTrauma Effectiveness Research in TBI (CENTER-TBI). Subj

Tracheal intubation in traumatic brain injury

Background: We aimed to study the associations between pre- and in-hospital tracheal intubation and outcomes in traumatic brain injury (TBI), and whether the association varied according to injury severity. Methods: Data from the international prospective pan-European cohort study, Collaborative European NeuroTrauma Effectiveness Research for TBI (CENTER-TBI), were used (n=4509). For prehospital intubation, we excluded self-presenters. For in-hospital intubation, patients whose tracheas were intubated on-scene were excluded. The association between intubation and outcome was analysed with ordinal regression with adjustment for the International Mission for Prognosis and Analysis of Clinical Trials in TBI variables and extracranial injury. We assessed whether the effect of intubation varied by injury severity by testing the added value of an interaction term with likelihood ratio tests. Results: In the prehospital analysis, 890/3736 (24%) patients had their tracheas intubated at scene. In the in-hospital analysis, 460/2930 (16%) patients had their tracheas intubated in the emergency department. There was no adjusted overall effect on functional outcome of prehospital intubation (odds ratio=1.01; 95% confidence interval, 0.79–1.28; P=0.96), and the adjusted overall effect of in-hospital intubation was not significant (odds ratio=0.86; 95% confidence interval, 0.65–1.13; P=0.28). However, prehospital intubation was associated with better functional outcome in patients with higher thorax and abdominal Abbreviated Injury Scale scores (P=0.009 and P=0.02, respectively), whereas in-hospital intubation was associated with better outcome in patients with lower Glasgow Coma Scale scores (P=0.01): in-hospital intubation was associated with better functional outcome in patients with Glasgow Coma Scale scores of 10 or lower. Conclusion: The benefits and harms of tracheal intubation should be carefully evaluated in patients with TBI to optimise benefit. This study suggests that extracranial injury should influence the decision in the prehospital setting, and level of consciousness in the in-hospital setting. Clinical trial registration: NCT02210221