Sepsis is a systemic inflammatory response to infection, which may ultimately lead to multi-organ failure. Sepsis causes millions of deaths each year and creates tremendous financial burdens on the health care system, yet there is no effective cure for sepsis. Even years after the onset of sepsis, patients who have clinically recovered still die from sepsis-related complications due to chronic immune dysfunction. Neutrophils, the most dominant leukocytes in human circulatory systems, play a critical role in not only promoting inflammation to fight against microbe invasion but also facilitating inflammation resolution to restore immune homeostasis. While dysfunctional/exhausted neutrophils have been implicated in the long-term morbidity and mortality of sepsis, the cause of neutrophil exhaustion and the system to rejuvenate the dysregulated immunity are understudied. To fill in the missing piece here, we conducted our trilogy-like projects. First, we established an in vitro culture system to mimic sepsis-like conditions: murine neutrophils prolonged-stimulated with LPS exhibit exhaustion-related phenotype with the elevated expression of both proinflammatory and immunosuppressive makers on the cell surface as well as dysregulated swarming patterns. We found that by knocking out TRAM (TICAM2), an adaptive molecule regulating TLR4 downstream MyD88-independent signaling pathway, neutrophils exhibit attenuated exhaustion on both phenotypic and functional levels. Of note, TRAM contributes to the development of exhausted neutrophils through activating Src family kinases (SFKs)-STAT1 cascade, and deficiency in TRAM provides protective effects on systemic inflammation, reduces tissue injury, and improves survival in a murine colitis-induced sepsis model. Next, in my second project, we reported that neutrophils can be clustered into three subpopulations even at their naïve state based on the single-cell RNA sequencing (scRNAseq) analyses. Of note, neutrophils in one of the clusters are more mature but less apoptotic with the elevated expression of resolving-associated markers Cd86 and Cd200r, hence we termed these neutrophils as 'resolving neutrophils'. We found that the resolving neutrophil population can be expanded via pharmacologically reprogramming with sodium 4-phenylbutyrate (4-PBA) or genetic deletion of TRAM. Resolving neutrophils not only secrete more pro-resolving mediators, such as ResolvinD1 and SerpinB1, but also exert enhanced phagocytic and bactericidal capacities. Mechanistically, we discovered that the development of resolving phenotype in neutrophils is mediated by the PPARγ/LMO4/STAT3 signaling circuitry, which is constitutively suppressed by TRAM. To explore the translational applications of resolving neutrophils, in my third and final project, we conducted adoptive transfer experiments to examine the effects of TRAM-deficient resolving neutrophils in cecal slurry (CS)-induced septic mice. We found that TRAM-deficient mice are more resilient to severe sepsis with reduced tissue injury and less compromised lung integrity as compared to wild-type (WT) mice, and splenic neutrophils from TRAM deficient septic mice better preserve resolving-related features. Moreover, transfusing TRAM deficient neutrophils in WT septic mice renders therapeutic effects with alleviated lung and kidney damage. We also observed TRAM-deficient neutrophil-mediated resolving memory propagation in vitro to promote resolving features of neutrophils, monocytes, and T cells, as well as to strengthen endothelial cell barrier function. In terms of the mechanism, we reported that TRAM is critical for the secretion of neutrophil elastase, a potent protein to compromise endothelium; hence, endothelial cells cocultured with TRAM deficient neutrophils maintain higher levels of adhesion/tight junction markers than cocultured with WT neutrophils. Taken together, our trilogy projects better define exhausted and resolving neutrophils. And most importantly, our works demonstrate that TRAM, an underappreciated molecule, is responsible for inducing neutrophil exhaustion and suppressing resolving neutrophil generation. / Doctor of Philosophy / A 'good' inflammation upon the infection should include two steps: the initial proinflammatory response to combat invading pathogens followed by the later resolution process to repair damage and restore the balance of the host's immune system. The harmony of these reactions is essential to maintain immune homeostasis, and the disruption of immune homeostasis may lead to different pathogenic conditions, including sepsis. Neutrophils are the most dominant white blood cells in human circulation, and they play a critical role in both promoting proinflammatory response and facilitating inflammation resolution. While the dysfunction of neutrophils is associated with the pathogenesis of sepsis and implicated in long-term sepsis-related death, approaches to rejuvenate dysregulated/exhausted neutrophils to restore immune homeostasis in septic patients are still lacking. In our projects, we better defined the characteristics of exhausted neutrophils in a sepsis-mimicking condition and unveiled the underlying mechanisms of neutrophil exhaustion. In addition, we demonstrated that neutrophils with pro-resolving features can be expanded concurrently with the decrease of exhausted neutrophils by a genetic modification approach. Finally, we showed that neutrophils with pro-resolving features can offer therapeutic effects in sepsis mice to alleviate tissue injury and organ dysfunction.

Sepsis is a life-threatening condition characterized by organ dysfunction due to an uncontrolled response to infection. Despite decades of research, the mortality rate remains high, emphasizing the need for an improved understanding of sepsis pathophysiology and improvements in preclinical animal research. Recently, extracellular histones, major mediators of organ dysfunction and death, have emerged as a potential therapeutic target for sepsis. In this thesis, we reported that the ability of heparin to neutralize the cytotoxic and procoagulant effects of histones is size-dependent but independent of the antithrombin- binding pentasaccharide. In contrast, the ability of heparin to neutralize histone-mediated impairment of activated protein C generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular disease states that are associated with elevated levels of histones. Before testing the therapeutic efficacy of the heparin variants in vivo, we aimed to develop and standardize a murine model of sepsis that can be utilized in a multi-center platform. As one of the lead sites for the National Preclinical Sepsis Platform (NPSP), we optimized a 72-hour model of abdominal sepsis using supportive treatments. As sepsis predominately impacts the elderly, we also explored the impact of aging on the host response to sepsis using our fecal induced peritonitis (FIP) model. Aged FIP mice exhibited a higher mortality rate compared to young FIP mice. The worsened organ injury and poor survival in aged mice may be attributed to heightened inflammation in aged mice. We also observed trends in increased bacterial loads, increased coagulation, elevated cell free DNA, and decreased ADAMTS13 activity in aged septic mice. These findings help to improve our understanding of how aging impacts the host response to sepsis, which may be translated into therapeutic strategies that considers advanced age as a risk factor for sepsis. / Thesis / Candidate in Philosophy

Bakgrund: Sepsis är ett allvarligt tillstånd som obehandlat kan leda till organsvikt och död. Infektioner kan utvecklas till sepsis hos såväl tidigare fullt friska som multisjuka personer, i alla åldersgrupper och samhällsskikt. Kombinationen av diffusa symtom och ett snabbt sjukdomsförlopp ställer krav på sjuksköterskors kompetens, eftersom tidig identifiering och behandling är avgörande för patienternas överlevnad. Syfte: Syftet med studien är att kartlägga olika interventioners effekt på sjuksköterskors tidiga identifiering och omvårdnad av patienter med sepsis. Metod: Litteraturöversikt baserad på 12 kvantitativa interventionsstudier från PubMed och Cinahl. Artiklarna kvalitetsgranskades och analyserades därefter med kvantitativ ansats. Resultatet presenteras i text samt med deskriptiv statistik. Resultat: Studiens resultat presenteras i tre kategorier; Identifiering, Åtgärder och Patientutfall. Statistiskt signifikanta förbättringar identifierades i samtliga tre kategorier i form av bland annat minskad dödlighet och LOS samt förbättrad identifiering och följsamhet för sepsis bundles. Konklusion: Studien indikerar att testade interventioner har potential att förbättra identifiering, omvårdnad och utfall för sepsispatienter. Litteraturstudien betonar behovet av screeningverktyg och omvårdnadsåtgärder med högre evidensgrad, mer tydliga riktlinjer och en arbetsmiljö som ger sjuksköterskor möjlighet att arbeta med hela sin kompetens. / Background: Sepsis is a serious condition that untreated may cause organ failure and death. Infections can develop into sepsis in previously healthy people as well as multimorbid patients, in all ages and social strata. The combination of diffuse symptoms and quick progression of disease make demands on nurses’ competence, thus early identification and treatment is crucial for patients’ survival. Aim: The aim of the study was to examine the effect of different interventions on nurses’ early identification and care of patients with sepsis. Methods: Literature review of 12 quantitative intervention studies from PubMed and Cinahl. The articles were quality checked and analyzed with a quantitative approach. Results are presented in written text and descriptive statistics. Results: The results are presented in three categories; Identification, Nursing interventions and Patient outcomes. This study demonstrated statistically significant improvements in all three categories including reduced mortality and LOS, improved identification and compliance to sepsis bundles. Conclusion: This study indicates that examined interventions have potential to improve identification, care and outcome for septic patients. It further emphasizes the need for higher evidence screening tools and nursing interventions, clearer guidelines and a working environment which gives nurses the opportunity to carry out their competencies.

Bakgrund: Infektionsorsakad sepsis är en av världens dödligaste sjukdomar. Varje år dör cirka 11 miljoner människor av sepsis i världen. Vem som helst kan drabbas och sjukdomen orsakar omfattande lidande för patienten som kräver snabb handläggning från sjuksköterskan. Syfte: Var att belysa sjuksköterskans erfarenhet av att vårda patienter med sepsis inom akutsjukvård och på vårdavdelning. Metod: En allmän litteraturöversiktav kvantitativ och kvalitativ vetenskaplig forskning har använts i studien. Där både kvantitativa och kvalitativa artiklar systematiskt har sökts fram, kritiskt granskats, analyserats och sammanställts till ett nytt resultat. Resultat: Fyra teman med tio underteman framkom. Huvudtemana benämndes: Komplex och erfarenhets krävande vårdform, Kommunikations- och samarbetssvårigheter, Bristande resurser och hög arbetsbelastnings påverkan på sepsisvården, Sjuksköterskans erfarenhet av kompetens och kunskap i sepsisvården. Resultatet visar att sjuksköterskor anser att sepsisvården är en krävande vårdform som också betonar vikten av att tidigt identifiera sepsis och påbörja behandling för att rädda liv. Konklusion: Studien visar att sepsis är en utmanande och svår sjukdom att både identifiera och behandla som sjuksköterska. Patienter med sepsis kräver dessutom ett starkt fungerande team omkring sig, för att klara av de utmaningar som både patienter och sjukvårdspersonalen står inför. / Background: Sepsis caused by infection is one of the world's deadliest diseases. Every year around 11 million people die from sepsis around the world. Anyone can be affected, the disease causes extensive suffering to the patient which requires prompt management by the nurse. Purpose: Was to shed light on nurse experience of caring for patients with sepsis in emergency care and in the nursing department. Method: A general literature review of quantitative and qualitative scientific research has been used in the study. Where both quantitative and qualitative articles have been systematically sought out, critically reviewed, analyzed and compiled into a new result. Results: Four themes with tensub-themes emerged. The main themes were: Complex and experience-demanding form of care, Communication and cooperation difficulties, Lack of resources and high workload impact on sepsis care, The nurse's competence and knowledge. The results show that the nurses believe that sepsis care is a demanding form of care that also emphasizes the importance of identifying sepsis early and starting treatment to save lives. Conclusion:The study shows that sepsis is a challenging and difficult disease to both identify and treat as a nurse. Patients with sepsis also require a strong functioning team around them, to cope with the challenges that both patients and healthcare professionals face.

Les sepsis sévères et chocs septiques constituent des causes croissantes de morbidité et de mortalité chez les patients hospitalisés. La dysfonction cardio-circulatoire lors d'un choc septique s'avère un élément majeur et initial dans l'induction et l'entretien des défaillances organiques subséquentes. En effet, certaines bactéries et endotoxines déclenchent une séquence d'événements cellulaires qui mènent à une altération des performances cardiaques. Le macrophage migration inhibitory factor (MIF) a récemment été identifié en tant que facteur de dépression cardiaque lors de choc septique. Ainsi, la présente étude révèle que la neutralisation du MIF prévient la dysfonction myocardique induite par l'endotoxine dans un modèle expérimental chez le rat. De façon sous-jacente, le blocage du MIF empêche la hausse d'expression et de production dans le coeur de cytokines inflammatoires paracrines et autocrines (IL-1, IL-6 et TNF-[alpha]) en réponse au LPS. D'un point de vue mécanistique, cette étude démontre que la neutralisation du MIF inhibe l'apoptose des cardiomyocytes provoquée par l'endotoxine. Ainsi, le présent travail met en évidence un mécanisme précis par lequel le MIF influence la dépression cardiaque endotoxinique. Ce mécanisme implique que le blocage du MIF induit une augmentation du ratio protéique cardiaque de Bcl-2/Bax contribuant ainsi à prévenir le relâchement du cytochrome c mitochondrial induit par l'endotoxine. Cette inhibition de la perte de cytochrome c mitochondrial entraîne une réduction de l'activation de la caspase-3. La neutralisation du MIF rétablie la déficience provoquée par le LPS au niveau de la translocation nucléaire de phospho-Akt et par conséquent l'expression du facteur nucléaire de survie cardiaque GATA-4. Cette baisse d'activité caspase-3 et le rétablissement de la translocation/expression des facteurs de survie par le blocage du MIF résultent en une diminution de la fragmentation de l'ADN caractéristique de l'apoptose tardive. Globalement, l'inactivation du MIF lors d'un choc endotoxinique prévient le déséquilibre inflammatoire et apoptotique dans le coeur protégeant ainsi contre la dysfonction myocardique subséquente.

Sepsis-related multiple organ dysfunction syndrome is a leading cause of death in intensive care units. There is overwhelming evidence that oxidative stress plays a significant role in the pathogenesis of sepsis-associated multiple organ failure; however, reactive oxygen species (ROS)-associated biomarkers and/or diagnostics that define mortality or predict survival in sepsis are lacking. Lung or peripheral blood gene expression analysis has gained increasing recognition as a potential prognostic and/or diagnostic tool. The objective of this study was to identify ROS-associated biomarkers predictive of survival in patients with sepsis. In-silico analyses of expression profiles allowed the identification of a 21-gene ROS-associated molecular signature that predicts survival in sepsis patients. Importantly, this signature performed well in a validation cohort consisting of sepsis patients aggregated from distinct patient populations recruited from different sites. Our signature outperforms randomly generated signatures of the same signature gene size. Our findings further validate the critical role of ROSs in the pathogenesis of sepsis and provide a novel gene signature that predicts survival in sepsis patients. These results also highlight the utility of peripheral blood molecular signatures as biomarkers for predicting mortality risk in patients with sepsis, which could facilitate the development of personalized therapies.

Sepsis is commonly caused by bacteria, fungi or both present in the blood stream during inflammation. In response, inflammatory cascades are released in multiple organ systems which if prolonged causes sepsis and can eventually lead to organ failure and death. The major diagnostic technique of sepsis is blood culturing. However, the technique is time consuming and lacks sensitivity; especially in patients under antimicrobial therapy. Molecular techniques particularly PCR could possibly become implemented in sepsis diagnostics in the future. The aim of the thesis was to compare the effect on PCR sensitivity by different PCR kits, with optimized PCR conditions to find an ideal Real-time PCR applicable for direct detection of rRNA or rDNA in whole blood, using the 16S rDNA gene. The study also surveyed the overall background flora of bacterial species circulating in the blood. During the optimization Haemophillus influenzae and Streptococcus pneumoniae were added to whole blood, rRNA or rDNA was isolated and extracted and subsequently processed by Real-time PCR. Four commercially available PCR kits were compared. Attempts using rRNA did not significantly increase the PCR sensitivity. LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics) used for rDNA, generated low cp-values, the cleanest sequences and the finest separation between amplification curves. Twenty whole blood and pre-cultured patient samples were processed by the optimized PCR. The effect on PCR sensitivity by pre-culturing patient blood samples was studied and no statistical difference was noted. Increased PCR sensitivity is essential for implementation of PCR techniques in sepsis diagnostics.

Bakgrund: Sepsis är en livshotande diagnos som idag drabbar många patienter i Sverige och runt om i världen. Forskning visar att det är av största vikt med en tidig identifiering av diagnosen för att patienten ska få adekvat behandling och därmed öka chansen för dennes överlevnad. Sjuksköterskan har en nyckelroll vad gäller tidig identifiering och initial behandling av sepsis. Syfte: Syftet var att belysa sjuksköterskans erfarenhet av att identifiera patienter med sepsis. Metod: En kvalitativ metod där sju sjuksköterskor med erfarenhet av att identifiera sepsis intervjuades med hjälp av en semistrukturerad intervjuguide. Intervjuerna spelades in, transkriberades och analyserades utifrån Graneheim och Lundmans kvalitativa innehållsanalys. Resultat: Tre huvudkategorier framkom: Rutiner och skattningsinstrument, Samarbete och kommunikation och Kunskap och kompetens. Informanterna upplevde det svårt att följa rutiner för NEWS-kontroller. För att identifiera misstänka sepsis- patienter krävdes att NEWS kompletterades med en helhetsbedömning av patientens tillstånd med hjälp av den kliniska blicken. Samarbete och kommunikation ansågs viktig både i arbetet med tidig identifiering av sepsis och i akuta situationer.   Slutsats: Sepsis upplevs som svårt att identifiera om det inte uppvisas tydliga symtom hos patienten, som feber och frossa. Utbildning och erfarenhet bidrar till ökad kunskap kring sepsis och hur tidig identifiering kan ske.

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