Neutrophils play an important, yet poorly understood role, in complement mediated pathologies. Here we identified that neutrophils contain key components from the complement alternative pathway: properdin (CFP), complement component 3 (C3), complement factor B (CFB), and complement factor H (CFH). Activation of neutrophils resulted in secretion of these complement components. When neutrophils are further activated to form neutrophil extracellular traps (NETs), CFP is deposited onto the surfaces of the NETs. In addition, CFP is able to bind to Pseudomonas aeruginosa, an opportunistic bacterium which can activate neutrophils to form NETs. Furthermore, NETs activate complement and increase formation of the terminal complement complex. The activation of complement on NETs can be initiated through multiple pathways, however, activation of the alternative pathway is dependent on CFP. This mechanism, potentially required for effective host defense, may also contribute to complement activation and disease.

Neutrophils play an important, yet poorly understood role, in complement mediated pathologies. Here we identified that neutrophils contain key components from the complement alternative pathway: properdin (CFP), complement component 3 (C3), complement factor B (CFB), and complement factor H (CFH). Activation of neutrophils resulted in secretion of these complement components. When neutrophils are further activated to form neutrophil extracellular traps (NETs), CFP is deposited onto the surfaces of the NETs. In addition, CFP is able to bind to Pseudomonas aeruginosa, an opportunistic bacterium which can activate neutrophils to form NETs. Furthermore, NETs activate complement and increase formation of the terminal complement complex. The activation of complement on NETs can be initiated through multiple pathways, however, activation of the alternative pathway is dependent on CFP. This mechanism, potentially required for effective host defense, may also contribute to complement activation and disease.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18857号 / 医博第3968号 / 新制||医||1008(附属図書館) / 31808 / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 江藤 浩之, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Neutrophil extracellular traps (NETs) are a link between infection and coagulation in sepsis. The major structural component of NETs is nucleosomes, consisting of DNA and histones. NETs not only act as a scaffold to trap platelets, but NET components also promote coagulation and impair fibrinolysis. Thus, removal of extracellular DNA by DNases may be a potential therapeutic strategy for sepsis. Since heparin is used for thromboprophylaxis in sepsis and may also be a potential anti-sepsis therapy, we investigated the mechanisms by which various forms of heparins modulate DNase function. There are two types of DNases in vivo: DNase I (produced by exocrine and endocrine glands) and DNase1L3 (secreted by immune cells). DNase I cleaves free DNA, whereas DNase1L3 preferentially cleaves DNA in complex with proteins such as histones. In this study, we investigated how DNase I and DNase1L3 activities are modulated by the following heparins: unfractionated heparin (UFH), enoxaparin (a low-molecular-weight heparin), Vasoflux (a low-molecular-weight, non-anticoagulant heparin), and fondaparinux (the pentasaccharide unit). Using agarose gel experiments, we showed that UFH, enoxaparin, and Vasoflux enhance the ability of DNase I to digest DNA-histone complexes (presumably by displacing DNA from histones), whereas fondaparinux does not. These findings are consistent with the KD values of the binding of heparin variants to histones, with fondaparinux having >1000-fold lower affinity for histones compared to the other heparins. Taken together, our data suggests that the ability of heparin to enhance DNase I-mediated digestion of DNA-histone complexes is size-dependent and independent of the pentasaccharide region of heparin. With respect to DNase1L3, we observed that it is able to digest histone-bound DNA, and that all heparins, except fondaparinux, inhibited DNase1L3-mediated digestion of histone-bound DNA. Next, we visualized the degradation of NETs by fluorescence microscopy. DNase I (± heparin variants) completely degraded NETs, presumably by digesting extracellular chromatin at histone-free linker regions, thereby releasing nucleosome units. DNase1L3 also degraded NETs, but not as effectively as DNase I, and was inhibited by all heparins except fondaparinux. Finally, we showed that DNase I levels are decreased and DNase1L3 levels are elevated in septic patients. Taken together, our findings demonstrate that heparin modulates the function of DNases, and that endogenous DNase levels are altered in sepsis pathophysiology. / Thesis / Master of Science (MSc) / Sepsis, a life-threatening condition due to hyperactivation of the immune system in response to infection, results in widespread inflammation and blood clotting. During sepsis, immune cells release sticky strands of DNA that block blood vessels and damage organs. Two different enzymes in the blood (DNase I and DNase1L3) can digest these DNA strands, and may represent a new class of anti-sepsis drugs. Our goal was to determine how heparins, commonly used blood thinners, alter the function of these enzymes. We found that (a) larger-sized heparins improved the activity of DNase I towards DNA-histone complexes and do not require any specific portion of heparin, (b) DNase I is more efficient than DNase1L3 in digesting DNA strands released from immune cells, and (c) levels of DNase I and DNase1L3 are altered in septic patients. Taken together, our studies provide new insights into how these enzymes function.

The core of the dissertation lies in developing two novel systems bioengineering approaches, a synthetic Escherichia coli killer-prey microecology, and a combined infection-inflammation NET-array system, to investigate the role of the mechanochemical complexity of the microenvironment in driving the microbe-microbe and host-microbe interactions, respectively. Herein, the first part of the dissertation includes designing and engineering a synthetic E. coli killer-prey microecological system where we quantified the quorum-sensing mediated interactions between the engineered killer and prey E. coli bacterial strains plated on nutrient-rich media. In this work, we developed the plate assay followed by plasmid sequencing and computational modeling that emphasizes the concept of the constant evolution of species or acquired resistance in the prey E. coli, in the vicinity of the killer strain. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. Through time-lapse imaging on petri plates automated using a scanner, we recorded the "kill wave" that originates outside the killer colony and travels outward as the prey dies. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. However, some of the prey colonies evolve to be resistant to the effects of AHL secreted by the killer. These prey colonies resistant to the killer were then selected and confirmed by plasmid sequencing. Using this empirical data, we developed the first ecological model emphasizing the concept of the constant evolution of species, where the survival of the prey species is dependent on the location (distance from the killer) or the evolution of resistance. The importance of this work lies in the context of the evolution of antibiotic-resistant bacterial strains and in understanding the communication between the microbial consortia, such as in the gut microbiome. Further, the second part of the dissertation includes quantifying the interactions between immune cells (primary healthy human neutrophils) and motile Pseudomonas aeruginosa bacteria in an inflammation-rich microenvironment. Neutrophils, being the first responding immune cells to infection, defend by deploying various defense mechanisms either by phagocytosing and killing the pathogen intracellularly or through a suicidal mechanism of releasing their DNA to the extracellular space in the form of Neutrophil Extracellular Traps (NETs) to trap the invading pathogens. Although the release of NETs is originally considered a protective mechanism, it is shown to increase the inflammation levels in the host if unchecked, ultimately resulting in end-organ damage (especially lung and kidney damage), as with the severe cases of sepsis and COVID-19. In our work, we developed a combined infection-inflammation NET-array system integrated with a live imaging assay to quantify the spatiotemporal dynamics of NET release in response to P. aeruginosa infection in an inflammatory milieu at a single-cell resolution. Importantly, we found increased NET release to P. aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. Our device platform is unique in that the nanoliter well-assisted individual neutrophil trapping enables us to quantify NET release with single-cell precision. Besides, incorporating confined side loops in the device helped us study the role of mechanical confinement on NET release, showing reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our microsystem. In summary, our work emphasizes the importance of studying the heterogeneity of NET release in host defense and inflammation. In the future, our system can be used for screening novel neutrophil-based immunotherapies and serve as a valuable research tool in precision medicine. / Doctor of Philosophy / The microenvironment plays a vital role in shaping the interactions within microbes and between the host and the microbes. Microbes use quorum-sensing-based chemical signaling to adapt to the environmental stresses in a microecology (be it a soil microecology or the gut microbiome). They communicate with each other with the help of these chemicals to regulate their population density (to mutual benefit in the case of a biofilm formation or to compete for resources in the case of a predator-prey model). In the first part of the dissertation, we utilize this quorum-sensing approach to study the spatiotemporal dynamics of the interactions between two engineered killer and prey Escherichia coli bacterial strains on a nutrient-rich agar plate in real-time. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. Further, through sequencing, we found that some of the prey colonies acquired resistance to the effects of AHL secreted by the killer. We then developed a computational model that recapitulates our experimental results, emphasizing the concept of the constant evolution of species or acquired resistance. The importance of this work lies in using experimental and computational approaches to better understand the evolution of multidrug-resistant (MDR) bacterial strains. Next, we investigated the interactions between primary human neutrophils (first responding immune cell type to infection) and motile Pseudomonas aeruginosa bacteria in the second part of the dissertation, explicitly focusing on quantifying neutrophil extracellular traps (NETs) release. With increasing concerns regarding the role of the dysregulated NET release in exaggerated inflammatory responses in the host, it is imperative to quantify NET release precisely at a single-cell level in a controlled microenvironment. To this end, we engineered a combined infection-inflammation NET-array device with 1024 nanoliter wells per device and achieved single-cell level trapping of neutrophils in these wells. Our device platform is unique in that the individual wells of the device have constricted side loops, which helps us better understand the role of mechanical confinement on NET release from an engineering standpoint. We then used the NET-array system to quantify the spatiotemporal dynamics of NET release to P. aeruginosa in an inflammatory mediator-rich microenvironment. Importantly, we found heightened NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. We also demonstrated reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our combined infection-inflammation microsystem. Especially with the increasing complexity of the intercellular cues at the site of infection, by integrating our microfluidic method with the conventional reductionist approaches, we can better solve the intricate puzzles of the immune cell decision-making processes at a single-cell level. Our study highlights the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation.

Orientador: Luciane Alarcão Dias-Melicio / Resumo: Recentemente, a deficiência de vitamina D vem se tornando um problema de abrangência mundial em virtude de hábitos rotineiros da população, como o trabalho por períodos prolongados em ambientes fechados e diminuição da exposição solar. Trabalhos recentes demonstram que a vitamina D age não somente na homeostase do cálcio, mas também na regulação do sistema imune. Diante da multiplicidade de funções dessa vitamina, sua deficiência tem sido associada ao risco de desenvolvimento de uma série de doenças, entre elas doenças infecciosas como as causadas por S. aureus. As infecções por essa bactéria têm trazido expressiva preocupação para a população humana em decorrência do aumento da prevalência de cepas resistentes aos fármacos antibacterianos, dificultando, dessa maneira, o tratamento e contribuindo para a busca de métodos alternativos para combater esse tipo de infecção. Além disso, o S. aureus conta com um potente arsenal de fatores de virulência que contribuem para a evasão da resposta imune do hospedeiro. Nesse contexto, torna-se importante avaliar se a vitamina D pode modular os efeitos bactericidas de neutrófilos humanos através de mecanismos intra e extracelulares, favorecendo, portanto, o combate a infecções, especialmente aquelas causadas por microrganismos resistentes aos principais tratamentos. Dessa maneira, nós demonstramos que neutrófilos tratados com vitamina D e desafiados com duas cepas de S. aureus tiveram um aumento nas taxas de fagocitose e atividade bacteric... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the last years, vitamin D deficiency has become a worldwide problem due to routine population habits, such as prolonged work indoors and increased use of sunscreen/decreased sun exposure in attempt to avoid high rates of skin cancer. Recent studies demonstrated that vitamin D acts not only on calcium homeostasis, but also on the regulation and function of the immune system. Facing the countless functions of vitamin D, its deficiency has been associated with the risk of development of many diseases, including infectious diseases such as those caused by S. aureus. Infections caused by these bacteria have brought significant concern to the human population due to the increased prevalence of strains resistant to antibiotics, thus making it difficult to treat and contributing to the search for alternative methods to combat this type of infection. In addition, S. aureus have a variety of virulence factors, which confer the ability to evade host immune responses. In this context, it is important to evaluate whether vitamin D can modulate the bactericidal effects of human neutrophils through intra- and extracellular mechanisms, such as phagocytosis, bacterial killing and release of Neutrophil Extracellular Traps (NETs), thus contributing to the response against infections, especially those caused by microorganisms resistant to the main treatments. Thus, we demonstrated that neutrophils treated with Vitamin D and challenged with two strains of S. aureus had an increase in phagocyti... (Complete abstract click electronic access below) / Doutor

Sepsis is life-threatening organ dysfunction produced by a dysregulated host response to infection in which neutrophils release neutrophil extracellular traps (NETs). NETs consist of DNA, histones, and antimicrobial peptides which kill pathogens. However, DNA and histones also exert damage by activating the intrinsic pathway of coagulation and inducing endothelial cell death, respectively. AADH, a 15kDa non-anticoagulant unfractionated heparin (UFH), prevents histone-mediated cytotoxicity in vitro and improves survival in septic mice. We explored the effectiveness of Vasoflux, a 5.5kDa low-molecular-weight-heparin as an anti-sepsis treatment as compared to enoxaparin and UFH. Vasoflux has reduced anticoagulant functions and hence reduces the risk of bleeding as compared to enoxaparin or UFH. We showed that UFH, enoxaparin, or Vasoflux at concentrations of up to 13.3uM, 40uM, or 40uM, neutralize histone-mediated cytotoxicity. These results suggest that these glycosaminoglycans (GAGs) are able to neutralize histone-mediated cytotoxicity independent of the AT-binding pentasaccharide. To quantitate the binding affinity between GAGs and histones, surface plasmon resonance was conducted. UFH is a more potent inhibitor of histone-mediated cytotoxicity compared to Vasoflux as UFH has a 10-fold greater binding affinity to histones compared to Vasoflux. To translate our in vitro findings to in vivo, Vasoflux, enoxaparin, and UFH were administered in a murine model of sepsis. Vasoflux at 8mg/kg - 50mg/kg reduced survival and exhibited damage in the lung, liver, and kidney in septic mice compared to 10 mg/kg of UFH or 8mg/kg of enoxaparin. This may be due to Vasoflux and UFH disrupting the DNA-histone complex, thereby releasing free procoagulant DNA. This is evident by our gel electrophoresis experiments, where addition of 1uM Vasoflux or 3.3uM UFH to DNA-histone complexes lead to histone dissociation from DNA. UFH bound to histones may be able to inhibit DNA-mediated thrombin generation, as it retains its anticoagulant properties, demonstrated by UFH-histone complexes attenuating DNA and TF-mediated thrombin generation. In contrast, Vasoflux may not neutralize the procoagulant DNA leading to a hypercoagulable state in the mice. Our study may have important clinical implications as there is an ongoing trial, HALO, which will administer intravenous UFH to patients suspected to have septic shock to reduce mortality. Based on our results, future clinical trials should consider the antithrombin-dependent anticoagulant activity of UFH being used as a sepsis treatment. / Thesis / Master of Science (MSc) / Sepsis is a life threatening condition caused by the body’s extreme response to microbial infection of the blood, whereby neutrophils release traps composed of cell-free DNA (cfDNA), histones, and antimicrobial proteins. In addition to fighting off infections, these traps also exert harmful effects like triggering clotting and killing host cells. Currently, no specific anti-septic drugs exist. Studies have shown that DNase1 (a recombinant protein that digests double stranded cfDNA) or a modified form of heparin (neutralizes histones) improves survival in septic mice. Our goal was to explore the protective effects of Vasoflux, (a non-anticoagulant heparin) and DNase1 in a mouse model of sepsis. We hypothesize that the combined therapy of DNase1 and Vasoflux will improve survival. We found that Vasoflux has minimal blood thinning activity and can prevent histones from killing cells. However, Vasoflux administered into septic mice worsened organ damage and decreased survival. We hypothesize that this damage may be due to Vasoflux’s ability to displace histones from histone-DNA complexes, thereby releasing free DNA, which promotes excessive blood clotting in sepsis.

Les polynucléaires neutrophiles (PN) sont des cellules essentielles au cours de la réponse immunitaire innée ; recrutés rapidement au site inflammatoire où ils participent à la phase aigüe, ils vont aussi contribuer à la résolution de l'inflammation. Ils peuvent en effet moduler la réponse adaptative par interaction avec les lymphocytes (Ly) ou les cellules dendritiques (DC) via des médiateurs solubles ou des interactions cellulaires directes. Les Neutrophil Extracellular Traps (NETs) libérés par les PN activés pourraient jouer un rôle important dans ce contexte. Les NETs sont des filaments de chromatine décondensée associés à des protéines issues principalement des granulations et du cytoplasme. Ils sont essentiels dans la réponse anti-infectieuse mais semblent également impliqués dans la physiopathologie de certaines maladies auto-immunes et inflammatoires. L'objectif de ce travail a été d'évaluer les effets des NETs sur la maturation des DC dans un contexte inflammatoire au cours duquel les PN et les DC peuvent co-exister, assurant ainsi un pont entre immunité innée et immunité adaptative. La première partie de ce travail a consisté à développer un modèle de production, isolement et caractérisation des NETs issus de PN sanguins humains. L'ionophore de calcium A23187 a été choisi pour induire les NETs et l'enzyme de restriction AluI a permis la récupération de fragments de NETs de taille hétérogène. Certains des composants de ces NETs sont quantifiables (ADN, élastase, histone 3 en particulier), et nous avons montré qu'ils conservaient leurs capacités bactéricides in vitro. Ces échantillons de NETs constituent donc un outil biologique standardisé, permettant d'évaluer leurs effets sur des cellules ou des tissus. Dans la deuxième partie de ce travail, nous avons mis en évidence que ces NETs purifiés régulaient négativement la maturation de moDC induites par le LPS (expression de HLA-DR, CD80, CD83, CD86 et production de TNFα, IL-12, IL-6, IL-23). De plus, les NETs diminuent la capacité de ces moDC à induire la prolifération des LyT, et leur polarisation est modulée en favorisant la production de cytokines de type Th2 (IL-5 et IL-13) aux dépens de cytokines de types Th1 (INFγ) et Th17 (IL-17). De manière intéressante, la capacité de migration des moDC activées par le LPS n'est pas modifiée en présence de NETs. En résumé, ces résultats suggèrent que les NETs pourraient jouer un rôle immunorégulateur sur la maturation des moDC dans des conditions inflammatoires. Les NETs produits par les PN activés pourraient ainsi participer à la régulation indispensable de la réponse inflammatoire.

Hemostasis, thrombosis, and inflammation are tightly interconnected processes which may give rise to thrombo-inflammation, involved in infectious and non-infectious acute and chronic diseases, including cardiovascular diseases (CVD). Traditionally, due to its hemostatic role, blood coagulation is isolated from the inflammation, and its critical contribution in the progressing CVD is underrated, until the full occlusion of a critical vessel occurs. Underlying vascular injury exposes extracellular matrix to deposit platelets and inflammatory cells. Platelets being key effector cells, bridge all the three key processes (hemostasis, thrombosis, and inflammation) associated with thrombo-inflammation. Under physiological conditions, platelets remain in an inert state despite the proximity to the endothelium and other cells which are decorated with glycosaminoglycan (GAG)-rich glycocalyx (GAGs). A pathological insult to the endothelium results in an imbalanced blood coagulation system hallmarked by increased thrombin generation due to losses of anticoagulant and cytoprotective mechanisms, i.e., the endothelial GAGs enhancing antithrombin, tissue factor pathwayinhibitor (TFPI) and thrombomodulin-protein C system. Moreover, the loss of GAGs promotes the release of mediators, such as von Willebrand factor (VWF), platelet factor 4 (PF4), and P-selectin, both locally on vascular surfaces and to circulation, further enhancing the adhesion of platelets to the affected sites. Platelet-neutrophil interaction and formation of neutrophil extracellular traps foster thrombo-inflammatory mechanisms exacerbating the cardiovascular disease course. Therefore, therapies which not only target the clotting mechanisms but simultaneously or independently convey potent cytoprotective effects hemming the inflammatory mechanisms are expected to provide clinical benefits. In this regard, we review the cytoprotective protease activated protein C (aPC) and its strong anti-inflammatory effects thereby preventing the ensuing thrombotic complications in CVD. Furthermore, restoring GAGlike vasculo-protection, such as providing heparin-proteoglycan mimetics to improve regulation of platelet and coagulation activity and to suppress of endothelial perturbance and leukocyte-derived pro-inflammatory cytokines, may provide a path to alleviate thrombo-inflammatory disorders in the future. The vascular tissue-modeled heparin proteoglycan mimic, antiplatelet and anticoagulant compound (APAC), dual antiplatelet and anticoagulant, is an injury-targeting and locally acting arterial antithrombotic which downplays collagen- and thrombin-induced and complement-induced activation and protects from organ injury.

Diabetes mellitus is a metabolic disease largely due to lifestyle and nutritional imbalance, resulting in insulin resistance, hyperglycemia and vascular complications. Diabetic kidney disease (DKD) is a major cause of end-stage renal failure contributing to morbidity and mortality worldwide. Therapeutic options to prevent or reverse DKD progression are limited. Endothelial and glomerular filtration barrier (GFB) dysfunction and sterile inflammation are associated with DKD. Neutrophil extracellular traps (NETs), originally identified as an innate immune mechanism to combat infection, have been implicated in sterile inflammatory responses in non-communicable diseases. However, the contribution of NETs in DKD remains unknown. Here, we show that biomarkers of NETs are increased in diabetic mice and diabetic patients and that these changes correlate with DKD severity. Mechanistically, NETs promote NLRP3 inflammasome activation and glomerular endothelial dysfunction under high glucose stress in vitro and in vivo. Inhibition of NETs (PAD4 inhibitor) ameliorate endothelial dysfunction and renal injury in DKD. Taken together, NET-induced sterile inflammation promotes diabetes-associated endothelial dysfunction, identifying a new pathomechanism contributing to DKD. Inhibition of NETs may be a promising therapeutic strategy in DKD.