Combining protein structure prediction with experiments and functional information

Proteins are key players in all cells of living organisms. In particular, knowledge of the spatial protein structure may give fundamental insights into protein function and disease processes. For many years, the successful prediction of the structural and functional properties of proteins has been a major research field in bioinformatics. This field is also addressed in this work, which comprises an applied biomedical and a methodological part. Comprehensive application studies of bioinformatics approaches were performed, which primarily targeted autoinflammatory and neurodegenerative diseases. A variety of computational tools was used to analyze medically relevant proteins and to evaluate experimental data. Many bioinformatics methods were applied to predict the molecular structure and function of proteins. The results provided a rationale for the design, prioritization, and interpretation of experiments performed by cooperation partners. Some of the generated biological hypotheses were tested and confirmed by experiments. In addition, the application studies revealed limitations of current bioinformatics techniques, which led to suggestions for novel approaches. Three new computational methods were developed to support the prediction of the secondary and tertiary structure of proteins and the investigation of their interaction networks. First, consensus formation between three different methods for secondary structure prediction was shown to considerably improve the prediction quality and reliability. Second, in order to utilize experimental measurements in tertiary structure prediction, scoring functions were implemented that incorporate distance constraints into the alignment evaluation, thus increasing the fold recognition rate. Third, an automatic procedure for decomposing protein networks into interacting domains was designed to obtain a more detailed molecular view of protein-protein interactions, facilitating further functional and structural analyses.Proteinen kommt in allen Zellen lebender Organismen eine Schlüsselrolle zu. Insbesondere die Kenntnis der Raumstruktur von Proteinen kann fundamentale Einsichten in ihre Funktion und in Krankheitsprozesse liefern. Seit vielen Jahren ist die erfolgreiche Vorhersage struktureller und funktioneller Eigenschaften von Proteinen ein wichtiges Forschungsgebiet in der Bioinformatik. Dieses Gebiet ist auch Gegenstand der vorliegenden Arbeit, welche einen angewandten biomedizinischen und einen methodischen Teil umfasst. Es wurden umfangreiche Applikationsstudien von bioinformatischen Verfahren durchgeführt, die sich vornehmlich mit autoinflammatorischen und neurodegenerativen Erkrankungen befassten. Verschiedene Computerwerkzeuge wurden verwendet, um medizinisch relevante Proteine zu analysieren und experimentelle Daten auszuwerten. Es kamen viele Bioinformatikmethoden zur Anwendung, um die molekulare Struktur und Funktion von Proteinen vorherzusagen. Die Ergebnisse dienten als Grundlage für die Planung, Priorisierung und Interpretation von Experimenten, die von Kooperationspartnern durchgeführt wurden. Einige der generierten biologischen Hypothesen wurden durch Experimente überprüft und bestätigt. Zusätzlich deckten die Applikationsstudien Grenzen von Bioinformatikmethoden auf, was zu Vorschlägen für neuartige Verfahren führte. So wurden drei neue rechnerbasierte Methoden entwickelt, um die Vorhersage der Sekundär- und Tertiärstruktur von Proteinen sowie die Untersuchung ihrer Interaktionsnetzwerke zu unterstützen. Erstens wurde gezeigt, dass die Bildung eines Konsensus zwischen drei verschiedenen Methoden der Sekundärstrukturvorhersage die Vorhersagequalität und -verlässlichkeit erheblich verbessert. Zweitens wurden zur Nutzung experimenteller Messungen in der Tertiärstrukturvorhersage Bewertungsfunktionen implementiert, die Distanzbeschränkungen in die Alignmentevaluation einbinden, um die Faltungserkennungsrate zu erhöhen. Drittens wurde eine automatische Prozedur zur Dekomposition von Proteinnetzwerken in interagierende Domänen entworfen, um eine detailliertere molekulare Sicht von Interaktionen zwischen Proteinen zu erhalten. Hierdurch werden weitere Analysen zu Funktion und Struktur erleichtert

Alternative splicing as a regulatory mechanism of the NLRP3 inflammasome

The innate immune system relies on germ-line encoded pattern recognition receptors and is critically involved in the early sensing of pathogens and disturbances of cell homeostasis. Upon activation by pathogenic or sterile danger signals, several cytosolic receptors of the innate immune system (e.g. NLRP3) can recruit multi- protein signaling platforms, so called inflammasomes. Inflammasome formation leads to the activation of caspase-1, causing pyroptosis as well as maturation and release of IL-1β and IL-18. NLRP3 is critically involved in several cardiovascular, neurodegenerative and inflammatory diseases. This study aimed to decipher whether alternative splicing (AS) might act as a regulator of NLRP3 inflammasome activation, similarly to what is described for other vertebrate immune receptors and plant R-proteins. I could show that the LRR of NLRP3 is encoded by multiple repetitive and highly conserved exons, a feature which is shared by other LRR encoding genes. This strict exonic modularity of LRR domains of several human gene families serves as a prerequisite for non-destructive AS. Indeed, I could show AS of the LRR of several NOD-like receptors, most prominently in NLRP3. Human NLRP3, but not mouse NLRP3, could be detected as two major isoforms: The fully active NLRP3 full-length variant and a variant lacking exon 5. By use of several different model systems and readouts, NLRP3 ∆ exon 5 could be shown to be inert to common NLRP3 activators. Furthermore, I could show that alternative splicing is stochastically regulated on a single-cell level. Mechanistically, I could provide evidence that NLRP3 ∆ exon 5 is inactive due to the absence of a necessary interaction surface for NEK7 binding, required for NLRP3 activation. Surprisingly, a prolonged priming for over 10 h rendered NLRP3 ∆ exon 5 activatable. In combination with the stochastic isoform expression, this allows for a backup pool of cells, which do not become pyroptotic in the first round of NLRP3 inflammasome activation, but rather enable a sustained inflammatory response. The data presented here provide evidence for a not yet described regulatory role of AS in NLRP3 inflammasome activation through differential utilization of highly conserved LRR modules. Moreover, the species differences described here might hold therapeutic potential that could not have been revealed in mouse models

Biochemical and Functional Characterization of the Mitochondrial Immune Signaling Protein Complex

The mitochondrion has emerged as a crucial organelle where key anti-viral responses including apoptosis, type 1 interferon (IFN-I) production and autophagy are regulated. A prime example is the intersection of the mitochondrial protein MAVS with the RLR (RIG-I-like receptors) to induce IFN-I. Here we show that MAVS is a pro-apoptotic protein independent of its function in initiating IFN-I production. Viral proteins such as NS3/4A encoded by HCV and NSP15 encoded by SARS-CoV inhibit this response. MAVS-mediated IFN-I is tightly regulated by an NLR (nucleotide-binding domain, leucine-rich repeats containing) protein NLRX1. More in-depth analysis utilizing cells from gene-deletion mice indicates that NLRX1 not only attenuates IFN-I production, it additionally promotes autophagy during viral infection. This dual regulatory function of NLRX1 parallels the previously described functions of Atg5-Atg12, although NLRX1 does not associate with Atg5-Atg12. High throughput quantitative mass spectrometry and biochemical analysis revealed a novel NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM/P43/EF-Tu/COXPD4/ EF-TuMT), which does interact with Atg5-Atg12. Similar to NLRX1, TUFM potently inhibits RLR signaling and promotes autophagy during a viral infection. This thesis demonstrates the dual roles of MAVS in controlling both IFN-I and apoptosis, and establishes the first link between an NLR protein and a viral-induced autophagic machinery via TUFM

The innate regulatory protein NLRP12 maintains commensal bacterial symbiosis, and mitigates intestinal inflammation and obesity

The nucleotide-binding domain, leucine-rich repeat containing proteins, also known as NOD-like receptors (NLRs), are pattern recognition receptors that play important roles in innate immunity (Aganna et al., 2002; Bertin et al., 1999; Harton et al., 2002; Hoffman et al., 2001; Inohara et al., 1999; Manji et al., 2002; Ting and Davis, 2005). Mounting evidence has expanded the known functions of innate immunity from the frontlines confronting infection to autoimmune and metabolic diseases. Therefore, studies on the function of NLRs have been extended to a much wider horizon, including transcriptional regulation (Bruchard et al., 2015), endoplasmic reticulum (ER) stress (Soares et al., 2014; Stokman et al., 2017), DNA damage repair (Hu et al., 2016; Wilson et al., 2015), protein ubiquitination (Abe et al., 2017; Allen et al., 2012b), and neurological disease (Lukens et al., 2015). Members of the NLR family can be categorized into two groups, the inflammasome-forming and non-inflammasome-forming NLRs. Inflammasomes are cytoplasmic multi-protein complexes that result in the catalytic cleavage of procaspase-1, generating caspase 1 necessary for the production of mature IL-1β and IL-18. These have been extensively studied due to their pro-inflammatory and anti-pathogen functions. However, mutations of non-inflammasome-forming NLRs, such as NLRP12 and NLRP14, have been reported to cause auto-inflammatory diseases (Abe et al., 2017; Allen, 2014; Borghini et al., 2011; Karki et al., 2016), which raises great interest in studying these non-inflammasome-forming NLRs (Allen, 2014; Ting et al., 2010). NLRP12, a negative regulator of innate immunity, suppresses colon inflammation and inflammation-driven colorectal cancer (Allen et al., 2012b; Zaki et al., 2011). Here, we identified that NLRP12 expression in mucosal tissue negatively correlates with colitis severity by performing gene expression analyses from multiple published colitis datasets. We also showed that exposure to commensal bacteria was required to differentiate colitis severity between WT and Nlrp12–/– mice, and the existence of commensal bacteria caused more basal inflammation attributed to the NLRP12-deficient hematopoietic cell components (such as CD11c+ macrophages and DCs). Meanwhile, this elevated basal inflammation promoted a dysbiotic microbiota manifested by the loss of diversity and protective stains, but increased abundance of colitogenic strains in the Nlrp12–/– mice. In this fashion, both the genetic defects of NLRP12 and the consequent dysbiotic microbiota worked together to achieve cumulative pathology. Finally, we found reconstitution of the absent beneficial bacteria in the Nlrp12–/– mice by cohousing with WT mice or inoculation of Lachnospiraceae can attenuate colitis severity. In the second part, we extended our knowledge of NLRP12 to diet induced obesity. We found NLRP12 expression was lower in patients with higher body mass index (BMI), and Nlrp12–/– mice were more susceptible to high-fat-diet (HFD) induced obesity and were insulin tolerant. These observations coincided with excessive systemic inflammation fueled by HFD-feeding, the NLRP12 mutation, and a skewed gut microbiota composition. We also identified that the gut microbiota was required to accelerate the process of weight gain in the Nlrp12–/– mice, and restoration of the decreased beneficial strains by cohousing with WT mice or Lachnospiraceae inoculation can attenuate the progression of obesity and improve insulin sensitivity. We found Lachnospiraceae, which were reduced by HFD-feeding and Nlrp12-deficiency, might produce anti-inflammatory short-chain-fatty-acid (SCFA) to promote IL10 secretion. Accordingly, Lachnospiraceae treatment restricts obesity and improves insulin sensitivity by suppressing the systemic inflammation caused by HFD and Nlrp12-deficiency.Doctor of Philosoph

Inflammasome biology, molecular pathology and therapeutic implications

Inflammasomes are intracellular multiprotein signaling complexes, mainly present in myeloid cells. They commonly assemble around a cytoplasmic receptor of the nucleotide-binding leucine-rich repeat containing receptor (NLR) family, although other cytoplasmic receptors like pyrin have been shown to forminflammasomes. The nucleation of the multiprotein scaffolding platform occurs upon detection of a microbial, a danger or a homeostasis pattern by the receptor that will, most commonly, associate with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) through homotypic domain interactions resulting in recruitment of procaspase-1. This will lead to the autoproteolytic activation of caspase-1, which regulates the secretion of proinflammatory IL1β and IL18 cytokines and pyroptosis, a caspase-1-mediated form of cell death. Pyroptosis occurs through cleavage of Gasdermin D, a membrane pore forming protein. Recently, non-canonical inflammasomes have been described, which directly sense intracellular pathogens through caspase-4 and -5 in humans, leading to pyroptosis. Inflammasomes are important in host defense; however, a deregulated activity is associated with a number of inflammatory, immune and metabolic disorders. Furthermore, mutations in inflammasome receptor coding genes are causal for an increasing number of rare autoinflammatory diseases. Biotherapies targeting the products of inflammasome activation aswell as molecules that directly or indirectly inhibit inflammasome nucleation and activation are promising therapeutic areas. This review discusses recent advances in inflammasome biology, the molecular pathology of several inflammasomes, and current therapeutic approaches in autoinflammatory diseases and in selected common multifactorial inflammasome-mediated disorders

Subcellular distribution of apoptosis associated speck-like protein mediates inflammasome assembly: A novel mechanism in the regulation of interleukin-1beta release

Inflammation is an essential component of the innate immune defense against pathogens. The process of inflammation is mediated by the rapid release of intercellular mediators, which function in the activation and recruitment of cellular components of both the innate and adaptive immune system. However, dysregulation of this process, resulting in sustained inflammation is the underlying cause of a number of common pathological conditions. These include hereditary autoinflammatory disorders, gout, arthritis, asthma, and cancer. Furthermore, IL-1beta is a key component of the tumor microenvironment, which serves to recruit tumor-associated macrophages, promote angiogenesis, enhance NF-kB activation, and suppress anti-tumor immunity. The studies presented here explore novel regulatory mechanisms of IL-1beta maturation through the modulation of the inflammasome adaptor protein ASC. ASC is an intriguing protein, which participates in both inflammatory and apoptotic pathways and is frequently silenced by aberrant methylation in a large number of cancers. It is a small protein, consisting of only two protein-interaction domains, an N-terminal PYD, and a C-terminal CARD. Numerous studies have found that it is required for inflammasome assembly following activation of a number of diverse cytoplasmic NLR receptors. Therefore, regulation of this protein has broad implications for IL-1beta maturation.;In the first study, we examined the endogenous localization of ASC. Several independent research groups had found that ectopic expression of ASC results in several distinct localization patterns including nuclear, cytoplasmic, and the formation of a characteristic perinuclear aggregate termed a \u27speck\u27. However, the subcellular distribution of endogenous ASC had never been evaluated. Therefore, we examined the subcellular localization pattern of ASC in several monocytic cell lines as well as in primary monocytes and macrophages. We discovered that in resting monocytes, ASC is localized diffusely throughout the nucleus. However, upon inflammatory stimulation of the cells, we determined that ASC was rapidly redistributed to the cytoplasm where it subsequently formed a perinuclear aggregate, reminiscent of the \u27specks\u27 observed upon overexpression of this protein. Furthermore, we determined that other core inflammasome proteins NLRP3 and caspase-1 co-localized with ASC in these aggregates, suggesting that they represent inflammasomes. Additionally, we found that nuclear export of ASC is necessary for proper inflammasome assembly and processing of IL-1beta.;In Study 2, we identified Mycoplasma sp. as a novel activator of the inflammasome. Infection with Mycoplasma sp. cause a number of organ-specific diseases in humans, and they have also been linked to a number of proinflammatory disorders. Therefore, we hypothesized that IL-beta played a role in the innate immune defense against these pathogens. We discovered that incubation of monocytes and macrophages with either live or heat-killed Mycoplasma sp. stimulated the secretion of mature IL-1beta at levels comparable to those observed with known inflammasome activators. Furthermore, we also determined that Mycoplasma infection induced the nuclear to cytoplasmic translocation of ASC, as well as the formation of perinuclear aggregates, just as we observed with known inflammasome activators in Study 1. Finally, we showed that Mycoplasma -induced release of IL-1beta was dependent upon ASC, further indicating the role of the inflammasome in this process. Based upon this data, we hypothesize that chronic infection with Mycoplasma sp. results in sustained production of IL-1beta, which leads to a prolonged inflammatory state.;In our third and final study, we identified and characterized three ASC isoforms with regards to their subcellular localization, and their ability to function as an inflammasome adaptor protein. ASCb lacks the linker domain, ASCc possesses an in-frame deletion in the PYD, which does not disrupt the linker region or the CARD, and ASCd expresses only the first 35 amino acids of the PYD, which is fused to a novel peptide. Upon ectopic expression, each isoform exhibited a different localization pattern, and none of them formed the classic perinuclear \u27speck\u27, which has become the hallmark of full-length ASCa. Upon co-expression with either NLRP3(R260W) or caspase-1, co-localization was only observed by those isoforms, which expressed a fully intact PYD or CARD, respectively. Functionally, only ASCb was able to mediate IL-1beta processing when co-expressed with NLRP3(R260W) and pro-caspase-1, although to a lesser extent, presumably because neither of its protein interaction domains were disrupted.;In summary, these studies show novel mechanisms by which IL-1beta release is regulated and induced. The discovery of novel ASC isoforms and the role its subcellular distribution plays in the regulation of IL-1beta processing provide us with new insights into how inflammasome assembly is regulated and fin-tuned. Furthermore, the nuclear to cytoplasmic redistribution of ASC provides us with a potential new therapeutic target that would prevent the release of IL-1beta without impacting the expression of ASC

Inflammasomes and the Innate Immune Response Against Yersinia Pestis: A Dissertation

Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection

Defining the Function of Pyrin, the Familial Mediterranean Fever-associated Protein, in Inflammation

Every day, the immune system makes decisions to differentiate harmful and inert stimuli, which allows protection against pathogens and prevents unnecessary or uncontrolled inflammation. Perturbations to the innate immune system can lead to autoinflammatory disorders such as Familial Mediterranean Fever (FMF), which is an inherited disorder characterized by unprovoked episodes of inflammation and fever. The genetic abnormality underlying FMF disorder is mutations in the gene MEFV (Mediterranean Fever), which encodes the protein Pyrin. Previous research indicates that Pyrin alters function of the inflammasome multiprotein complex that mediates post-translational IL-1 family cytokine processing. This work has led to disparate conclusions about the function of Pyrin. Interpretation of these results is ambiguous, in part, because mutations within the Pyrin protein are not defined as gain or loss of function. Previous research also indicates that the expression of MEFV is abundant in neutrophils, and that neutrophils from FMF patients display altered immune function and survival in comparison to neutrophils from healthy donors. However, there is no direct evidence that mutations in Pyrin affect neutrophil cell processes. We postulated that mice lacking Pyrin (Mefv-/-) would allow us to clarify the function of Pyrin in the regulation of the immune response and FMF pathophysiology. Characterization of naïve Mefv-/- mice revealed no deficits in immune cell development or distribution and no indication of unprovoked inflammation. In response to immune challenge in vitro, IL-1β cytokine levels were increased by the absence of Pyrin. However, neutrophil recruitment and survival were not affected by the loss of Pyrin. In vivo models of peritonitis demonstrated that Mefv-/- mice generate a normal hypothermic response and recruit and retain inflammatory cells normally. No difference in the physiological outcome of immune challenge was detected. These studies indicate that Pyrin negatively regulates the immune response by altering IL-1β secretion. Since IL-1β is at least in part responsible for FMF-associated inflammation, our findings support a model in which loss-of-function mutations in Pyrin can cause FMF.Doctor of Philosoph

Inflammasomi-tulehdussignalointireitin rooli valtimon rasvakovettumataudissa

Atherosclerosis is the underlying cause of myocardial infarction and stroke, the leading causes of death worldwide. It is a complex multifactorial disease closely linked with obesity, type II diabetes, and metabolic syndrome and, together, these conditions comprise the global epidemic of metabolic disorders that are becoming more and more prevalent, affecting adults and children alike. Atherosclerosis affects the large arteries that gradully loose their normal structure and function via a degenerative process involving lipid accumulation and chronic inflammation in the arterial wall. The lipid accumulation is driven by high circulating levels of cholesterol-carrying low density lipoproteins that become trapped and modified in the arterial wall. This causes an inflammatory reaction characterized by abundant immune cell infiltrates, mainly monocyte-derived macrophages. The macrophages scavenge large amounts of lipids and become activated to secrete a host of proinflammatory mediators and matrix-degrading enzymes that drive the progression of the disease. These processes result in the focal development of fatty lesions or plaques along the arteries. Over time, more complex lesions develop as a result of inflammatory and fibrotic responses, matrix remodeling, calcification, cholesterol crystallization, neovessel formation, and microhemorrhages. Ultimately, the plaques may rupture, causing thrombosis and acute complications. Although inflammation is recognized as a major driving force in atherosclerotic lesion development, the mechanisms triggering and maintaining the arterial wall inflammation remain incompletely understood. The aim of this thesis was to study the role of a key innate immune signaling pathway, the inflammasome, in atherosclerosis. The inflammasomes are large cytoplasmic signaling complexes that trigger the proteolytic maturation and secretion of two proinflammatory and proatherogenic cytokines, interleukin(IL)-1beta and -18. The inflammasome pathway can be triggered by microbial components or by sterile endogenous danger signals that elicit the activation of cytoplasmic sensor molecules from the NLR (nucleotide-binding domain and leucine-rich repeat containing) or PYHIN (pyrin and HIN domain containing) families. Despite the established roles of IL-1beta and -18 in driving atherosclerotic lesion development, the triggers of inflammasome activation in atherosclerotic plaques remained unknown. Macrophages are the prototypical inflammasome pathway-expressing cells, and thus cultured human macrophages were utilized to identify and characterize atherosclerosis-associated triggers of the inflammasome pathway. Cholesterol crystals and acidic environment were both found to trigger a strong inflammatory response via the activation of NLRP3 inflammasome and secretion of IL-1beta and IL-18. Cholesterol crystals are a hallmark of atherosclerotic lesions, yet they have been considered an inert material that merely acts as a sink for excess free cholesterol in the arterial wall. These new data suggested, however, that cholesterol crystals act as a potent sterile danger signal that may directly link pathological lipid accumulation and inflammation in the lesions. Local extracellular acidosis arises in the growing plaque due to the hindered diffusion of oxygen and the highly active glycolytic metabolism of macrophages. Acidic environment not only triggered the NLRP3 inflammasome, but even a very mild acidification from the physiological pH of 7.4 to 7.0 was sufficient to greatly amplify the IL-1beta response to other NLRP3 activators, including cholesterol crystals. Having showed that the atherosclerotic lesions harbour potent activators of the inflammasome pathway, we further analyzed the expression of this pathway in atherosclerotic human coronary specimens obtained from 10 explanted hearts. For this purpose, we utilized a quantitative PCR array targeting 88 inflammasome pathway-related molecules. Significant upregulation of 12 target genes was found in advanced coronary plaques compared to early lesions from the same coronary trees, including many of the very core components of the inflammasome pathway. Moreover, p38delta mitogen-activated protein kinase (MAPK), a poorly characterized isoform of the stress- and cytokine-activated p38 MAPK family, was consistently upregulated in advanced coronary plaques. Immunohistochemical stainings of human coronary lesions showed strong expression of NLRP3 inflammasome components and p38delta MAPK in macrophages surrounding the cholesterol crystal-rich lipid core. Furthermore, the p38delta MAPK was activated in cultured human macrophages upon NLRP3 inflammasome activation by cholesterol crystals and extracellular ATP, and required for NLRP3-mediated IL-1beta secretion. Taken together, the data presented in this thesis propose novel inflammasome-mediated mechanisms that may trigger sterile inflammation in atherosclerotic lesions and thus drive lesion progression.Tässä väitöskirjatyössä tutkittiin tulehdusreaktioiden merkitystä valtimon rasvakovettumataudin eli ateroskleroosin kehittymisessä. Ateroskleroosissa valtimoverisuonien seinämään kertyy sekä veren lipoproteiini-hiukkasten kantamaa kolesterolia että immuunipuolustuksen syöjäsoluja eli makrofageja. Makrofagit yrittävät poistaa liiallista kolesterolia, mutta juuttuvat lopulta rasvarakkuloiden täyttäminä valtimon seinämään ja laukaisevat ateroskleroosin etenemistä edistävän kroonisen tulehdusreaktion. Sen seurauksena rasva-aineiden ja tulehdussolujen muodostamat kertymät kehittyvät vähitellen monimuotoisiksi valtimoa ahtauttaviksi plakeiksi, jotka voivat revetessään aiheuttaa aivo- tai sydäninfarktin. Inflammasomi on makrofageista löydetty tulehdussignalointireitti, jonka aktivaatio laukaisee voimakkaan tulehdusreaktion käynnistämällä tulehdusvälittäjäaine interleukiini(IL)-1beta:n erityksen. Ateroskleroosin hiirimalleissa geneettinen IL-1beta-puutos vähentää huomattavasti plakkien kasvua, ja IL-1beta:n määrä lisääntyy myös ihmisen valtimon seinämässä plakkien kehittyessä. Plakkien inflammasomi-aktivaatiota ja IL-1beta-eritystä laukaisevia tekijöitä ei kuitenkaan aiemmin tunnettu ja väitöskirjatutkimuksen tavoitteena oli tunnistaa tällaisia tekijöitä. Tutkimus osoitti, että plakeissa yleisesti esiintyvät kolesterolikiteet sekä plakin kehittymiseen liittyvä kudosnesteen paikallinen happamoituminen laukaisevat makrofageissa voimakkaan inflammasomi-välitteisen tulehdusvasteen IL-1beta-erityksen kautta. Elimistön immuunipuolustus kykenee siis tunnistamaan kyseiset taudinkehitykseen liittyvät muutokset vaarasignaaleiksi ja reagoimaan niihin käynnistämällä tulehdusreaktion. Kolesterolikiteitä on vuosikymmenien ajan pidetty ateroskleroosin kehityksen kannalta merkityksettöminä sivutuotteina. Tutkimuksen tulokset haastoivat tämän käsityksen osoittamalla, että kolesterolikiteet ovat aktiivinen tekijä ateroskleroosin kehittymisessä ja voivat selittää häiriintyneen rasva-aineenvaihdunnan ja valtimon seinämän tulehduksen välistä yhteyttä. Lisäksi tutkimuksessa havaittiin, että jo hyvin lievä solunulkoinen happamoituminen voimistaa merkittävästi kolesterolikiteiden aiheuttamaa tulehdusvastetta makrofageissa, kun nämä ärsykkeet annetaan soluille samanaikaisesti. Väitöskirjatyössä inflammasomi-reitin toiminnallisuutta tutkittiin myös sydämen sepelvaltimonäytteissä laajan geeni-ilmentymisanalyysin ja vasta-ainevärjäysten avulla. Tulokset osoittivat, että kaikki inflammasomi-reitin keskeisimmät komponentit ilmentyvät sepelvaltimon seinämän makrofageissa ja useat niistä lisääntyvät merkittävästi ateroskleroottisten plakkien kehittymisen myötä. Geeni-ilmentymisanalyysissa havaittiin myös erään tulehdusta säätelevän molekyylin, p38delta MAP-kinaasin, lisääntyminen sepelvaltimoissa ateroskleroottisten plakkien kehittymisen myötä. Kyseistä molekyyliä tutkittiin tarkemmin viljellyissä ihmisen makrofageissa, mikä johti uuden tulehdusta säätelevän reitin löytymiseen. Tulokset osoittivat, että p38delta MAP-kinaasin aktivoituminen on keskeinen säätelijä kolesterolikiteiden laukaisemassa inflammasomi-aktivaatiossa. Ateroskleroottisten sairauksien hoito perustuu tällä hetkellä pääsääntöisesti veren kolesterolipitoisuutta alentavaan lääkitykseen. Valtimon seinämän tulehdusmekanismien tarkka selvittäminen luo perustan uudentyyppisen, kroonista tulehdustilaa hillitsevän lääkityksen kehittämiseen tämän kansanterveydellisesti merkittävän taudin hoitoon