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To induce an appropriate immune response, pathogen- or damage-associated molecular
patterns (PAMPs and DAMPs) are sensed by innate immune cells. One of the first
mechanisms after recognition is the assembly of an inflammasome leading to the cleavage of
pro-IL-1β and pro-IL-18 into the active cytokines IL-1β and IL-18, respectively. Hyperinflammatory
conditions and excessive activation of the inflammasome, however, are drivers
of inflammatory and autoimmune diseases. The NLR family pyrin domain-containing 3
(NLRP3) inflammasome is suggested to be involved in the development of gout,
artherosclerosis, type II diabetes, cryopyrin-associated periodic syndrome (CAPS), various
types of cancer, and inflammatory bowel disease (IBD). To tackle these diseases, drugs
interfering with IL-1 signaling are of great value. Anakinra, an IL-1 receptor antagonist,
inhibits binding of both IL-1α and IL-1β. Rilonacept consists of the Fc portion of human IgG1
with two IL-1 receptors grafted onto it. This allows neutralization of IL-1α and IL-1β. The
monoclonal IgG1 antibody Canakinumab specifically blocks IL-1β. Although well tolerated,
patients undergoing a therapy with these drugs are at a higher risk of developing bacterial
respiratory tract infections.
The human upper respiratory tract (URT) is commonly colonized asymptomatically by
bacteria like Staphylococcus aureus and Streptococcus pneumoniae. Only a limited number of
studies have so far addressed the role of the NLRP3 inflammasome during pneumococcal
infections. Imbalances in the immune system enhance the dissemination of these bacteria into
the lungs thereby causing life-threatening infections. Asides from immune-compromising
therapies such as IL-1 inhibition, viral infections are common factors leading to the
development of a secondary, bacterial infection. Influenza A virus infection of C57BL/6J
mice was characterized by a bi-phasic disease progression with a complete remission around
day 16 irrespectively of a previous Streptococcus pneumoniae 19F colonization. At this time
point, low amounts of bacteria were recovered from the lungs of 50% of co-infected mice.
This co-infection model was used for in-depth analyses of the innate immune responses.
Bioactive lipids, such as eicosanoids, are host-derived metabolites, which can heavily impact
the cellular immune response and skew it towards a pro- or anti-inflammatory phenotype.
Eicosanoid analyses revealed increased levels of different hydroxydocosahexaenoic acids
(HDHAs) and hydroxyeicosatetraenoic acids (HETEs) in the lung after single viral infection.
Staphylococcal pneumonia mainly resulted in high expression of 20-HETE. During a coinfection
with S. pneumoniae and influenza A virus (IAV), reduction of HETEs and HDHAs
was measured. Furthermore, ceramide-1-phosphate (C1P) was elevated in the lung, whereas
sphingosine-1-phosphate (S1P) was reduced. The tight control of an inflammatory response
by bioactive lipid mediators might be protective during later stages of an infection.
Cytokine analyses revealed high expression of inflammatory cytokines in lungs and plasma of
single bacteria- and virus infected as well as co-infected mice at early stages of an infection.
In all three infections monocyte chemoattractant protein-1 (MCP-1) dependent monocyte
recruitment was noted. The elevated expression of CC chemokine receptor 2 (CCR2) and
major histocompatibility complex II (MHCII) in the majority of analysed myeloid cells
indicated an activation of these cells. In single virus and co-infections, we further observed a
mainly neutrophil-independent immune response. During pneumococcal pneumonia, an influx
of neutrophils was observed.
Neutrophils, as first recruited responders during an inflammation, can release their granule
contents after activation in order to eliminate extracellular pathogens. However, excessive
neutrophil activation correlates with tissue damage and detrimental outcome of an infection.
As a result of excessive cell death, large amounts of ATP are released into the extracellular
space. Pneumolysin (Ply), a pore-forming toxin and major virulence factor of S. pneumoniae,
is a potent activator of human neutrophils. Stimulation of neutrophils with sublytic
concentrations of Ply readily resulted in the release of resistin and other granule proteins into
the supernatant. Microscale Thermophoresis (MST) analyses revealed that ATP neutralizes
Ply by binding it in the extracellular space. Co-stimulation of neutrophils with both lytic and
sublytic amounts of Ply and physiologically relevant concentrations of ATP resulted in
diminished neutrophil activation. We speculate that during hyper-inflammation (caused by
excessive neutrophil recruitment and activation), tissue injury results in the release of large
amounts of extracellular ATP which, in turn, will bind to Ply. Ultimately, binding of ATP to
Ply inhibits further neutrophil activation and might mitigate immunopathology.
Group A streptococcus (GAS) and Streptococcus pneumoniae are both Gram-positive bacteria that asymptomatically colonise various human body parts. Both microbes cause diseases ranging from mild to severe invasive infections. The later are associated with high mortality. GAS is the major microbial aetiology of type II necrotising skin and soft tissue infections (NSTIs). Type II NSTIs typically affect the lower and upper limbs of healthy young adults and often require debridement as a surgical intervention to prevent the spread of infection. S. pneumoniae is the major cause of respiratory tract infections including community-acquired pneumonia in young children and the elderly. Although most respiratory tract infections are successfully treated with antibiotics, emerging antibiotic resistance is a major cause of concern. Secreted virulence factors of Gram-positive bacteria play a major role in the successful invasion of host tissues causing different diseases. Additionally, they facilitate the spread of infection, contribute to tissue pathology, and potentially act as immune evasion mechanisms. This thesis summarises the consequences of streptococcal pyrogenic exotoxin B (SpeB), a potent cysteine protease secreted by GAS and pneumococci-derived hydrogen peroxide (H2O2) on host responses.
GAS have developed genetic or phenotypic ways of adapting to the immune response to escape immune clearance. Analysis of GAS clones recovered from NSTI patient biopsies exhibit a mixed SpeB phenotype, with most clones being SpeB negative. SpeB negative clones have been associated with hyper-virulence. In Paper II, we showed that SpeB negative GAS clones recovered from tissue exhibit reversible impaired SpeB secretion due to environmental factors. In addition, mutations in covS and ropB, the major transcriptional regulators of SpeB expression, were responsible for the irreversible loss of SpeB expression. Immunohistochemistry analysis demonstrated that neutrophil degranulation, necrosis and excessive inflammation observed in NSTIs patient biopsies correlated with bacterial load and SpeB negativity of clones. Proteomic data analysis showed that SpeB negative GAS recovered from neutrophil infection harboured the protease intracellularly suggesting that the bacteria expressed but did not secrete SpeB. We have also shown that neutrophil-derived reactive oxygen species, H2O2 and hypochlorous acid, drive the SpeB negative phenotype. The SpeB negative clones survived neutrophil-mediated antimicrobial killing and induced excessive degranulation when compared with SpeB positive clones. These results provide new insights into GAS fitness induced by host factors in tissue and may be useful for the development of new treatment strategies in NSTIs.
Pneumococci produce H2O2 as a by-product of carbohydrate metabolism in a reaction catalysed by pyruvate oxidase SpxB. However, very little is known about the effects of pneumococcal H2O2 as a virulence factor. Our study aimed to investigate the role of H2O2 in initiating epithelial cell death, focusing on apoptosis and pyroptosis. In Paper III, we showed that pneumococci-derived H2O2 caused epithelial cell cytotoxicity by priming and activating the NLRP3 inflammasome resulting in subsequent IL-1β production and release. Additionally, H2O2 caused apoptotic and pyroptotic cell death as evidenced by activation of caspase-3/7 and caspase-1, respectively. However, the release of IL-1β was dependent on apoptosis and not pyroptosis since inactive gasdermin D was detected post-infection. These observations were not detected in the absence of H2O2. Overall, we showed the damaging effects of pneumococci-derived H2O2 on human bronchial epithelial cells.
The human innate response plays a pivotal role in detection of pathogen- or damage-associated molecular patterns (PAMPs and DAMPs) and contributes to a crucial inflammatory response. PAMPs or DAMPs are recognized by the host immune system via pattern recognition receptors (PRRs). NLR family pyrin domain-containing 3 (NLRP3) inflammasome is one of these PRRs. NLRP3 is a cytoplasmic immune sensor that upon activation produce pro-inflammatory cytokines such as IL-1β and IL-18. These cytokines induce a diverse range of protective host pathways aiming to eradicate the pathogen. However, excessive or chronic inflammasome activation are implicated in the pathogenesis of several autoimmune and auto-inflammatory disorders. Pharmacologic inhibitors of IL-1 are commonly used to combat these disorders. In paper I, we explore the currently available IL-1β inhibiting therapies and how patients undergoing these treatments are at a disproportionate risk to experience invasive bacterial infections. We also summarize the limited knowledge on the role of NLRP3 inflammasome in pneumococcal pathogenesis.
Hydrogen peroxide (H2O2) is a physiological metabolite and an important virulence determinant produced by pneumococci. It is highly cytotoxic to host cells. However, not much is known about its impact on host cell death pathways such as NLRP3 inflammasome mediated pyroptosis. In Paper II, we examined the effect of pneumococci-derived H2O2 on epithelial cells by analyzing the interplay between two key cell death pathways, namely apoptosis and pyroptosis. We show that H2O2 can prime as well as activate the NLRP3 inflammasome. Furthermore, we demonstrate that pneumococcal H2O2 initiates cell death via the activation of both apoptotic as well as pyroptotic pathways, mediated by the activation of caspase-3/7 and caspase-1, respectively. H2O2 mediated inflammasome activation results in caspase-1 dependent IL 1β production. However, we show that the final IL-1β release is independent of gasdermin-D (GSDMD) and mainly dependent on the apoptotic cell lysis.
In paper III, we focused on understanding the host metabolic responses to infections with pathogens which cause respiratory diseases. We performed metabolome profiling of in vitro single bacterial and viral as well as co-infections of bronchial epithelial cells with Influenza A virus (IAV), Streptococcus pneumoniae, and Staphylococcus aureus. We show that IAV and S. aureus use the host resources for survival and multiplication and have minimal effects on the host metabolome. In contrast, pneumococci significantly alter various host metabolome pathways, including glycolysis, tricarboxylic acid (TCA) cycle and amino-acid metabolism. A hallmark of pneumococcal infections was the intracellular citrate accumulation, which was directly attributed to the action of pneumococci-derived H2O2.
Host cell death during an infection results in the release of pro-inflammatory cytokines and danger signals such as ATP. Released ATP can induce neutrophil chemotaxis mediated via purinergic signaling. Neutrophils are typically the first leukocytes to be recruited to the site of infection and are key players in bacterial clearance. However, excessive neutrophil activation is associated with further tissue injury. In paper IV, we investigated the role of ATP in neutrophil response to pneumococcal infections. We show that pneumolysin (Ply), a highly effective pore-forming toxin produced by pneumococci, is a potent activator of neutrophils. Microscale Thermophoresis analysis revealed that Ply and ATP bind to each other. Subsequently, ATP binding neutralizes Ply-mediated neutrophil degranulation, suggesting that Ply-ATP interactions are potentially beneficial during the course of the infection as this could limit the lung injury resulting from excessive Ply-mediated neutrophil activation.
During infections, innate immune cells are crucial for initiating a pro-inflammatory immune response and clearing the invading pathogen. Delay in pathogen clearance or initiation of an immune response due to impaired functionality of immune cells can result in devastating consequences. The cellular compartment of the innate immune system comprises an array of specialized cell types: Macrophages are tissue-resident professional phagocytes that clear cellular debris, pathogens, and foreign objects. Dendritic cells (DCs) are immune sentinels specialized in antigen uptake and subsequent T cell priming. They are primary sources of cytokines in response to infection. Neutrophils are efficient effector cells that respond rapidly to infection and clear bacteria by different mechanisms. If effector mechanisms of these cells are affected by either bacterial or other factors, infections might not be resolved and can spread throughout the host. Cobalt-chromium-molybdenum biomaterial is widely used in arthroplasty. Implant-derived wear particles and ions lead to macrophage-driven adverse local tissue reactions: Such reactions have been linked to an increased risk of periprosthetic joint infection after revision arthroplasty. While metal-induced cytotoxicity is well characterized in human macrophages, direct effects on their functionality remain elusive. In Paper I, we show that local peri-implant tissue is exposed to Co and Cr in situ. Influx of macrophages is also evident. Exposure of isolated human monocytes/macrophages to Cr3+ in vitro had only minor effects. However, exposure of monocytes/macrophages to pathologic concentrations of Co2+ significantly impaired both phenotype and functionality. High concentrations of Co2+ induced loss of surface markers, including CD14 and CD16. Both Co2+ and Cr3+ impaired macrophage responses to Staphylococcus aureus infection. Co2+ -exposed macrophages, in particular, showed decreased phagocytic activity. These findings demonstrate the immunosuppressive effects of locally elevated metal ions on the innate immune response. Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild to severe necrotizing soft tissue infections (NSTIs). In the host environment hypervirulent GAS variants carrying mutations within the genes encoding for control of virulence (Cov)R/S two component system are enriched. This adaptation is associated with loss of SpeB secretion. In Paper II, we show that in vitro infections with hyper-virulent GAS variants harboring dysfunctional CovR/S suppress secretion of IL-8 and IL-18 by human monocytic cells. This phenotype was mediated by a caspase-8 dependent mechanism. Knockout of streptococcal SLO in a GAS strain carrying functional CovR/S even increased secretion of IL1β and IL-18 by moDCs. Of 67 fully sequenced GAS NSTI isolates, 28 contained covS or covR mutations that rendered the TCS dysfunctional. However, no differences in systemic IL-8 and IL-18 were detected in these patients. GAS isolates recovered from patients often display a mixed phenotype, consisting of SpeB positive (SpeB+ ) and SpeB negative (SpeB- ) clones. Irreversible loss of SpeB expression is often caused by loss of function mutations in regulatory components (CovR/S, RopB). Loss of SpeB is often associated with hyper-virulence. In Paper III, we show that the host environment induces transiently abrogated secretion of SpeB by GAS. Tissue inflammation, neutrophil influx, and degranulation correlated with increased frequencies of SpeB- GAS clones. Isolates recovered from tissue expressed but did not secrete SpeB, which was reversible. Neutrophilderived ROS were identified as the main factor responsible for abrogated SpeB secretion. Hyper-virulent SpeB- clones also exhibit better survival within and induce excessive degranulation of neutrophils.