Novel Approaches to Study the Haematological System During Inflammation

Physiological Science

Investigating Molecular Mechanisms behind Bacterial Chondronecrosis with Osteomyelitis (BCO) Pathogenesis in Modern Broilers

Bacterial chondronecrosis with osteomyelitis (BCO), a leading cause of lameness in broiler chickens, is characterized by infection, inflammation, and bone attrition. There are currently no effective treatments and positive diagnosis is only possible through necropsy evaluations. Lameness is also a rising animal welfare and economic concern, making prevention and detection of BCO all the more critical. These challenges are exacerbated by a lack of mechanistic understanding of BCO’s etiology. The question I asked during my dissertation was how bacteria induce bone attrition in BCO pathology. My research has shown that mitochondrial dysfunction is characteristic of BCO conditions along with autophagy machinery dysregulation. This autophagy dysregulation is also seen to a result of in vitro infection with known BCO-isolates and affecting bone cell viability. The local bone and systemic blood profile of cytokines, chemokines, inflammasomes, and relevant FGFs were also evaluated. This revealed a unique signature of BCO detectable within circulation and in local bone. Additionally, this signature was made up of factors which negatively affect bone cell viability. It was also shown that primary avian chondrocytes exhibiting optimal phenotypes could be successfully isolated form chicks. These primary cells could provide an improved, highly relevant model for in vitro analysis of avian bone diseases and infections. Finally, the potential roles of two factors regulating energy and lipid metabolism were preliminarily explored as a future target for BCO research. These findings provide novel insight into mechanisms of etiology and means of non-invasive detection while also improving upon current methods of avian growth-plate researc

Inflammasomes in Alveolar Bone Loss

Bone remodeling is tightly controlled by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Fine tuning of the osteoclast–osteoblast balance results in strict synchronization of bone resorption and formation, which maintains structural integrity and bone tissue homeostasis; in contrast, dysregulated bone remodeling may cause pathological osteolysis, in which inflammation plays a vital role in promoting bone destruction. The alveolar bone presents high turnover rate, complex associations with the tooth and periodontium, and susceptibility to oral pathogenic insults and mechanical stress, which enhance its complexity in host defense and bone remodeling. Alveolar bone loss is also involved in systemic bone destruction and is affected by medication or systemic pathological factors. Therefore, it is essential to investigate the osteoimmunological mechanisms involved in the dysregulation of alveolar bone remodeling. The inflammasome is a supramolecular protein complex assembled in response to pattern recognition receptors and damage-associated molecular patterns, leading to the maturation and secretion of pro-inflammatory cytokines and activation of inflammatory responses. Pyroptosis downstream of inflammasome activation also facilitates the clearance of intracellular pathogens and irritants. However, inadequate or excessive activity of the inflammasome may allow for persistent infection and infection spreading or uncontrolled destruction of the alveolar bone, as commonly observed in periodontitis, periapical periodontitis, peri-implantitis, orthodontic tooth movement, medication-related osteonecrosis of the jaw, nonsterile or sterile osteomyelitis of the jaw, and osteoporosis. In this review, we present a framework for understanding the role and mechanism of canonical and noncanonical inflammasomes in the pathogenesis and development of etiologically diverse diseases associated with alveolar bone loss. Inappropriate inflammasome activation may drive alveolar osteolysis by regulating cellular players, including osteoclasts, osteoblasts, osteocytes, periodontal ligament cells, macrophages, monocytes, neutrophils, and adaptive immune cells, such as T helper 17 cells, causing increased osteoclast activity, decreased osteoblast activity, and enhanced periodontium inflammation by creating a pro-inflammatory milieu in a context- and cell type-dependent manner. We also discuss promising therapeutic strategies targeting inappropriate inflammasome activity in the treatment of alveolar bone loss. Novel strategies for inhibiting inflammasome signaling may facilitate the development of versatile drugs that carefully balance the beneficial contributions of inflammasomes to host defense

Analysis of Cell Signaling Perturbations in Response to Chronic Localized Infections

The tissue-level response to pathogens involves an intricate series of signal transduction events, influenced by immune and healing mediators that alert the host to danger and eliminate the infection. Disruptions to normal signaling events can compromise the host’s ability to respond and lead to the development of chronic infections that cannot be resolved without clinical intervention. Prolonged inflammation due to chronic infection can damage tissues and compromise healing processes, thus, the interactions of immune and healing mediators in signaling cascades are intimately linked to tissue health outcomes. Studying signaling networks relevant to these responses provided a more thorough understanding of localized tissue health to identify the drivers of disruptions to signaling cascades, and this knowledge can lead to the development of improved diagnostic and therapeutic biomarkers to combat chronic infections. The work presented here focused on elucidating the relationships between immune and wound healing factors in an in vivo rodent model and a clinical cohort to understand the tissue-level responses to chronic inflammation and infection. Specifically, extracellular inflammatory immune responses (i.e., cytokines and chemokines) related to intracellular signaling (i.e., phosphorylation of proteins) were investigated to identify alterations in native responses compared to those provoked by chronic inflammation and infection. Reponses in native tissues were compared to tissues with inflammatory and infectious stimuli to test if levels of immune related cytokines were elevated in response to chronic joint infections. Wound healing phosphoproteins were also included to look for shifts in wound healing-related processes across groups. Traditional statistical approaches and network analysis were used to dissect these complex biological datasets and identified drivers of network disruptions in response to inflammation and infection. The spatial analysis suggested that changes in biological responses were related to proximity to inflammation and infection, and the degree of response differed across spatial gradients, which demonstrated the ability for these chronic insults to affect disparate tissues in a clinically-relevant manner. The objective of this research and future related research is to facilitate new clinical strategies to combat chronic infection, and monitoring alterations to cell signaling pathways in this work highlighted the value of using network analysis to approach biological interrogation of signal disruptions related to these insults

Staphylococcal trafficking and infection-from 'nose to gut' and back

Staphylococcus aureus is an opportunistic human pathogen, which is a leading cause of infections worldwide. The challenge in treating S. aureus infection is linked to the development of multidrug-resistant strains and the mechanisms employed by this pathogen to evade the human immune defenses. In addition, S. aureus can hide asymptomatically in particular ‘protective’ niches of the human body for prolonged periods of time. In the present review, we highlight recently gained insights in the role of the human gut as an endogenous S. aureus reservoir next to the nasopharynx and oral cavity. In addition, we address the contribution of these ecological niches to staphylococcal transmission, including the roles of particular triggers as modulators of the bacterial dissemination. In this context, we present recent advances concerning the interactions between S. aureus and immune cells to understand their possible roles as vehicles of dissemination from the gut to other body sites. Lastly, we discuss the factors that contribute to the switch from colonization to infection. Altogether, we conclude that an important key to uncovering the pathogenesis of S. aureus infection lies hidden in the endogenous staphylococcal reservoirs, the trafficking of this bacterium through the human body and the subsequent immune responses

Effects of electronic cigarettes on respiratory immune homeostasis using translational in vitro and in vivo approaches

Millions of people are current e-cigarette users. E-cigarettes are commonly perceived to be safer than cigarettes, but their inhalation toxicity has not been fully described. Previous work in cells, rodents, and humans has demonstrated that e-liquids, e-liquid chemical components, and e-cigarette aerosols can be cytotoxic and modulate cellular and respiratory host defense function. However, with only a decade of research on e-cigarettes, many knowledge gaps remain, and the variety of e-cigarette devices and e-liquid formulations constantly being introduced to the market presents additional challenges when investigating the effects of e-cigarettes on respiratory health. The goal of this dissertation was to address specific knowledge gaps pertaining to the effects of e-cigarettes on respiratory host defense, including whether e-cigarette flavoring chemicals affect neutrophil function, whether e-cigarette use is associated with respiratory microbiome dysbiosis, and whether different e-cigarette device types are associated with different central airway immune phenotypes using cells and clinical samples from human subjects. Our data demonstrate that aromatic aldehyde e-cigarette flavoring chemicals can impair neutrophil phagocytosis and oxidative burst. We also observed unique nasal microbiome dysbiosis in e-cigarette users relative to smokers and non-smokers, and this signature was associated with changes in proteins that are associated with the host-microbiome interaction. Furthermore, we found that users of newer generation e-cigarettes such as JUUL had significantly lower expression of soluble immune mediators in cell-free induced sputum in comparison with smokers, non-smokers, and users of older generation devices. Taken together, these findings demonstrate dysregulated immune homeostasis in association with e-cigarette use, with a trend toward impaired immune responses. Additional contributions of this dissertation include development and characterization of a human monocyte-derived macrophage cell culture model for use in air-liquid interface exposures, assessment of sex differences in neutrophil function, and creation of tools (high school biology lessons, clinical vaping questionnaire) to disseminate e-cigarette science to youth and facilitate conversations about vaping. Overall, these findings highlight the need for continued investigation of the mechanisms underlying the effects of e-cigarettes and have direct implications for e-cigarette regulation, including the importance of device type in e-cigarette toxicity.Doctor of Philosoph

Bacterial biofilm interactions with human immune cells

The human immune system is vast, and ranges from physical barriers to cellular mediators which coordinate to protect the host from the environment and pathogens. Bacterial cells have been shown to interact with various immune receptors which contribute to pathogen recognition, clearance and memory. Importantly, C-type lectin receptors (CLRs) interact with polysaccharides and are involved in the innate stages of immune recognition and decision-making during infection. Specifically, the CLRs MR, Dectin-2 and DC-SIGN all interact with high-mannose structures, which are abundant in multiple pathogens. Pseudomonas aeruginosa is a ubiquitous, Gram-negative bacterium that causes opportunistic infection in immunocompromised hosts, and is particularly associated with both diabetes and cystic fibrosis patients. Additionally, Staphylococcus aureus is a commensal organism and a common resident of the skin microbiome. P. aeruginosa and S. aureus pose a burden to the global healthcare system as ESKAPE organisms, and possess the ability to switch between a planktonic, free-swimming growth mode to biofilm, characterised by development of bacterial communities within an extracellular matrix (ECM). Our lab has previously demonstrated that both P. aeruginosa biofilms and purified biofilm carbohydrates from PAO1 biofilm-overproducing mutants, are able to interact with CLRs DC-SIGN, MR and Dectin-2, which all play a role in early immune decision making and modulation. During this study, we were able to optimise assays to confirm binding of these lectins to ECM polysaccharides from P. aeruginosa, namely Pel and Psl, as well as introduce a new species, S. aureus (SH1000), into our in vitro biofilm model which displayed potential GalNAc ligands. Due to binding artifacts identified in the culture medium, P. aeruginosa biofilm growth was optimised in DMEM-BSA. Binding of all three CLRs was maintained for whole biofilms in this new medium, but to purified product, only DC-SIGN binding was observed. Optimisation of this work is ongoing. To investigate cellular interactions during infection, a 3D co-culture model using a skin-like collagen matrix was designed and optimised to investigate interaction of human immune cells (monocyte-derived dendritic cells, moDCs) with P. aeruginosa (PAO1) biofilms in the context of cytotoxicity, cytokine production and surface marker expression. In this model, moDCs maintained high viability up to 24 h when cultured with live bacteria, although cytokine profiles were altered in the presence of bacteria. Specifically, chemoattractant protein MCP-1 was specifically downregulated in the presence of bacteria compared to other inflammatory cytokines (TNF-α, IL-1β) which were upregulated in the same conditions. Neither cytotoxicity nor cytokine profile was altered in the absence of DC-SIGN, indicating a certain level of redundancy in this context, contrary to earlier binding data. Additionally, surface markers (HLA-DR, CD11b, DC-SIGN, MR) were downregulated in the presence of PAO1 compared to uninfected cells when analysed by flow cytometry. After further investigation, this reduction was shown to be independent of secreted bacterial proteases, LPS stimulation or the general interaction of collagen with the immune cells. The findings displayed during this study have provided novel contributions to our understanding of the immune response to bacterial biofilms, in context of polysaccharide interaction with CLRs and whole biofilms with immune cells in a wound-like setting, and have given us some exciting insight into how bacteria modulate immune responses during chronic infection. Although not without limitations, the work conducted here provides some interesting routes for future investigation, potentially highlighting new avenues for therapeutic research and disease control