The brain and spinal cord make up the central nervous system (CNS), and as an immune-privileged site, it requires special immune surveillance and regulation. The complement system is a component of innate immunity produced locally in the CNS, since size restrictions from the blood brain barrier prevent complement proteins from easily passing through from the rest of the body. The complement pathway contributes to inflammatory cell recruitment, cell lysis, and opsonization, and thus requires regulation to avoid inappropriate activation. Despite its important role in innate immunity, very little is known about complement production, regulation, and function in the CNS of healthy or diseased individuals. For this dissertation, the central goal was to investigate and characterize the regulation of complement factor H (CFH), a regulator of the alternative pathway of complement activation. CFH polymorphisms have been associated with a number of diseases including atypical hemolytic syndrome, age-related macular degeneration, and Alzheimer's disease, but the regulation of CFH is not well understood, especially in the CNS. To investigate the role of CFH in the CNS, mRNA and protein production in glial cells was first established. The murine CFH (mCFH) promoter was cloned and the transcription start site was identified in astrocytes, microglia, and liver tissue. The mCFH promoter was truncated and different regions were investigated for enhancer or silencer activity. Database mining identified potential transcription factor binding sites, and mutagenesis studies and binding assays identified transcription factor binding candidates. Specifically, the activating protein-1 (AP-1) transcription factors c-Jun and c-Fos bound to a region of the mCFH promoter between – 416 base pairs (bp) and – 175 bp in an electrophoretic mobility supershift assay. Cytokine stimulation increased mCFH mRNA and protein production, as well as the mRNA production of c-Jun and c-Fos and the protein production of c-Jun. These results suggest a relationship between cell cycle and complement regulation, and the investigation of how these transcription factors and CFH affect disease will be a valuable area of research for CNS immune regulation.

Indiana University-Purdue University Indianapolis (IUPUI) / Rationale: CD55 down-regulation on airway epithelium correlates with local complement activation observed in hypoxia-associated pulmonary diseases. Therefore, we hypothesized that induction of hypoxia inducible factor 1 alpha (HIF-1α) in hypoxic airway epithelium, mediates CD55 down-regulation. Methods: Chetomin and HIF-1α siRNA inhibited HIF-1α in hypoxic SAECs (1% O2), and mice lungs (10% O2). DMOG mediated HIF-1α stabilization in normoxic SAECs and mice lungs (21% O2). Transduction of SAECs with AdCA5 also stabilized HIF-1α. CD55 and CA9 transcripts were measured by RT-PCR. CD55 and HIF-1α protein expression was assessed by western blots. In vivo, immunohistochemistry (IHC) confirmed CD55 and HIF-1α expression. C3a and C5a levels in bronchoalveolar lavage fluid (BALF) were measured by ELISA. Results: HIF-1α was induced in 6 hour hypoxic SAECs (p<0.05), but CD55 transcripts were repressed (p&lt;0.05). CD55 protein was down-regulated by 72 hours (p<0.05). CA9 transcripts were elevated by 48 -72 hours (p<0.05 and p<0.01, respectively). In vivo, CD55 transcripts and protein were down- regulated by 24 hours post-hypoxia (p<0.01) which corresponded to complement activation (p<0.05) in BALF. However, CA9 was increased (p<0.01). Chetomin (100nM) treatment in 6 hour hypoxic SAECs, recovered CD55 transcripts (p<0.01) and protein (p<0.05), but down-regulated CA9 (p<0.05). Similarly, in vivo chetomin (1mg/ml) treatment recovered CD55 protein (p<0.01) and down-regulated CA9 (p<0.01). Silencing HIF-1α (50nM) in hypoxic SAECs restored CD55 transcripts by 6 hours (p<0.05), and protein expression by 24 hours (p<0.05). However, CA9 was repressed (p<0.01). In vivo silencing of HIF-1α (50µg) restored CD55 protein expression (p<0.05) but down-regulated CA9 (p<0.05). Stabilizing HIF-1α in normoxic SAECs via DMOG (1µM), down-regulated CD55 transcripts and protein (p<0.01), but increased CA9 within 6-24 hours (p<0.05 and p<0.01, respectively). HIF-1α induction by DMOG (1mg/ml) in normoxic mice lungs down-regulated CD55 transcripts (p<0.01) and protein (p<0.01), but increased CA9 (p<0.05). Induction of HIF-1α in AdCA5 (50 PFUs/cell) transduced normoxic SAECs, resulted in CD55 protein down-regulation (p<0.05), but increased CA9 (p<0.001). Conclusions: HIF-1α down-regulates CD55 on airway epithelium. Targeting this mechanism may be a potential therapeutic intervention for attenuating complement activation in hypoxic pulmonary diseases.

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The body’s humoral immune response plays a larger role in the body’s defenses beyond screening for invading pathogens. Modulation of this response is also vital for tissue regeneration, drug delivery, and vaccine development. The immune system operates within a complicated feedback loop and as such, altering the strength of the immune response can be approached from an engineering perspective. While a strong initial input can direct the response to either a pro- or anti-inflammatory bias, extreme responses can be deleterious, as in the case of allergic reactions or sepsis. Therefore, the objective of this thesis was to develop a novel biomaterials platform that can be used to alter the immune response in a tunable manner. Antibodies are not only the workhorses of the adaptive immune response but are also powerful immunomodulators through their Fc (constant fragment) regions. By coating microparticles with Fc ligands in variable surface densities, we were able to utilize the sensitivity of multivalent signaling to tune the response of the immune response. Microparticle size was also varied to decouple the effects of physical versus biochemical signaling. The goal of this thesis was to analyze the effects of Fc coated particles on two major components of the humoral immune responses: macrophages and the complement system. We first looked at the mechanical response of macrophages through phagocytosis and found that both Fc density and microparticle size had significant impacts on macrophage phagocytosis. These results also provide a particle delivery “toolbox” for future applications. We then analyzed the downstream effects of Fc particles on macrophage phenotype and on phenotype plasticity. This showed that the addition of Fc particles lead to increased production of TNFα and IL-12 and inverted the response of LPS treated macrophages. Finally, we applied our particles to activate the complement system, an often overlooked cascade of serum protein activation that results in bacterial cell lysis. Cleaved components of the complement system are also powerful chemokines and can act as a vaccine adjuvant. Fc density on particles played a large role in complement system activation, both through the classical and alternative pathway, as it lead to a binary response for smaller particles and a tunable response for larger particles. We then applied these results to create a novel form of antibiotic by using Fc particles to direct complement-mediated bacterial cytotoxicity. The use of immune activation by Fc particles was also applied to better understand and improve the tuberculosis vaccine. Our findings are significant to the biomaterials and immunology fields as we showed that Fc microparticles can generally be used to alter the immune response in a tunable manner for a broad range of applications, as well answering fundamental immunology questions.

One of the differentiating features of PARAFAC decompositions is that, under certain conditions, unique solutions are possible. The search for uniqueness conditions for the PARAFAC Decomposition has a limited past, spanning only three decades. The complex structure of the problem and the need for tensor algebras or other similarly abstract characterizations provided a roadblock to the development of uniqueness conditions. Theoretically, the PARAFAC decomposition surpasses its bilinear counterparts in that it is possible to obtain solutions that do not suffer from the rotational problem. However, not all PARAFAC solutions will be constrained sufficiently so that the resulting decomposition is unique. The work of Kruskal, 1977, provides the most in depth investigation into the conditions for uniqueness, so much so that many have assumed, without formal proof, that his sufficient conditions were also necessary. Aided by the introduction of Khatri-Rao products to represent the PARAFAC decomposition, ten Berge and Sidiropoulos (2002) used the column spaces of Khatri-Rao products to provide the first evidence for countering the claim of necessity, identifying PARAFAC decompositions that were unique when Kruskals condition was not met. Moreover, ten Berge and Sidiropoulos conjectured that, with additional k-rank restrictions, a class of decompositions could be formed where Kruskals condition would be necessary and sufficient. Unfortunately, the column space argument of ten Berge and Sidiropoulos was limited in its application and failed to provide an explanation of why uniqueness occurred. On the other hand, the use of orthogonal complement spaces provided an alternative approach to evaluate uniqueness that would provide a much richer return than the use of column spaces for the investigation of uniqueness. The Orthogonal Complement Space Approach (OCSA), adopted here, would provide: (1) the answers to lingering questions about the occurrence of uniqueness, (2) evidence that necessity would require more than a restriction on k-rank, and (3) an approach that could be extended to cases beyond those investigated by ten Berge and Sidiropoulos.

Factor H (FH) is a key regulator of the complement system, the principal molecular component of innate immunity in humans. The tight regulation of the alternative pathway (AP) of complement by FH occurs on host cells as well as in fluid phase. FH regulation of AP is achieved through its C3b. Bb-decay accelerating activity and cofactor activity for C3b proteolysis by factor I. This study presents evidence that the first three CCP modules, i.e. FH~1-3, constitute the minimal unit with cofactor activity for factor I. The work presented in this thesis describes the recombinant protein expression and NMR-derived structure determination of two overlapping pairs, FH~1-2 and FH~2-3, together with the use of these structures to build a model of the FH~1-3 structure. A structural comparison with other C3bengaging proteins (namely factor B, complement receptor type 1 and decay accelerating factor) is presented and used to devise hypotheses as to the respective roles of the three modules during an encounter with the convertase. This thesis further describes an investigation of the structural effects of two disease-associated sequence variants in the context of FH~1-2: namely the single nucleotide polymorphism V62I linked to age-related macular degeneration, and the R53H mutation linked to atypical haemolytic uraemic syndrome.

Biomaterials are commonly used in the medical clinic today; however, artificial materials can activate the cascade systems in the blood (complement-, coagulation-, contact- and fibrinolytic systems) as well as the platelets to various degrees. When an artificial surface comes in contact with blood, plasma proteins will be adsorbed to the surface within seconds. The composition of the layer of proteins differs between materials and is crucial for the hemocompatibility of the material. This thesis includes five projects. In Paper I the anticoagulants heparin and the thrombin inhibitor hirudin were evaluated in a whole blood model. Hirudin was found to be superior to low dose heparin since it did not affect the activation of the complement system nor the leukocytes. The most interesting observation was that expression of TF was seen on surface-attached monocytes in hirudin- treated blood but not heparin blood. In Paper II peptides from the streptococcal M-protein, which has affinity for the human complement inhibitor C4BP, were attached to a polymeric surface. When being exposed to blood the endogenous complement regulator was enriched at the surface of the material, via the M-peptides. With this new approach we created a self-regulatory surface, showing significant lowered material-induced complement activation. In Paper III apyrase, an enzyme which hydrolyzes nucleoside ATP and ADP, was immobilized on a polymer surface. Lower platelet activation and platelet-induced coagulation activation was seen for the apyrase-coated surface compared to control surfaces after exposure to whole human blood, due to the enzymes capability to degrade ADP released from activated platelets. In Paper IV and V we synthesized an array of polymeric materials which were characterized regarding physical-chemical properties, adsorption of plasma proteins, and hemocompatibility. The polymers showed widely heterogeneous protein adsorption. Furthermore, when the polymers were exposed to whole blood, two of the materials showed superior hemocompatibility (monitored as complement- and coagulation activation), compared to the reference poly(vinyl chloride).

Periodontitis is a polymicrobially-induced, chronic inflammatory disease of the tissues that support and surround the tooth. While greater than 500 organisms are found in dental plaque, Treponema denticola is one of only a few species associated with disease. It has been hypothesized that oral bacteria disrupt host homeostasis through manipulation of the innate immune system. In this study, we examine the impact of binding and subsequent cleavage of factor H, a complement regulator, by T. denticola in the evasion and subversion of complement. The molecular interaction between the sole FH-binding protein, FhbB, and FH was detailed by x-ray crystallography, site-directed mutagenesis, and inhibition analyses. Negatively-charged amino acids of FhbB formed salt-bridges with positively-charged residues of FH within the complement control protein domains (CCP) 6 and 7. In support of its critical role in disease, FhbB was universal among T. denticola isolates, although, different strains produce highly divergent FhbB proteins. Despite extensive sequence variation, predominantly within the FH-binding determinant, all FhbB proteins have similar structure and interact with FH using nearly identical molecular mechanisms. In addition to differences in FhbB, many isolates of T. denticola displayed significant variation in the activity of the chymotrypsin-like protease, dentilisin. Neither FhbB type produced nor dentilisin activity influenced serum resistance, as all strains tested were highly serum resistant. While sequence diversity of FhbB did not influence the interaction with FH or serum resistance, FhbB proteins elicited type-specific antibodies that blocked FH binding. Collectively, these analyses indicate that FH binding is an essential complement evasion mechanism of T. denticola and characterizes the uniquely complex interaction between T. denticola and complement. The data presented here provide novel insight into the pathogenesis of disease and begins to explore a hypothetical molecular mechanism by which this key periopathogen disrupts the host innate immune system, leading to periodontitis.

Periodontitis is a chronic inflammatory disease that affects over 116 million adults in the United States. A shift in the normal microflora occurs as periodontal disease develops resulting in a larger number of Gram-negative anaerobes and spirochetes. An increase in the oral spirochete, Treponema denticola, is highly correlated with periodontal disease progression and severity. The ability of this periopathogen to thrive in the subgingival crevice is dependent on complement evasion mechanisms. Earlier analyses demonstrated that the primary mechanism of T. denticola serum resistance is binding of the human complement regulatory protein, Factor H (FH), to the factor H-binding protein (FhbB). FH serves as cofactor in the Factor-I mediated cleavage of C3b and accelerates the decay of the C3 convertase complex, leading to downregulation of C3b production. Several pathogens bind FH, and a number of these bacterial binding proteins have been shown to bind plasminogen. Plasminogen is a plasma glycoprotein that circulates as a zymogen. Its active form, plasmin, degrades components of the extracellular matrix and cleaves complement proteins C3b and C5 inhibiting complement pathway progression. Through molecular and biochemical analyses, this study demonstrates that FhbB simultaneously binds plasminogen and FH to residues located on its positively and negatively charged surfaces, respectively, and that the two ligands do not compete for binding. This study also shows that the surface-bound plasminogen is available for proteolytic cleavage into the active serine protease plasmin. The activated plasmin could break down components of the periodontal tissue leading to increased nutrient availability and creation of a larger anaerobic environment where the bacteria can flourish, thereby promoting periodontal disease.