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Connective tissue extracellular matrix (ECM) consists of an interwoven network of contiguous collagen fibers that inform cell activity, direct biological function, and guide tissue homeostasis throughout life. Recently, ECM analogs have emerged as a unique ex vivo culture platform for studying healthy and diseased tissues and in the latter, enabling the screening for and development of therapeutic regimen. Unfortunately, current commonly used platforms, such as tissue-culture polystyrene (TCPS) or the basement membrane matrix, Matrigel, fail to fully recapitulate the physical and biochemical properties of the ECM. Tissue-culture polystyrene is significantly stiffer than typical ECM tissues and lacks the composition and 3-dimensional architecture that is critical for ECM function. Improving upon TCPS’s shortcomings, Matrigel retains a natural ECM structure and is comprised of native biopolymers. However, it is derived from mouse sarcomas, and thus, has significant batch-to-batch variability and often contains growth factors at non-physiologic concentrations. Moreover, despite being biopolymer based, Matrigel has relatively low amounts of type I collagen and high levels of type IV collagen, and as such, compositionally does not match the predominantly type I collagen matrix intrinsic to connective tissues. Thus, it is clear that new and improved models of the ECM are needed for in vitro culture. In pursuit of developing a highly biomimetic ECM analog, the objectives of this work were three-fold— first, to fabricate collagen-based ECM analogs with nanoscale mimicry, second, to systematically optimize crosslinking protocols in order to produce a stable substrate with continuous fibrous architecture, and third, to evaluate the substrate’s biocompatibility and utility as a platform for studying biomineralization. It was hypothesized that an architecturally and chemically relevant fibrous substrate could be prepared from gelatin and provide an optimal ex vivo platform for cell culture and new therapy screening and development. Thus, the ECM analog will be collagen-like, biocompatible, consist of continuous fibers, demonstrate both viscoelastic and elastic behavior, exhibit relevant mechanical properties, and remain stable for at least 14 days at cell culture conditions. To this end, first, a “green” electrospinning method was developed for preparing fibrous meshes from gelatin, which avoids typical electrospinning solvents that present significant health risks and barriers to large scale production. Next, crosslinking methods were developed using the reactive dialdehyde, glutaraldehyde (GTA), and the naturally derived enzyme, transglutaminase (TGase). These methods stabilized the meshes for over 28 days under cell culture conditions without disrupting its biomimetic architectures and chemical properties. In addition, a third approach to mesh fabrication using gelatin methacryloyl (gelMA) was developed to overcome the shortcomings of GTA and TGase crosslinking. With gelMA, the number of crosslinking sites were customized and, by taking advantage of its ability to undergo free radical polymerization, stable fibrous meshes were prepared with reproducible architecture, chemistry, and tunable mechanical properties. Following fabrication, the biocompatibility of the meshes was evaluated through macrophage, stem cell, and differentiated cell cultures. During culture, the macrophages maintained a naïve, non-polarized state, indicating they were not triggered towards an inflammatory response by the meshes. In addition, fibrochondrocytes, a cell critical for maintaining the collagen-based matrices where ligaments attach to bone, remained viable and maintained phenotypic expression on the meshes, as evident by their enhanced proteoglycan and collagen production relative to TCPS cultures. After demonstrating biocompatibility, the gelatin platform was coupled with a synthetic matrix vesicle (SMV) system and successfully acted as a mineralization platform in the presence of human osteoblast-like cells. Additionally, the platform supported mesenchymal stem cell expansion and mineralization when cultured with an alkaline phosphate conjugated SMV. In this work, three unique methods were developed for preparing ECM analogs. These efforts led to the production of a collagen-like mesh with nano- and micro-scale cues, fibrous continuity with little batch-to-batch variability, and proven stability in both dry and wet conditions. Importantly, these meshes did not instigate any inflammatory responses and supported fibrochondrocyte, osteoblast, and stem cell culture. Furthermore, the mesh successfully functioned as a template for biomineralization using both human osteoblast-like cells and stem cells. Collectively these findings demonstrate the potential of a collagen-like ECM analog with physiological relevance for ex vivo cell culture studies; and furthermore, its potential as a high-fidelity platform for studying cell-mediated biomineralization, cell-matrix interactions, and developing new therapeutic approaches for the treatment of connective tissue disorders.

Long non-coding RNAs (lncRNAs) have been implicated as important regulators of inflammation through various mechanisms in both the innate and adaptive immune systems of mice and humans. The majority of SNPs identified by GWAS to be associated with autoimmune disorders lie within non-coding areas of the genome, including genes for lncRNAs. To identify lncRNAs with relevancy to inflammation and autoimmunity, a discovery pipeline was used to find lncRNAs differentially expressed in TLR4 activated murine macrophages, conserved between mice and humans, and harboring GWAS identified SNPs associated with autoimmune disorders. Two of the six candidate lncRNAs identified, Lnc15 and Lnc13, are decreased in activated macrophages and are associated with both celiac and Crohn’s disease. To further explore the regulation and influence of these two lncRNAs during inflammation and its resolution, a variety of in vitro and in vivo techniques were utilized, including novel mouse knockout models. An investigation of Lnc15 was conducted in cells of both the innate and adaptive immune system, where the dominant isoform of Lnc15 was identified to be a ~1.4 kb transcript localized to the cytoplasm in both murine macrophages and T cells. Analysis of Lnc15 regulation was conducted in activated murine macrophages, focused on TLR signaling. Through stimulating macrophages with specific TLR ligands, Lnc15 was found to be decreased by TLR2, TLR3, and TLR4 signaling, likely dependent upon both MYD88 and TRIF. While not dependent upon NF-κB, protein synthesis is required for TLR induced decreases in Lnc15 levels. Conversely, activated neutrophils significantly increase Lnc15 levels, although the mechanism of regulation is not yet known. Mice lacking Lnc15 globally were found to be more susceptible to DSS induced colitis, which is likely dependent upon a defect in the innate immune system. In the adaptive immune system, Lnc15 was found to be specifically upregulated in Tregs compared to other T cell subsets. Lnc15 deficient Tregs had a reduced suppressive capacity in vitro, but not in vivo in a T cell induced model of colitis. These findings suggest Lnc15 plays a role in Treg suppressive capacity under certain conditions, but the exact mechanism influenced remains to be identified. Additionally, overexpression of Lnc15 in a murine T cell line resulted in a decrease in Rorc expression. A Lnc15 RNA pulldown experiment identified USF2, a transcription factor known to regulate Rorc expression, and UBR5, a ubiquitin-protein ligase known to influence RORyt stability, as protein interactors of Lnc15. These data indicate that Lnc15 can influence aspects of RORyt biology, which implicates Lnc15 as a regulator of either the plasticity between Tregs and Th17 cells, or Treg ability to suppress inflammatory Th17 cells. An investigation into Lnc13 regulation by disease relevant cytokines was conducted with a series of macrophage stimulation experiments. Lnc13 was found to be positively regulated by cytokines with an anti-inflammatory capacity, including IL-6, IL-4 and IL-10. When Lnc13 deficient macrophages were polarized, a higher expression of Il6 was detected in both M1 and M2 macrophages, suggesting a regulatory connection between Lnc13 and IL-6 across macrophage activation states. Previously identified Lnc13 target genes displayed a quicker transcriptional response to LPS stimulation in Lnc13 deficient macrophages. Additionally, when the Lnc13 mouse was crossed with the DQ8 transgenic mouse model and challenged to gluten, the ileum tissue of Lnc13 deficient mice expressed higher amounts of Il12 and Ifng, cytokines directly relevant to celiac disease. These findings provide support for Lnc13 as a novel regulator of macrophage response and cytokine expression in response to disease relevant stimuli.

During tumor growth there are a series of phenotypic and functional changes that occur in macrophages (M_Φ) that ultimately lead to the immunosuppression of the tumor-bearing host (TBH). To investigate the phenotypic changes of M_Φ during tumor growth, we examined the expression of the M_Φ surface antigens, Mac-1, Mac-2, Mac-3, and Ia on peritoneal and splenic M_Φ. In the peritoneal cavity there was no change in the percentage of Mac-1⁺ M_Φ but a decrease in the percentage of Mac-2⁺, -3⁺, and Ia⁺ M_Φ during tumor growth. In addition, three distinctly sized populations of peritoneal M_Φ, showing differential antigen expression, also shifted during tumor growth. In the peritoneal cavity there was a decrease in the percentage of M_Φ co-expressing the Mac-2, -3, and Ia antigens, leading to a shift towards Mac-1⁺ 2⁻ 3⁻ Ia⁻ TBH M_Φ. In splenic M_Φ, the percentage of Mac-1⁺, -2⁺, and -3⁺ M_Φ increased, while the percentage of Ia⁺ M_Φ decreased. Splenic M_Φ showed an increase in the percentage of M_Φ co-expressing Mac-1, -2, and -3 antigens and a decrease in the percentage of M_Φ co-expressing Ia, leading to a shift towards a Mac-1⁺ 2⁺ 3⁺ Ia⁻ TBH M_Φ. Taken together, these data suggest that tumor growth alters the phenotype of M_Φ and causes a shift in M_Φ subpopulations. After measuring the phenotypic changes in M_Φ during tumor growth, changes in M_Φ accessory function to T cells were assessed. TBH M_Φ have significantly reduced accessory activity for autoreactive T cells. This reduction is caused by decreased Ia antigen expression and increased production of the suppressor molecule, prostaglandin (PG). TBH M_Φ down-regulated autoreactive T cell responsiveness to interleukin (IL)-1, IL-2, and IL-4. In addition to TBH M_Φ reducing T cell responsiveness to cytokines, TBH CD4⁺ T cells alone were less responsive to the cytokines IL-1, IL-2, and IL-4. To examine the responsiveness of M_Φ to activation signals, lipopolysaccharide (LPS) was incubated with normal and TBH splenic M_Φ and assessed for their phenotypic, functional, and cell-cycle changes. The data showed that TBH M_Φ had a decreased responsiveness to LPS. We showed that there was a shift from an Ia⁺ M_Φ in the normal host to an Ia⁻ M_Φ in the TBH. Concomitant with the shift in TBH M_Φ Ia⁻ phenotype was a change in TBH M_Φ function. Normal and TBH Ia⁻ M_Φ were suppressor M_Φ. TBH Ia⁻ M_Φ, however, suppressed autoreactive and alloreactive CD4⁺ T cells significantly more than could their normal counterparts. Tumor growth causes quantitative and qualitative changes in Ia⁻ suppressor M_Φ. Although Ia⁻ M_Φ-mediated suppression seemed to be the major source of down-regulation of CD4⁺ T cells, CD8⁺ T cells were not without fault. In the TBH, there was an increase in the percentage of CD8⁺ T cells and an increase in CD8⁺ T cell-mediated suppression. In conclusion, tumor growth leads to a change in immunoregulation that causes suppression of the immune response. / Ph. D.

L'objectif de cette étude est d'établir un lien possible entre la physiopathologie de la péri-implantite et la présence de cellules immunitaires (lymphocytes T (CD3) et macrophages (CD68)) présents dans le tissu conjonctif de lésions de péri-implantite comparativement au tissu péridentaire sain. Une analyse morphologique des échantillons colorés à l'hématoxyline et l'éosine ainsi qu'un marquage immunohistochimique de ces cellules ont été réalisés sur des biopsies de tissus péri-implantaires prélevées autour d'implants affectés par une péri-implantite sévère, et de tissus contrôles sains provenant de gingivectomies. L'identification des cellules positives a été réalisée à l'aide de la technique de marquage en immunofluorescence. À l'analyse histologique, les coupes ne contiennent pas les structures d'attache. Les résultats montrent une augmentation non significative de la densité de CD3 et de CD68. La densité des lymphocytes T et des macrophages dans les lésions de péri-implantite serait similaire à celle du tissu gingival sain.

Le VIH-1 est un virus enveloppé qui pénètre ses cellules cibles par fusion. Bien qu'on ait longtemps cru que la fusion du VIH-1 se faisait uniquement à la membrane plasmique, plusieurs études ont montré que celui-ci pouvait fusionner avec les membranes endosomales dans certains types cellulaires. Ce processus se produirait lorsque le VIH-1 est rapidement internalisé par endocytose après la liaison de son récepteur, et serait dépendant de l'efficacité d'endocytose et de dégradation du virus par la cellule cible, ainsi que de la cinétique de fusion du virus. De plus, deux études indépendantes ont montré que l'endocytose du VIH-1 peut mener à une infection productive dans les macrophages dérivés de monocytes humains (MDM), et ce, malgré leur grande capacité à internaliser et à dégrader des cargos. Dans le cadre de cette thèse, nous avons étudié l'endocytose du VIH-1 dans le modèle des MDM afin de mieux comprendre les facteurs permettant l'infection productive de ces cellules par la voie endosomale. Globalement, nos résultats ont montré que, bien que la macropinocytose ne soit pas la seule voie impliquée dans l'endocytose du VIH-1 dans les MDM, celle-ci présente des caractéristiques qui favorisent la fusion endosomale. En effet, elle requiert la liaison du CCR5 à la surface cellulaire, ce qui implique que le CCR5 soit présent dans l'environnement endosomal en même temps que le VIH-1. De plus, l'étude du transport du virus a révélé que sa dégradation débute tardivement. La situation est cependant différente dans les MDM activés. En effet, l'activation des macrophages a un impact différentiel sur l'efficacité d'endocytose du VIH- 1, selon le type d'activation. De plus, l'activation des MDM est couplée à une augmentation de l'endocytose du VIH-1 par la voie clathrine-dépendante, qui ne nécessite pas la liaison au CCR5, et à une augmentation de l'efficacité de la dégradation du virus. Ces résultats indiquent que la fusion suite à l'endocytose pourrait être restreinte dans les macrophages activés. Une meilleure connaissance des facteurs permettant la fusion endosomale du VIH-1 est essentielle afin d'élaborer de nouvelles stratégies pour contrer le virus et pourrait mener au développement de nouveaux antirétroviraux plus efficaces.

Les macrophages jouent un rôle important dans l’infection par le virus d’immunodéficience humaine de type-1 (VIH-1) et sont suspectés d’être des réservoirs viraux, empêchant ainsi l’élimination complète du virus chez les individus infectés. Cinquante gènes ont été sélectionnés à la suite d’une étude transcriptomique comparant l’expression génique entre des cellules spectatrices (bystanders) ou infectées par le VIH-1. Parmi ces gènes, l’expression d’une enzyme impliquée dans le cycle du folate (Gamma-Glutamyl Hydrolase ; GGH), est augmentée rapidement à la suite de l’infection. Nous suggérons que dans les macrophages dérivés de monocytes (MDM), une concentration faible de folate pourrait jouer un rôle protecteur en limitant la disponibilité des nucléotides nécessaire au VIH-1 lors de l’infection. Notre modèle expérimental est basé sur l’identification de MDM productivement infectés par un clone moléculaire de tropisme R5 du VIH-1 exprimant tous les gènes viraux ainsi qu’une petite protéine murine membranaire (Heat Stable Antigen; HSA). Ce virus a servi à infecter des MDM transfectés avec de petits ARN interférents (siRNA) ou exposés à des inhibiteurs chimiques. Ces expériences ont permis d’observer l’effet de l’extinction génique de ces enzymes durant l’infection par le VIH-1. Après 3 à 18 jours avec le virus, le pourcentage de cellules productivement infectées est évalué par cytométrie en flux ainsi que par ELISA ciblant la capside virale (p24). La diminution de l’expression de certaines enzymes régulant le niveau de folate intracellulaire (ex. GGH, FPGS et MTHFR) fait augmenter le nombre de cellules productivement infectées par le virus. De plus, le traitement au Raltitrexed (RTX), un inhibiteur de la Thymidylate Synthase (TYMS), avant l’infection mène à un blocage de la réplication virale. Les résultats montrent donc que des interactions réciproques entre le virus et le cycle du folate pourraient être des éléments déterminants dans la susceptibilité des MDM à l’infection par le VIH-1. / Macrophages plays an important role in HIV-1 infection. These cells are suspected to act as a viral reservoir preventing complete virus eradication in infected individuals. Following a transcriptomic study, fifty genes were selected based upon their differential expression between non-infected, infected and bystander populations. One of those genes, coding for Gamma-Glutamyl Hydrolase (GGH), an enzyme involved in folate metabolism, was upregulated rapidly after HIV-1 infection before returning to a basal state. We propose that in monocyte-derived macrophages (MDM), a low folate concentration may play a protective role by limiting nucleotide availability for HIV-1 during the process of infection. We have developed an experimental model based on MDM identification productively infected with a R5 tropism HIV-1 molecular clone expressing all viral genes and a small membrane murine protein (Heat Stable Antigen; HSA). This virus was used to infect MDM transfected with small interfering RNAs (siRNA) or exposed to chemical inhibitors. The purpose of those experiments was to assess the effect of genetic downregulation of important folate proteins during HIV-1 infection. Between 3 and 18 days post-infection, percentage of productively infected cells was evaluated with flow cytometry or ELISA targeting the viral capsid protein p24. Downregulation of important enzymes involved in intracellular folate retention (e.g. GGH, FPGS and MTHFR) increased the number of cells productively infected with HIV-1. Also, Raltitrexed (RTX), a specific inhibitor of Tymidylate Synthase (TYMS), was able to inhibit viral replication when used before infection. Those results show that an interplay between HIV-1 and folate cycle may play a decisive role in MDM susceptibility to virus infection.

Lors d'une exposition au VIH-1, bien qu'une seule petite proportion des macrophages soit infectée, il est proposé que ces cellules jouent un rôle important dans l'infection et la propagation du VIH-1. Pour approfondir nos connaissances dans ce domaine, des analyses transcriptomiques et protéomiques ont été effectuées afin de comparer les MDMs (Macrophages Dérivés de Monocytes) infectés aux non infectés. Ces analyses ont mené à la sélection de 50 gènes dont l'expression est modulée chez les cellules infectées pour effectuer un criblage par siRNA pour leurs rôles fonctionnels dans le cycle viral. Huit cibles ont été identifiées comme des régulateurs de l'infection chez les MDMs, mais seulement le gène MDM2 agissait comme un facteur de susceptibilité. Ce gène a donc été l'objet d'études plus approfondies. L'inhibition de l'expression de MDM2 induit une diminution de moitié de l'expression virale. Nos résultats indiquent que la résistance accrue au VIH-1 associée à l’interférence de MDM2 est maintenue même si le niveau d'ARNm est rétabli, suggérant que cette protéine serait impliquée indirectement dans l'infection par le VIH-1. L'identification des cofacteurs viraux régulés par MDM2 mènera à une compréhension des évènements signalétiques contrôlant la réplication du VIH-1 dans les macrophages. / Upon exposure to HIV-1, only a small proportion of macrophages are infected whereas most remain uninfected. It is proposed that these cells play an important role in the establishment and propagation of HIV-1 infection. To further our knowledge in this field, transcriptomic and proteomic comparative analyses of uninfected and HIV-1-infected MDMs (Monocyte-derived macrophages) were performed. These analyses led to the selection of 50 genes that were tested for their functional roles in HIV-1 replication by siRNA screen. Eight genes were identified as regulators of HIV-1 infection in MDMs, but only MDM2 acted as a susceptibility factor. The knockdown of MDM2 decreased HIV-1 expression by two folds. Our results indicate that the resistance to HIV-1 upon MDM2 silencing is maintained in MDMs even if MDM2 mRNA level is restored, thus suggesting that this protein might be indirectly involved in HIV-1 infection. Identification of viral cofactors regulated by MDM2 will bring a new understanding of signaling events controlling HIV-1 replication in macrophages.

Traumatic brain injury (TBI) is a major health concern in the United States and worldwide and effective treatment options are limited. Differences in the magnitude and characteristics of the peripheral-derived immune cell response to TBI are key contributors to the secondary cascades of damage following brain trauma, and means of modifying this response to improve clinical outcome are a current area of active research. Our work elucidated the peripheral immune response to TBI by characterizing the transcriptomic profile of juvenile vs adult peripheral immune cells following TBI as well as discovering a novel role for the tyrosine kinase receptor EphA4 in the peripheral-derived immune response to brain trauma. Previous work has demonstrated significant differences in recovery from TBI in young vs adult animals, and some studies have indicated that the immune response contributes to these differences. We utilized next-generation sequencing to compare gene expression profiles of blood cell fraction samples in juvenile and adult mice. Our work demonstrated that juvenile peripheral immune cells show a more dynamic response to TBI than adult and that pattern recognition receptor signaling is a cornerstone of these differences. To assess the specific mechanisms involved in the peripheral response to TBI, we utilized a bone marrow chimeric mouse model lacking EphA4 in the hematopoietic compartment. These studies found decreased lesion infiltration of peripheral immune cells, specifically activated macrophages, in the absence of EphA4. We also showed that EphA4 interacts with the Tie2/Angiopoietin signaling axis to regulate macrophage phenotype on the M1/2 continuum. Overall, our work demonstrated a novel role for EphA4, mediated by Tie2, as a pro-inflammatory regulator of the peripheral-derived immune cell response to TBI. / Traumatic brain injury (TBI) is a major health concern in the United States and worldwide and effective treatment options are limited. While the blood-brain barrier (BBB) excludes immune cells in the blood from entering the healthy brain, brain trauma compromises BBB integrity and allows massive infiltration of peripheral neutrophils, macrophages, and other immune cells. This circulating immune cell response to TBI contributes to damage following brain trauma, and means of modifying this response to improve recovery are a current area of active research. Our work explored the circulating immune cell response to TBI by comparing the gene expression profile of young vs adult circulating immune cells following TBI as well as discovering a novel role for the EphA4 protein in the circulating immune cell response to brain trauma. Previous work has found significant differences in recovery from TBI in young vs adult animals and that the immune response contributes to these differences. To explore this, we compared gene expression profiles of blood immune cells in young and adult mice and found that young immune cells show a more dynamic response to TBI than adult. To assess the specific pathways involved in the circulating immune cell response, we used a mouse model lacking EphA4 in these cells. Our studies found decreased numbers of immune cells, specifically macrophages, entering the injury area in the absence of EphA4. We also showed that EphA4 interacts with the Tie2 protein and its Angiopoietin protein binding partners. Originally studied as an important contributor to blood vessel function, Tie2 has recently been found to play a role in the function of macrophages. Our work demonstrated that EphA4 interacts with Tie2 to regulate pro-recovery vs proinflammatory characteristics in macrophages. Overall, our work demonstrated a novel role for EphA4, mediated by Tie2, as a pro-inflammatory regulator of the circulating immune cell response to TBI.