Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Notre laboratoire a préalablement démontré que les macrophages alvéolaires de rat allergique, lorsque cultivés 18 h ex vivo et réinstillés dans les poumons d'un rat allergique et dépiété de ses propres macrophages alvéolaires, inhibent l'hyperréactivité bronchique de ce rat receveur à la suite d'une stimulation allergénique. Le présent projet avait pour but d'identifier les mécanismes par lesquels la culture cellulaire conférait cette propriété de protection contre l'hyperréactivité bronchique. La production de cytokines par les macrophages alvéolaires, cultivés ou non, a été mesurée par PCR multiplex. Le nombre de cellules et le niveau de cytokines dans les lavages bronchoalvéolaires ont été mesurés chez des rats ayant reçu des macrophages alvéolaires fraîchement isolés et chez des rats ayant reçu des macrophages alvéolaires cultivés pendant 18 h. À la lumière des résultats obtenus, ce projet démontre que le mécanisme probablement sous-jacent à cette inhibition de l'hyperréactivité bronchique est lié à l'augmentation de la production de cytokines de type Th 1.

Osteoarthritis (OA) can be debilitating and career-ending for horses. Current treatments offer temporary and symptomatic relief, but potentially deleterious side effects. Bone marrow mononuclear cells (BMNC) are a rich source of macrophage progenitors that are anti-inflammatory and promote inflammation resolution. The objective of this study was to evaluate the ability of intra-articular BMNC therapy to improve clinical signs of naturally occurring equine OA. Horses presenting with clinical and radiographic evidence of moderate OA in a single joint were randomly assigned to 1 of 3 treatments: saline (negative control), triamcinolone (positive control), or BMNC (treatment group). Horses were subjectively and objectively evaluated for lameness and synovial fluid collected (cytology and cytokine/growth factor quantification) at 0, 7, and 21 days post-injection. Data were analyzed using General Estimating Equations with significance set at P<0.05. There were no adverse effects noted in any treatment group. No significant differences in synovial fluid cytology parameters, objective/subjective lameness scores, nor joint circumference were found between treatment groups at any time point. Within treatment groups, joint circumference did not change over time for saline- and triamcinolone-treated horses. However, joint circumference and objective lameness decreased significantly within BMNC-treated horses between Days 0 and 21 and Days 7 and 21. Lameness improved in saline-treated horses from 0 to 21 days, but did not improve in triamcinolone-treated horses. The decreased lameness and lack of adverse effects in the BMNC-treated horses in our study support a larger clinical trial using BMNC. / Master of Science / Osteoarthritis (OA) is a common source of joint pain in people and horses. Current treatments provide only partial and/or temporary relief. As a result, there is an urgent need for more effective and long-lasting treatment options. Arthritis is characterized by uncontrolled joint inflammation and progressive cartilage and bone destruction. Macrophages are cells within normal joints that function to resolve mild inflammation, maintaining joint health. However, when physiologic functions are overwhelmed, macrophages perpetuate inflammation through the recruitment of additional cell types to cope with the increased demands for repair. If this process is appropriately accomplished, macrophages resolve the inflammation, thereby enabling recovery and repair within the joint. Bone marrow aspirate is an excellent source of bone marrow-derived macrophage precursors (bone marrow mononuclear cells or BMNC) that have been shown to reduce joint inflammation and lameness in people and horses. The objective of our clinical trial was to evaluate the ability of intra-articular BMNC to improve clinical signs of naturally occurring OA in horses. BMNC treatment was compared to a placebo injection of saline and a standard-of-care in horses, corticosteroids. There were no adverse effects of BMNC treatment and BMNC-treated horses had significantly reduced joint circumference and lameness after 21 days. Synovial fluid cytology parameters did not differ significantly between treatment groups at any time point. In summary, BMNC are exciting because a horse can be treated with its own cells without the need for specialized equipment, and have the potential to naturally benefit thousands of people and horses suffering from arthritis.

One-way mixed-lymphocyte reactions (MLR) were performed to determine qualitatively and quantitatively how macrophages and macrophage-derived factors (MDF), interacting with mouse spleen nonadherent (NA) subpopulations, affected the ability of NA cells to respond to allogeneic lymphocytes. Comparative reactivity was measured using normal cells and cells from mice in various stages of tumorigenesis. In MLR's using normal NA cells, recognition of allogeneic cells was dependent on addition of an optimal (2-3%) concentration of macrophages. Beyond this optimum concentration, macrophage addition became inhibitory to NA cell stimulation. Macrophage enhancement or inhibition was not contact dependent, since cell-free macrophage supernatants (depending on their concentration) also possessed a similar capacity for enhancement or depression of the thymus-derived (T) cell response to allo-antigens. In the tumor-bearing host, initial phases of tumorigenesis caused in vitro NA cell activation in the absence of macrophages or MDF; however in advanced stages of tumorigenesis, NA cells required macrophages in order to respond to histoincompatibility antigens. Macrophages and MDF from normal or one week palpable tumor-bearers did not differ in their ability to enhance or depress in vitro T cell activity. Macrophages and MDF from two-week palpable tumor-bearing hosts exhibited a decreased capacity to stimulate. As tumorigenesis progressed, MLR activity of the NA population decreased, in response to the suppressor action of macrophages and macrophage supernatants. To explain the dual role of the macrophage as both an enhancing cell and a repressor cell, possession of two macrophage supernatant factors has been proposed. / Master of Science

M.S.

L'avènement de la trithérapie dès le milieu des années 90 combinées aux mesures de prévention a permis de minimiser les nouvelles contaminations au VIH-1 en plus de reconsidérer l'infection comme une pathologie chronique n'engageant plus le pronostic vital. Cependant, de nombreuses contraintes persistent; la méconnaissance du statut sérologique et l'accès limité ou inexistant à la médication et à la prévention pour certaines populations engendrent encore plus d'un million de nouvelles contaminations et autant de morts chaque année. En outre, les effets secondaires des traitements ainsi que le poids social de vivre avec le virus demeurent problématiques et démontrent la nécessité de poursuivre les efforts de recherche vers une guérison totale du VIH-1. Néanmoins, cet objectif est encore hors de notre portée. En effet, et ce malgré une charge virale indétectable, l'arrêt des traitements entraîne inexorablement une reprise de la propagation virale. La persistance du VIH-1 serait la conséquence de l'établissement précoce de réservoirs viraux anatomiques et cellulaires dans lesquels le virus se réplique à bas bruit ou demeure dans un état latent. Cette latence cellulaire est caractérisée par une présence du génome viral intégré au génome cellulaire, mais ne produisant pas de particules virales. Cette particularité confère aux cellules dites latentes une protection face aux effets toxiques associés à la production virale, ainsi qu'un moyen d'échapper à leur propre élimination parle système immunitaire de l'hôte. Dotées d'une longue durée de vie, ces cellules latentes sont considérées comme les principaux responsables de la reprise de l'infection lors de l'arrêt de la médication. La réactivation de la production virale des cellules latentes permettrait, en théorie, de lever leurs protections menant ainsi à leur élimination. Appelée "shock and kill", cette stratégie, combinée aux traitements pour limiter la propagation virale, représente un atout majeur dans l'éradication du VIH-1. Afin de réactiver la production virale dans les cellules latentes, de nombreuses molécules dénommées agents réactivateurs de la latence (LRA) sont à l'étude depuis plus d'une décennie. Cependant, les agents actuellement étudiés sont non discriminants et l'étude de leurs impacts sont majoritairement limités à la population de lymphocytes T CD4⁺, première population cellulaire identifiée comme infectée de façon latente. L'implication d'autres sous-populations cellulaires, dont notamment les macrophages, dans l'établissement et la progression de l'infection est pourtant avérée. En effet, il est admis que ces cellules contribuent à la formation des réservoirs viraux, la latence virale y étant fortement soupçonnée. La problématique des effets des LRA sur cette sous population cellulaire est ainsi cruciale. Les travaux présentés dans cette thèse visent à étudier l'impact de 3 classes différentes de LRA sur la physiologie des macrophages, leur sensibilité à l'infection par le VIH-1 et leur production virale. Nos résultats ont montré que le traitement des macrophages primaires humains avec certains LRA n'est pas toxique, mais que ces agents sont à même de moduler le transcriptome et le sécrétome de ces cellules. La bryostatine-1, un activateur de la voie PKC, est par exemple associée à des augmentations importantes de médiateurs pro-inflammatoires tels CCL2, CCL5, l'IL-8 et le TNF. Les autres LRA testés induisent des modulations mineures de ces médiateurs alors que cette sécrétion est absente chez les lymphocytes T CD4⁺. De plus, les effets des LRA sur les fonctions physiologiques des macrophages sont minimes, à l'exception d'une diminution de l'efferocytose pour la romidepsine et de l'endocytose dépendante de la transferrine pour la bryostatine-1. La stimulation des macrophages avec les deux molécules précédentes diminue fortement l'expression des récepteurs de surface CCR5 et CD4. Ces modulations sont associées à la diminution de l'infection des macrophages parle VIH-1 sous la dépendance de la modulation de CD4 pour la bryostatine-1 et par l'augmentation de l'activité antivirale de SAMHD1 pour la romidepsine. Le traitement des macrophages infectés aux LRA n'entraîne pas d'augmentation de la transcription ou de la production virale. Néanmoins et de façon surprenante, la bryostatine-1 est associée à une diminution importante de la détection des protéines matures du gène viral codant pour gag sans modulation de leurs précurseurs, et ce, uniquement dans les cellules myéloïdes. Cette étude suggère ainsi que l'impact des LRA diffère selon le type cellulaire, démontrant la nécessité d'étudier les différentes cibles du virus lors des stratégies de cures. / The advent of cART in the mid-1990s combined with preventive measures made it possible to minimize new HIV-1 infections and to reconsider this infection as a chronic no longer life-threatening pathology. However, the constraints remain numerous; the ignorance of the serological status and the limited or nonexistent access to medication and prevention for certain population generates even more than a million of new infections and deaths each year. In addition, the side effects of the treatments and the social burden of living with the virus remain problematic and demonstrate the need to continue research efforts towards a complete cure for HIV-1. However, this concept is still beyond our reach. Indeed, despite an undetectable viral load, treatment interruption ultimately leads to a rebound of viral spread. HIV-1 persistence is believed to be the result of the early establishment of anatomical and cellular viral reservoirs in which the virus replicates at low noise or remains in a latent state. This HIV-1 latency is characterized by an integration of the viral genome into the cell genome without production of viral particles. This peculiarity gives so-called latent cells protection against the toxic effects associated with viral production as well as escaping elimination by the host's immune system. With a long lifespan, these latent cells are considered to be the main culprit in the resumption of infection after medication's termination. The reactivation of the viral production of these latent cells would, in theory, make it possible to lift their protections thus leading to their elimination. Called "shock and kill", this strategy, combined with therapeutic treatments to limit viral spread, represents a major asset in the eradication of HIV-1. To reactivate viral production in latent cells, many molecules known as latency-reversing agents (LRAs) have been under study for more than a decade. However, agents currently studied are non-discriminating and their impacts is mainly limited to the population of CD4⁺ T cells, the first cell population identified as latently infected. Yet, the involvement of many cell populations, including macrophages, in the establishment and progression of the infection is acknowledged. In addition, these cells participate to the HIV-1 reservoirs establishment and are highly suspected to harbor latency. Thus, the problematic of LRAs' effect on this cell population arises. The work presented in this thesis aims to monitor the impact of 3 different classes of LRAs on the physiology of macrophages, their susceptibility to HIV-1 infection and their viral production. Our results have shown that the treatment of primary human macrophages with some LRAs are not toxic but are able to modulate the transcriptome and secretome of these cells. Bryostatin-1, an activator of the PKC pathway, is for example associated with significant increases in proinflammatory mediators such as CCL2, CCL5, IL-8 and TNF. The other LRAs tested induce minor modulations of these mediators while this secretion is absent in CD4⁺ T lymphocytes. Moreover, physiologic features were mostly unchanged by treatment with the studied LRAs except for a downregulation of efferocytosis for romidepsin and transferrin dependent endocytosis for bryostatin-1. Treatment of macrophages with these agents reduces the surface expression of CD4 and CCR5 receptors on macrophages. These modulations were associated with an impairment in HIV-1 infection which relies on CD4 downregulation for bryostatin-1 and SAMHD1 antiviral activity upregulation for romidepsin. Treatment of HIV-1-infected macrophages with LRAs does not increase neither transcription nor viral production. However, and surprisingly, bryostatin-1 is associated with a significant decrease in the production of mature Gag proteins while their precursor level remained unchanged, a mechanism which seemed specific to the myeloid cell lineage. Hence, this study suggests that the impact of LRAs differ depending on the cell type, emphasizing the need to study the different targets of the virus during treatment strategies.

Four macrophage (Mφ) surface antigens (Ia, Mac-1, -2, and -3) were examined for their association with Mφ regulatory functions. Observations of antigen expression on Mφ derived from normal or tumor-bearing hosts (TBH) showed that changes occurred in the antigen-defined phenotypes of Mφ which evolve during tumor growth. These changes in antigen expression were correlated with notable changes in Mφ immunoregulatory functions. Experiments using only normal host-derived Mφ showed that in the presence of complement (C), monoclonal antibodies (mAb) directed against Mφ could lyse targeted Mφ and that enrichment of the remaining cells provided populations of Mφ that were altered in their regulatory functions. Analysis of mAb-treated Mφ in the absence of C, suggested that the alterations observed in the presence of C were not due to ligand-receptor activation of peritoneal Mφ and that antibodies alone were not altering Mφ viability. When anti-Mac- I, -2, and -3 antibodies, were used to modify accessory cell activity of whole spleen cell (WSC) or splenic adherent cell (SAC) preparations from normal or TBII, differential susceptibilities of the Mφ were noted. Ligand-receptor activation of WSC by anti-Mac-I was observed in normal but not TBH WSC. With C, anti-Mac-I and -3 each reduced normal and TBH WSC proliferation. To evaluate the possible role of different types of SAC in T cell lectin responsiveness, adherent cells were collected and depleted by antibody plus C treatment and added back to normal T cells. Removal of Mac-1⁺ normal host SAC stimulated the supportive accessory function of the remaining SAC. Enhancing accessory cell function diminished after removal of normal host Mac-2⁺ or TBH Mac-1⁺ SAC. In summary, SAC from normal host demonstrated an accessory cell function corresponding to a Mac-1⁻ phenotype, which was either replaced or obscured by the predominance of a Mac-1⁺ phenotype in TBH. Variable Ia antigen expression by Mφ was examined during tumor growth. Tumor growth induced progressive loss of Ia antigen expression on Mφ. TBH splenic Mφ supported Concanavalin A-induced proliferation of syngeneic T cells (Ia antigen-independent) but did not support syngeneic T cell proliferation in the mixed lymphocyte reaction (MLR) (Ia antigen-dependent). Irrespective of tissue source, normal and TBH Mφ differed in their MLR stimulatory capabilities. In general, splenic Mφ preparations were better stimulators of allogeneic T cell blastogenesis in the MLR than thioglycollate-elicited peritoneal Mφ. Expression of Ia antigens by normal but not TBH Mφ were diminished by 24-hr in vitro plating of the peritoneal Mφ. Indomethacin treatment showed Prostaglandin E₂ was not a direct in vitro factor in Ia antigen-mediated reduction of splenic Mφ MLR stimulatory activity. Taken together, this data suggested a loss of Mφ Ia antigen expression, resulting in a decrease in Ia antigen-mediated functional activities during tumor growth. To continue the assessment of Mφ phenotypes and to determine if alterations in Mφ function during tumor growth included changes in the secretion of soluble regulators of T cell activities, anti-Mac-1, -2, and -3 mAb were used to modulate monokine-mediated regulation of T lymphocyte proliferation. The mAb anti-Mac-1, -2, and -3 (plus C) exhibited differential depletion of normal and TBH Mφ. There was a distinct increase in the number of peroxidase-positive Mφ during tumor growth. Peroxidase-positive TBH Mφ were susceptible to C-mediated lysis by anti-Mac-1 and -3 but not anti-Mac-2, whereas no direct relationship was observed among normal host-derived Mφ. Immunofluorescence of mAb-binding showed a decrease in Mac-2⁺ cells in TBH Mφ populations that was accompanied by an increase in Mac-3 expression. Anti-Mac-2 treatment significantly reduced the ability of TBH Mφ to produce a soluble suppressor(s) but did not alter normal host Mφ-derived suppressor production. In contrast, anti-Mac-1 and -3 treatment of normal host Mφ significantly reduced suppressor production but had diminished effects on TBH Mφ. Anti-Ia plus C treatment of splenic or peritoneal Mφ derived from normal or TBH showed that selection of Ia Mφ increased the secretion of PGE and also increased the T cell suppressor activity in Mφ culture supernatants. Collectively, these data suggest that tumor-induced aberrations in immunoregulation can be attributed to differences in Mφ subpopulations which were discriminated by their surface membrane components. / Ph. D.

Yes / The IL-1 family member cytokine IL-36γ is recognised as key mediator in the immunopathology of psoriasis, hallmarks of which involve the activation of both resident and infiltrating inflammatory myeloid cells and aberrant angiogenesis. This research demonstrates a role for IL-36γ in both myeloid activation and angiogenesis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23 and TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. IL-36γ was also demonstrated to induce endothelial tube formation and branching, in a VEGF-A-dependent manner. Furthermore, IL-36γ-stimulated macrophages potently activated endothelial cells and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, psoriasis monocytes showed increased adherence to both the stimulated and unstimulated endothelium when compared with monocytes from healthy individuals. Collectively, these findings show that IL-36γ has the potential to enhance endothelium directed leucocyte infiltration into the skin and strengthen the IL-23/IL-17 pathway adding to the growing evidence of pathogenetic roles for IL-36γ in psoriatic responses. Our findings also point to a cellular response, which could potentially explain cardiovascular comorbidities in psoriasis in the form of endothelial activation and increased monocyte adherence. / Faculty of Life Sciences, University of Bradford. MRC, Grant/Award Number: MR/M01942X/1; British Skin Foundation, Grant/Award Number: BSF 5035.

Les blessures musculaires sont fréquentes et engendrent un coût social élevé en hausse constante. Suite à une blessure, une réadaptation musculaire efficace s'appuyant sur des évidences scientifiques est souhaitable. Les processus de résolution de l'inflammation et de réparation sont loin d'être compris dans leur ensemble. Le but de cette thèse était donc de contribuer à l'élargissement des connaissances se rapportant aux mécanismes moléculaires et cellulaires utilisés par les différents acteurs impliqués dans ces processus. Dans un premier temps, nous avons démontré in vitro que les mastocytes, reconnus pour leurs rôles inflammatoires, stimulent la prolifération des cellules musculaires. De façon plus précise, il a été établi que la tryptase exogène stimule la prolifération des myoblastes L6 via l'activation du récepteur activé par une protease de type 2 {protease-activated receptor, PAR-2) et la modulation subséquente de l'activité de la cyclooxygénase-2 (COX-2). De plus, la production de la prostaglandine 15-deoxy-delta-¹², ¹⁴-prostaglandine h (I5A-PGJ₂) serait potentiellement impliquée. Ensuite, l'effet direct des mastocytes sur la stimulation de la prolifération des myoblastes L6 a été confirmé in vitro. Parallèlement, il a été établi in vivo que les mastocytes sont activés suite à une blessure musculaire induite par l'injection intramusculaire de bupivacaïne et qu'en plus d'être modulée immédiatement après la blessure, l'expression de la tryptase augmente ultérieurement à un stade correspondant au début de la myogenèse. Contrairement à nos observations in vitro, l'inhibition de l'activité des mastocytes a induit une stimulation de la prolifération des myoblastes in vivo associée à une augmentation de la densité de cellules immunitaires (neutrophiles, macrophages Ml et M2) dont certaines sont reconnues pour stimuler la myogenèse. Les mastocytes sont impliqués dans plusieurs processus inter-reliés et l'isolation d'un seul effet est expérimentalement difficile. Finalement, les mécanismes assurant l'accumulation des macrophages dans le muscle post-blessure ont été investigués. Il a été établi que les macrophages Ml prolifèrent davantage lorsque leurs précurseurs sanguins sont éliminés alors que l'accumulation des macrophages M2 repose autant sur la prolifération locale que sur l'infiltration de précurseurs. En conclusion, plusieurs acteurs présents dans la phase inflammatoire jouent également un rôle essentiel dans la réparation musculaire.

Le rôle des macrophages dans le développement des gliomes, des tumeurs attaquant le système nerveux central, est controversé. Nous avons étudié le développement des gliomes chez des souris transgéniques permettant la déplétion spécifique des macrophages par l'administration d'un pro-médicament. Nos résultats indiquent qu'une réduction de la densité de macrophages augmente la croissance tumorale. Selon le modèle, leur activité est apparue dépendante ou non du recrutement des lymphocytes T. Nous avons ensuite émis l'hypothèse que les anti-inflammatoires pourraient favoriser le développement des gliomes. Au contraire, nous avons observé que l'administration de dexaméthasone à des souris implantées avec des cellules gliomales diminuait la progression des tumeurs. Nous avons établi que cet agent affectait le profil d'expression génique des cellules endothéliales en diminuant notamment l'expression de l'angiopoiétine-2, un facteur pro-angiogénique. L'utilisation d'un anticorps synthétique inhibant l'Angpt2 est apparue plus efficace que la dexaméthasone pour réduire la croissance des gliomes. Les inhibiteurs de l'Angpt-2 pourraient donc représenter des outils intéressants pour le traitement des gliomes.