Osteoarthritis (OA) is the leading cause of musculoskeletal disability in people and horses, and is characterized by progressive joint degeneration. There is a critical need for a better understanding of disease processes leading to OA in order to develop more efficient therapies. A shared feature among different arthritic conditions is chronic synovitis. Macrophages are the main drivers of synovitis and can display pro-inflammatory (M1) or pro-resolving responses (M2). Macrophages promote joint health through phagocytic and secretory activities; however, when these functions are overwhelmed, macrophages upregulate inflammation, recruiting more cells to counteract damage. Once cell recruitment is efficiently accomplished, macrophages coordinate tissue repair and further resolution of inflammation. Bone marrow mononuclear cells (BMNC) are a source of macrophages used to treat inflammation and produce essential molecules for cartilage metabolism; however, little information exists regarding their use in joints. The studies presented in this dissertation focus on understanding the dual role of macrophages in driving and resolving synovitis and how to harness their therapeutic potential. In the first study, patterns of macrophage phenotypes (M1:M2) in healthy and osteoarthritic equine synovium were compared and correlated with gross pathology, histology, and synovial fluid cytokines. M1 and M2 markers were co-expressed in normal and osteoarthritic joints, varying in intensity of expression according to degree of inflammation. Concentrations of synovial fluid IL-10, a macrophage-produced cytokine that is vital for chondrocyte recovery from injury, was lower in OA joints. The combined findings of this study suggest homeostatic mechanisms from synovial macrophages in OA may be overwhelmed, preventing inflammation resolution. In the second study we investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). BMNC cultured in autologous SF or ISF developed into macrophage cultures that were more confluent in ISF (~100%) than SF (~25%), and exhibited phenotypes that were ultimately similar to cells native to normal joints. BMNC cultured in SF or ISF were neither M1 nor M2, but exhibited aspects of both phenotypes and a regulatory response, characterized by increasing counts of IL-10+ macrophages, decreasing concentrations of IL-1β, and progressively increasing concentrations of IL-10 and IGF-1, all more marked in ISF. These findings suggest that homeostatic mechanisms were preserved over time, and potentially favored by macrophage proliferation. Our data suggest that BMNC therapy could potentiate the macrophage- and IL-10-associated mechanisms of joint homeostasis lost in OA. Finally, using an equine model of synovitis, the last study investigated the response of normal and inflamed joints to autologous BMNC injection. Inflamed joints treated with BMNC showed gross and analytical improvements in synovial fluid and synovial membrane, with increasing numbers of regulatory macrophages and synovial fluid concentrations of IL-10, not observed in saline-treated controls. Autologous BMNC are readily available, downregulate synovitis through macrophage-associated effects, and can benefit thousands of patients with OA. Combined, the results of these studies support the role of macrophage-driven synovial homeostasis and identified a therapeutic way to recover homeostatic mechanisms of synovial macrophages lost during chronic inflammation. Our findings also uncover new research directions and methods for future studies targeting modulation of joint inflammation. / Doctor of Philosophy / Osteoarthritis (OA) is a common cause of joint deterioration in people and horses. Current treatments provide limited recovery of joint function, creating an urgent need for more efficient therapies; however, development of new treatments requires better understanding of the mechanism causing OA. A shared characteristic among many arthritic conditions is long-standing inflammation. Cells called macrophages are the main drivers of joint inflammation and can exert pro- and anti-inflammatory effects. Macrophages promote joint health by clearing aggressor agents and secreting molecules required for optimal joint function. However, when these housekeeping functions are overwhelmed by damage, macrophages drive inflammation recruiting more cells to cope with increased demands for repair. If this process is efficiently accomplished, macrophages then resolve inflammation, recovering joint health. Macrophages in the bone marrow (BMNC - bone marrow mononuclear cells) are used to treat inflammation in several tissues and are known to produce molecules essential for joint health. Although little information exists regarding their use in joints, studies treating different organs suggest it can provide high rewards. The studies presented in this dissertation focused on understanding the dual function of macrophages in driving and controlling joint inflammation, and harnessed their therapeutic potential. In the first study, macrophages were investigated in normal and OA-affected joints, and curiously exhibited a hybrid pro- and anti-inflammatory identity in both groups. The indicators of this mixed identity were more markedly expressed in arthritic joints showing gross inflammation. Low levels of a macrophage-derived anti-inflammatory protein called IL-10 were detected in OA joints. The results of this study suggest that anti-inflammatory mechanisms from macrophages may be overwhelmed in OA-affected joints, preventing inflammation to be resolved, and that recovering this anti-inflammatory function may aid in the treatment of OA. In the second study we investigated how the incubation of BMNC in fluid from normal and inflamed joints affects the response of macrophages. Similar to what we observed in the first study, BMNC incubated in both normal and inflamed joint fluid induced macrophages to develop a hybrid identity that was ultimately similar to native cells from normal joints. Macrophages proliferated more when incubated in fluid from inflamed joints. Macrophages in both groups produced anti-inflammatory effects with high levels of IL-10 that were highest in ISF cultures. These observations suggest that higher proliferation of macrophages in inflamed joint fluid helped preserve anti-inflammatory mechanisms. Therefore, our study suggests that joint injection with BMNC could maximize macrophage- and IL-10-associated mechanisms required to resolve joint inflammation. The third and final study investigated the response of normal and inflamed joints to BMNC injection using a model of joint inflammation in horses. Inflamed joints treated with BMNC showed visual and laboratorial markers of improvement, with increasing numbers of macrophages and concentrations of IL-10 in the joint fluid, which remained lower in joints treated with placebo. BMNC provide means to recover macrophage-associated effects required to control joint inflammation and can benefit thousands of patients with OA. Together, the results of these studies show that macrophages are biased promoters of joint health, leading to inflammation when their anti-inflammatory mechanisms are overwhelmed. Replenishing inflamed joints with healthy macrophages maximizes their anti-inflammatory effects, favoring the recovery of a healthy articular environment.

Orientador: Vânia Niéto Brito de Souza / Resumo: A infecção pelo Mycobacterium leprae (ML) estimula um processo de desdiferenciação e proliferação das células de Schwann (SCs) que pode contribuir para a disseminação do bacilo. Os macrófagos (MOs) são células efetoras da resposta imune que promovem a eliminação de patógenos, entretanto, na hanseníase são colonizados pelo ML. Sabe-se que os MOs podem apresentar uma polarização funcional na qual os MOs M1 apresentam características pró-inflamatórias e microbicidas enquanto os MOs M2 atuam na reparação tecidual e possuem perfil anti-inflamatório. SCs infectadas pelo ML produzem mediadores capazes de interferir com a função dos MOs aumentando sua sobrevida e promovendo sua migração. Embora diferentes programas funcionais tenham sido observados em MOs de pacientes com formas polares da hanseníase a influência de SCs nesse processo não é sabida. Neste estudo avaliamos se SCs infectadas pelo ML podem interferir na polarização de MOs murinos derivados de medula óssea. Para tanto, culturas primárias de SCs murinas foram infectadas experimentalmente com bacilos viáveis e cocultivadas com MOs. Nossos resultados indicam que a produção de óxido nítrico foi baixa nas culturas de MOs após a infecção com o bacilo, mas mostrou-se aumentada nas coculturas de MOs e SCs infectadas pelo ML. A infecção com ML não induziu produção significante das citocinas IL-6, IL-10 e TNF em culturas de MOs e SCs, entretanto, a interação entre MOs e SCs infectadas com o bacilo resultou em aumento na produção de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mycobacterium leprae (ML) infection stimulates dedifferentiation and proliferation of Schwann cells (SCs) that may contribute to the spread of the bacillus. Macrophages (MOs) are effector cells of the immune response that promote the elimination of pathogens, however, in leprosy they are colonized by ML. It is known that MOs can present a functional polarization in which M1 MOs show pro-inflammatory and microbicidal activities while M2 MOs act in tissue repair presenting an anti-inflammatory profile. SCs infected by ML produce mediators able to interfere with MOs function, increasing their survival and promoting their migration. Although different functional programs have been observed in MOs from patients with polar forms of leprosy, the influence of SCs in this process is not known. In this study we evaluated whether SCs infected with ML could interfere in the polarization of murine MOs derived from bone marrow. For this purpose, primary cultures of murine SCs were experimentally infected with viable bacilli and co-cultivated with MOs. Our results indicate that nitric oxide production was low in cultures of MOs after infection with the bacillus, but it was increased in the co-cultures of MOs and ML-infected SCs. The infection with ML did not induce significant production of IL-10, TNF and IL-6 in cultures of MOs and SCs, however, the interaction between MOs and ML infected-SCs resulted in increased production of cytokines, mainly IL-10, inducing a decrease in the TNF/IL-10 ... (Complete abstract click electronic access below) / Mestre

La pemphigoïde bulleuse (PB) est la plus fréquente et la plus grave des dermatoses bulleuses auto-immunes caractérisée par une cascade inflammatoire impliquant plusieurs cytokines et cellules inflammatoires, avec la libération de protéases (MMP-9, élastase) conduisant à la formation de la bulle. L’objectif de cette étude est de caractériser cette cascade inflammatoire en analysant la contribution de la chimiokine CXCL10 et des cellules inflammatoires comme les macrophages dans le mécanisme physiopathologique associé à la maladie. Nos résultats ont montré que CXCL10 est présent à des taux importants dans le sérum des patients et dans le liquide de bulle, de plus cette chimiokine reste fortement présente dans le sérum des patients qui rechutent. D’autre part nous avons montré pour la première fois que l’orientation des macrophages dans la PB est contrôlée dans un premier temps par les sérums de patients, qui préactivent les macrophages dérivés de monocytes, puis par le liquide de bulle qui différencient les macrophages vers une polarisation M2. Nous avons également montré que seulement les neutrophiles et les monocytes isolés de patients atteints de PB, mais pas leur lymphocytes, répondaient à une stimulation par CXCL10 par une augmentation de leur sécrétion en MMP-9 via l’activation des voies de signalisation ERK1/2, P38 et PI3K. Cette expression de la MMP-9 a été associée à une polarisation de type M2 dans les macrophages issus de monocytes de patients. Enfin, nous avons montré l’importance des activateurs sélectifs des récepteurs des glucocorticoïdes, tels que le composé A, qui était aussi efficace que le traitement avec la méthylprénisolone dans l'inhibition de la MMP-9, mais avec des effets distincts sur l'expression des marqueurs phénotypiques de macrophages. / Bullous pemphigoid (BP) is the most common and serious autoimmune bullous dermatosis characterized by inflammatory cascade involving many cytokines and inflammatory cells, with the release of proteases (MMP-9, HLE) leading to the formation of the blister. The objective of this study is to characterize the inflammatory cascade by analyzing the contribution of the CXCL10 chemokine and inflammatory cells such as macrophages in the pathophysiology associated with the disease. Our results showed that CXCL10 is present at significant levels in the serum of patients and in the blister fluid, this chemokine remains strongly present in the serum of patients who relapse. On the other hand we have shown for the first time that macrophages polarization in BP is controlled by the sera of patients, which activate macrophages derived from monocytes, and then blister fluid differentiate macrophages in an M2 polarization. We also showed that only neutrophils and monocytes isolated from patients, but not the lymphocytes responding to stimulation by CXCL10 by an increase in secretion of MMP-9 via the activation of signaling pathways ERK1 / 2, P38 and PI3K. This expression of MMP-9 has been associated with an M2 macrophages polarization derived from monocytes of patients. Finally, we have demonstrated the importance of selective glucocorticoid receptor activators, such as Compound A, which was as effective as treatment with methylprednisolone in inhibiting MMP-9, but with distinct effects on the expression of phenotypic markers of macrophages.

Notre équipe étudie les HSP, et notamment HSP110. Les HSP sont des chaperons impliqués dans le repliement des protéines nouvellement synthétisées et dénaturées. Les HSP sont surexprimées lors des stress et participent à la survie des cellules par leurs propriétés anti-apoptotiques et anti-agrégations. HSP110 est surexprimée dans le cancer colorectal et est associée à un mauvais pronostic. L’expression d’un mutant d’HSP110, nommé HSP110DE9, a été mise en évidence dans les cancers colorectaux de type MSI. Celui-ci y agit comme un dominant négatif, en se liant à HSP110 et en inhibant ses fonctions. Son expression sensibilise les cellules cancéreuses à la chimiothérapie et est associée à un bon pronostic chez les patients.Je me suis tout d’abord intéressé au rôle d’HSP110 dans la régulation de la voie oncogénique STAT3. Son activation est en effet associée à un mauvais pronostic, par l’induction de gènes impliqués dans la prolifération et la survie. La protéine HSP110 favorise la prolifération des cellules colorectales cancéreuses à travers cette voie. HSP110DE9 en revanche l’inhibe. Je me suis ensuite intéressé au rôle d’HSP110 sur la polarisation des macrophages dans le cancer colorectal. Celle-ci peut être sécrétée par les cellules cancéreuses et induit une polarisation pro-tumorale des macrophages. HSP110DE9, en bloquant la sécrétion d’HSP110, conduit en revanche à une polarisation pro-inflammatoire. L’effet d’HSP110 sur la polarisation implique le récepteur TLR4.L’ensemble de ces résultats montrent le rôle d’HSP110 dans la progression tumorale. HSP110 apparaît comme une cible thérapeutique dans le traitement du cancer colorectal. / Our team studies HSPs, including HSP110. HSPs are chaperones involved in the folding of newly synthesized and denatured proteins. HSPs are overexpressed under stress conditions and are involved in cell survival thanks to their anti-apoptotic and anti-aggregation functions. HSP110 is overexpressed in colorectal cancer and is associated with a poor prognosis. The expression of a mutant HSP110, named HSP110DE9, has been shown in MSI colorectal cancer. This one was shown to act there as a dominant negative, by binding HSP110 and inhibiting its functions. Its expression sensitizes cancer cells to chemotherapy and is associated with a better prognosis for patients.I was first interested in HSP110 role in regulating the oncogenic STAT3 pathway. Its activation is associated with a poor prognosis, as it induces the transcription of genes involved in proliferation and survival. HSP110 favors colorectal cancer cell proliferation through this pathway. Conversely, HSP110DE9 inhibited it.I then focused on the role of HSP110 on macrophage polarization in colorectal cancer. HSP110 can be secreted by cancer cells and induces a pro-tumoral macrophage polarization. In contrast, HSP110DE9, by inhibiting HSP110 release, leads to a pro-inflammatory polarization. HSP110 effect on macrophage polarization involve the TLR4 receptor.All these results show HSP110 role in tumor progression. HSP110 appear as a therapeutic target in the treatment of colorectal cancer.

Choline is necessary for the synthesis of phosphatidylcholine (PC), the predominant phospholipid species and an important lipid intermediate. Macrophages, critical mediators of innate immunity, have been implicated in lipid dysregulation associated with metabolic disease. Despite the importance of choline in lipid metabolism, few studies have investigated the relationship between choline metabolism and inflammation. My research revealed that macrophage polarization increased choline metabolism and the expression of the choline transporter CTL1. In addition, choline deficient macrophages showed altered cytokine secretion, suggesting choline metabolism may play an important role in regulating the immune response. This study also describes the generation of a novel CTL1-/- mouse, which showed decreased choline uptake and incorporation into lipids. As an in vivo model for choline deficiency, CTL1-/- mice represent an important model for the future study of choline metabolism. Altogether, these findings suggest an important relationship exists between choline metabolism and inflammation.

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Muscular dystrophies are a heterogeneous group of genetic diseases that involve mutations in genes leading to progressive muscular weakness. Limb-Girdle Muscular Dystrophy 2B (LGMD2B) is a subset of muscular dystrophy caused by mutations in the DYSF gene, which encodes for dysferlin protein and has an incidence of 1/100,000-1/200,000 people, or 1/300 people of Libyan Jewish descent. Since there is no effective treatment that can cure or reverse effects of LGMD2B once diagnosed, our goal is to investigate and develop a protein therapy that mitigates effects of this disease in patients. Galectin-1 (Gal-1) is a small, soluble 14.5 kDa protein with a carbohydrate recognition domain capable of stabilizing the sarcolemma. The exact role that Gal-1 plays in myogenic cells is not fully understood, however, it is known that Gal-1 possesses anti-inflammatory properties and increases the terminal differentiation of committed myogenic cells. Our hypothesis is that Gal-1 treatment increases myogenic potential, improves membrane repair capability, and modulates the immune response in models of LGMD2B by stabilizing muscle integrity, leading to decreased disease manifestation. To test this hypothesis and assess the effect of Gal-1 treatment on myogenesis, anti-inflammatory modulation, and membrane repair, we designed, produced, and purified recombinant human galectin-1 (rHsGal-1) to be used in LGMD2B models. Our in vitro results indicate that after 2-3 days of treatment with 0.11μM rHsGal-1, A/J-/- myotubes enhance expression of myogenic late markers and increase in size and alignment. Additionally, after short-term treatment, rHsGal-1 improves membrane repair capability in a Ca2+ independent manner through an activated carbohydrate recognition domain (CRD) in in vitro and in vivo models of LGMD2B. We give evidence that rHsGal-1 upregulates anti-inflammatory cytokines, increases functional activity, and modulates the canonical NF-κB inflammatory pathway in dysferlin-deficient models by decreasing expression of TAK-1 and the p65 and p50 subunits in vitro and short-term in vivo treatment. Similar effects of the rHsGal-1 treatment were observed in patient-derived dysferlin-deficient human myotubes. Exploratory results show a potential decrease in muscle fat deposition in Bla/J mice. Furthermore, Gal-1 contributes to immune modulation by helping to initiate muscle regeneration by shifting M2 macrophage polarization. Together, our novel discoveries provide direct evidence that Gal-1 is a promising candidate to treat LGMD2B disease pathologies by improving expression of late-stage myogenic markers, improving membrane repair in vitro and short-term in vivo studies, promoting muscle regeneration through immune modulation, and reducing canonical NF-κB inflammation.

Macrophages (MF) are known to exhibit distinct responses to viral and bacterial infection, but how they react when exposed to the pathogens in succession is less well understood. Accordingly, we determined the effect of a rubella virus (RV)-induced infection followed by an LPS-induced challenge on cytokine production, signal transduction and metabolic pathways in human GM (M1-like)- and M (M2-like)-MF. We found that infection of both subsets with RV resulted in a low TNF-a and a high interferon (IFN, type I and type III) release whereby M-MF produced far more IFNs than GM-MF. Thus, TNF-a production in contrast to IFN production is not a dominant feature of RV infection in these cells. Upon addition of LPS to RV-infected MF compared to the addition of LPS to the uninfected cells the TNF-a response only slightly increased, whereas the IFN-response of both subtypes was greatly enhanced. The subset specific cytokine expression pattern remained unchanged under these assay conditions. The priming effect of RV was also observed when replacing RV by IFN-b one putative priming stimulus induced by RV. Small amounts of IFN-b were sufficient for phosphorylation of Stat1 and to induce IFN-production in response to LPS. Analysis of signal transduction pathways activated by successive exposure of MF to RV and LPS revealed an increased phosphorylation of NFkB (MMF), but different to uninfected MF a reduced phosphorylation of ERK1/2 (both subtypes). Furthermore, metabolic pathways were affected; the LPS-induced increase in glycolysis was dampened in both subtypes after RV infection. In conclusion, we show that RV infection and exogenously added IFN-b can prime MF to produce high amounts of IFNs in response to LPS and that changes in glycolysis and signal transduction are associated with the priming effect. These findings will help to understand to what extent MF defense to viral infection is modulated by a following exposure to a bacterial infection.

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