Osteoporosis and fragility fractures are recognized complications of inflammatory rheumatic diseases. This is thought to result from the effects of chronic inflammation, relative immobility and corticosteroid use. A rare syndrome of osteoporosis in a patient with coeliac disease has been described which results from production of neutralizing antibodies to the bone protective protein osteoprotegerin (OPG). The aim of my thesis is to evaluate prevalence and clinical predictors of osteoporosis in a contemporary cohort of patients with rheumatoid arthritis (RA) and to investigate the role of OPG autoantibodies in the pathogenesis of osteoporosis in rheumatic diseases. In a retrospective cohort study, I found that the overall prevalence of osteoporosis in patients with RA was 29.9% which is in keeping with older reports that recorded a prevalence rate between 17% and 36%. In our contemporary cohort osteoporosis was significantly more common than in a gender and age matched control cohort (17.4%). Further analysis showed that only age and BMI were independent predictors of osteoporosis in RA. A predictive tool based on age and BMI was developed which had 91.4% sensitivity for the detection of osteoporosis in an independent RA population. I went on to screen for the presence of autoantibodies to OPG in patients with various rheumatic diseases. In a study of 75 patients with rheumatoid arthritis and 199 healthy controls OPG autoantibodies were detected in two controls (1%) compared with seven patients with RA (9.3%). The RA patients with detectable OPG antibodies had a longer disease duration, higher DAS28 scores and higher levels of the bone resorption marker CTX than RA patients who did not have autoantibodies. Purified IgG from patients with high levels of OPG antibodies blocked the ability of recombinant OPG to inhibit RANKL induced NFκB activation in a HEK293 cell based assay indicating that they were functional. In a further study of 134 patients with ankylosing spondylitis (AS), 16 patients (11.9%) had detectable OPG antibodies. The presence of OPG-Ab was independently associated with reduced hip bone mineral density and an increased risk of fractures in this population. In patients with a longer disease duration we have also observed that there was a higher discrepancy between spinal and hip BMD in OPG-Ab positive patients compared with OPG ab negative patients (p=0.003). In order to investigate if OPG antibodies affected measurement of serum RANKL concentrations as detected by ELISA using OPG as the capture reagent, I measured OPG ab and free RANKL concentrations in 55 rheumatic disease patients. Surprisingly there was a significant positive correlation between free RANKL and OPG Ab concentrations (r=0.430, p=0.001) which was the opposite to what I had expected. These findings reject the hypothesis that OPG ab block binding of synthetic OPG to RANKL in the ELISA. In conclusion, I have shown that osteoporosis is a common complication in RA and I have developed a new risk prediction tool for the use in clinical practice. I have also found that OPG antibodies are produced more commonly in patients with RA and AS than in healthy controls and that antibody levels correlate with bone resorption markers in RA and bone mineral density in AS patients. In vitro studies have shown that some OPG antibodies have functional effects on RANKL signalling. These findings raise the possibility that OPG antibodies may contribute to the pathogenesis of local and systemic bone loss in rheumatic diseases and signal the need to study the relationship between these antibodies and bone disease in large-scale longitudinal studies.

Resident tissue macrophages are an integral component of many tissues and are important in development, homeostasis and repair. Macrophages are present at sites of both pathologic bone deposition and loss, and can produce osteo-active factors. These observations link macrophages to bone disease, however their contribution to bone dynamics is poorly understood. The molecular and cellular mechanisms driving osteoblast differentiation, matrix deposition and mineralization in vivo are incompletely understood and this deficiency is translated to limited ability to clinically manipulate bone formation. The emerging understanding of the bi-directional interactions between the osseus and immune systems (osteoimmunology) provides a novel avenue to identify mechanisms involved in the regulation of bone formation. In this study, the presence and distribution of macrophages on bone surfaces was systematically analysed and their functional contribution to the bone microenvironment was investigated. Using immunohistochemistry a discrete population of mature resident tissue macrophages was demonstrated throughout resting murine osteal tissues, termed OsteoMacs. Utilising MacGreen mice (csf1r promoter drives eGFP transgene expression in macrophages and other myeloid cells), it was demonstrated that OsteoMacs were intercalated amongst other bone lining cells in both the endosteum and periosteum. OsteoMacs were TRAPneg in situ and had limited osteoclastogenic ability in vitro therefore they are unlikely to serve as the immediate physiologic osteoclast precursors in vivo. Microarray gene expression profiling demonstrated that macrophage gene expression was regulated in response to a characteristic feature of the bone microenvironment, elevated extracellular calcium. Quantitative PCR validated upregulation of sphingosine kinase 1, interleukin 1 receptor antagonise, progressive ankylosis, vascular endothelial growth factor c and dipepetidase 2 mRNA in response to elevated extracellular calcium, suggesting the potential roles of these genes in this unique niche. GNF Symatlas microarray and quantitative PCR demonstrated the expression of macrophage-restricted genes throughout a 21-day primary osteoblast differentiation time course, suggesting co-isolation of OsteoMacs with primary osteoblasts. Flow cytometry analysis confirmed that over all 15.9% of the digested primary calvarial cell preparations were OsteoMacs. Immunocytochemistry demonstrated that OsteoMacs persisted and expanded in standard 21-day osteoblast differentiation assays. Contrary to previous studies, we demonstrated it was the OsteoMacs, and not osteoblasts, within calvarial preparations that selectively detected patho-physiological concentrations of the bacterial product lipopolysaccharide (LPS). A protocol was developed to deplete OsteoMacs from calvarial digests to determine if their presence within these cultures facilitates osteoblast differentiation or function. OsteoMac removal did not affect expression of the early osteoblast differentiation marker genes collagen type I or alkaline phosphatase. However, OsteoMac removal significantly decreased gene expression of the osteoblast mineralisation marker osteocalcin and mineralisation function, assessed by von Kossa staining. Microarray gene expression profiling demonstrated that osteoblast enrichment had a broad impact on transcription within the culture, identifying both candidate OsteoMac marker genes as well as osteoblast expressed genes that are regulated by OsteoMacs. Potential OsteoMac-enriched candidate genes insulin-like growth factor a, dipepetidase 2, glycoprotein NMB, and macrophage expressed gene 1 as well as osteoblast-specific genes bone sialoprotein and thrombospondin 1 were selected based on their potential involvement in osteoblast function. In a transwell co-culture system of enriched osteoblasts and macrophages, it was demonstrated that macrophages were required for osteoblast mineralisation in response to the physiologic remodelling stimulus, elevated extracellular calcium. A blocking soluble receptor strategy provided evidence that this is mediated in a BMP-2 and -4 independent manner. To investigate the relevance of OsteoMacs to bone formation in vivo, immunohistochemistry staining for the mature tissue macrophage marker F4/80 was performed in long bone sections from rapidly growing mice. OsteoMacs were closely associated with areas of bone formation in situ, forming a distinctive canopy structure over mature cuboidal osteoblasts (collagen type I+, osteocalcin+) on endosteal cortical surfaces. Using adapted histomorphometic analysis, we determined that 77 ± 2.1% (n = 7) of the endosteal mature osteoblast surface was covered by the F4/80+ OsteoMac canopy. This observation suggested that OsteoMacs are optimally located to regulate osteoblast function in vivo. In summary, we have demonstrated that OsteoMacs are an integral component of bone lining tissues and play a novel role in bone dynamics through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics. Further delineation of OsteoMac functions is likely to provide new avenues for treating bone disease and assisting bone repair.

Resident tissue macrophages are an integral component of many tissues and are important in development, homeostasis and repair. Macrophages are present at sites of both pathologic bone deposition and loss, and can produce osteo-active factors. These observations link macrophages to bone disease, however their contribution to bone dynamics is poorly understood. The molecular and cellular mechanisms driving osteoblast differentiation, matrix deposition and mineralization in vivo are incompletely understood and this deficiency is translated to limited ability to clinically manipulate bone formation. The emerging understanding of the bi-directional interactions between the osseus and immune systems (osteoimmunology) provides a novel avenue to identify mechanisms involved in the regulation of bone formation. In this study, the presence and distribution of macrophages on bone surfaces was systematically analysed and their functional contribution to the bone microenvironment was investigated. Using immunohistochemistry a discrete population of mature resident tissue macrophages was demonstrated throughout resting murine osteal tissues, termed OsteoMacs. Utilising MacGreen mice (csf1r promoter drives eGFP transgene expression in macrophages and other myeloid cells), it was demonstrated that OsteoMacs were intercalated amongst other bone lining cells in both the endosteum and periosteum. OsteoMacs were TRAPneg in situ and had limited osteoclastogenic ability in vitro therefore they are unlikely to serve as the immediate physiologic osteoclast precursors in vivo. Microarray gene expression profiling demonstrated that macrophage gene expression was regulated in response to a characteristic feature of the bone microenvironment, elevated extracellular calcium. Quantitative PCR validated upregulation of sphingosine kinase 1, interleukin 1 receptor antagonise, progressive ankylosis, vascular endothelial growth factor c and dipepetidase 2 mRNA in response to elevated extracellular calcium, suggesting the potential roles of these genes in this unique niche. GNF Symatlas microarray and quantitative PCR demonstrated the expression of macrophage-restricted genes throughout a 21-day primary osteoblast differentiation time course, suggesting co-isolation of OsteoMacs with primary osteoblasts. Flow cytometry analysis confirmed that over all 15.9% of the digested primary calvarial cell preparations were OsteoMacs. Immunocytochemistry demonstrated that OsteoMacs persisted and expanded in standard 21-day osteoblast differentiation assays. Contrary to previous studies, we demonstrated it was the OsteoMacs, and not osteoblasts, within calvarial preparations that selectively detected patho-physiological concentrations of the bacterial product lipopolysaccharide (LPS). A protocol was developed to deplete OsteoMacs from calvarial digests to determine if their presence within these cultures facilitates osteoblast differentiation or function. OsteoMac removal did not affect expression of the early osteoblast differentiation marker genes collagen type I or alkaline phosphatase. However, OsteoMac removal significantly decreased gene expression of the osteoblast mineralisation marker osteocalcin and mineralisation function, assessed by von Kossa staining. Microarray gene expression profiling demonstrated that osteoblast enrichment had a broad impact on transcription within the culture, identifying both candidate OsteoMac marker genes as well as osteoblast expressed genes that are regulated by OsteoMacs. Potential OsteoMac-enriched candidate genes insulin-like growth factor a, dipepetidase 2, glycoprotein NMB, and macrophage expressed gene 1 as well as osteoblast-specific genes bone sialoprotein and thrombospondin 1 were selected based on their potential involvement in osteoblast function. In a transwell co-culture system of enriched osteoblasts and macrophages, it was demonstrated that macrophages were required for osteoblast mineralisation in response to the physiologic remodelling stimulus, elevated extracellular calcium. A blocking soluble receptor strategy provided evidence that this is mediated in a BMP-2 and -4 independent manner. To investigate the relevance of OsteoMacs to bone formation in vivo, immunohistochemistry staining for the mature tissue macrophage marker F4/80 was performed in long bone sections from rapidly growing mice. OsteoMacs were closely associated with areas of bone formation in situ, forming a distinctive canopy structure over mature cuboidal osteoblasts (collagen type I+, osteocalcin+) on endosteal cortical surfaces. Using adapted histomorphometic analysis, we determined that 77 ± 2.1% (n = 7) of the endosteal mature osteoblast surface was covered by the F4/80+ OsteoMac canopy. This observation suggested that OsteoMacs are optimally located to regulate osteoblast function in vivo. In summary, we have demonstrated that OsteoMacs are an integral component of bone lining tissues and play a novel role in bone dynamics through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics. Further delineation of OsteoMac functions is likely to provide new avenues for treating bone disease and assisting bone repair.

With the development of Highly Active Anti-Retroviral Therapy (HAART), Human Immunodeficiency Virus (HIV) infection has evolved from a fatal disease to a chronic condition with increased risk for non-infectious comorbidities, including reduced bone density. Bone density is maintained through the coupled activities of osteoblast matrix deposition and osteoclast resorption; while uncoupling this process can result in bone loss and increased fracture risk. CD4+ T cells are critical in regulating the activity of these cells. Relevant to this thesis, studies have shown that HAART treated patients experience higher levels of immune activation; possibly contributing to the observed bone loss. If osteoimmune dysregulation does occur, there is a need to develop therapeutics that target this process, especially in the context of HIV infection. To evaluate osteoblast differentiation, we developed a high-throughput screening method to identify osteo-regulatory compounds. By screening over 5,000 compounds, we identified 18 that robustly induced osteoblast differentiation, using a mouse mesenchymal stem cell. We validated two of these compounds, rapamycin and FK-506, which are known immunosuppressants. Secondly, we addressed the role of activated CD4+T cells and HIV-infected T cells in osteogenesis. We found that supernatants from activated T cells potently inhibit osteoblast differentiation. However, when osteoblasts were co-cultured with HIV-infected T cells, differentiation was inhibited regardless of activation status, suggesting intrinsic differences between HIV infected and uninfected T cells. Finally, to prevent the inhibition of osteogenesis by activated T cells, we evaluated rapamycin, our pro-osteogenic and T cell activation antagonist, as well as the novel compound JQ1, an inflammatory inhibitor that targets bromodomain-containing proteins. Both rapamycin and JQ1 efficiently blocked the cytotoxic effects of supernatants from non-infected activated T cells on osteoblasts, whereas only rapamycin prevented inhibition in the co-culture model. In contrast, neither rapamycin nor JQ1 were able to prevent inhibition by HIV infected, activated T cells. This suggests that HIV exacerbates the negative effects of T cell activation on osteoblastogenesis. These data support a mechanism for HIV infection and T cell activation mediating bone loss.

O receptor NLRP12 é um receptor intracelular que está envolvido no reconhecimento de PAMPs e DAMPs durante uma infecção. Foi visto que durante a osteoclastogênese, há uma diminuição da transcrição de NLRP12, e que este receptor inibe a reabsorção óssea através da supressão da via alternativa de NF-κB, exercendo um papel protetor sobre o tecido ósseo. Além disso, foi observado que a deficiência de NLRP12 em monócitos sob o estímulo de RANKL levou a maior estabilização de NIK e maior translocação de RelB para o núcleo, aumentando a formação dos osteoclastos in vitro. Portanto, o objetivo deste estudo foi avaliar o papel de NLRP12 no desenvolvimento e progressão da lesão periapical induzida em camundongos. Para isso, foi induzida a lesão periapical dos primeiros molares inferiores dos camundongos fêmeas C57/Bl6 (WT) e camundongos deficientes para o receptor NLRP12 (Nlrp12-/-). Após 14 e 21 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos; expressão gênica de marcadores osteoclastogênicos por qPCR; contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); avaliação enzimática das MMPs por Zimografia. Os linfonodos cervicais foram submetidos à análise da expressão dos fatores de transcrição T-bet, RORγt, GATA-3 e Foxp-3 por qPCR. Aos 14 dias, na análise histomorfométrica os animais Nlrp12-/- apresentaram uma maior lesão periapical quando comparados aos animais WT, associado com o aumento da expressão de Trap, Catepsina K e MMP-9 em amostras de hemi-mandíbulas com lesão. Além disso, o número de células TRAP positivas foi significantemente maior em Nlrp12-/- com lesão quando comparado com seu controle, enquanto no grupo WT com e sem lesão foram semelhantes. Assim como foi observado o maior aumento dos osteoclastos presentes no local da lesão dos animais Nlrp12-/-, estes também se mostraram com maior atividade gelatinolítica de MMP-9 e MMP-2 em relação ao seu controle, diferente dos animais WT que não apresentaram diferença entre os grupos controle e com lesão. Ainda nesse período, foi observado nas amostras de linfonodos cervicais que, no grupo Nlrp12-/- houve uma tendência à maior expressão de RORγt, seguidos de menor expressão de T-bet quando comparados com o grupo WT. Aos 21 dias, os animais WT e Nlrp12-/- apresentaram lesões periapicais de tamanhos semelhantes. Além disso, somente o grupo Nlrp12-/- com lesão apresentou um aumento significativo da expressão de Trap em relação ao seu controle e a expressão de Catepsina K foi semelhante em ambos os grupos. Neste período houve um aumento na quantidade de células TRAP positivas em ambos os grupos com lesão quando comparados com seus respectivos controles, entretanto também não houve diferença entre os animais WT e Nlrp12-/-. A atividade de MMP-9 e MMP-2 foram semelhantes entre os animais WT e Nlrp12-/- e entre seus respectivos controles aos 21 dias. Nossos dados sugerem que a deficiência de NLRP12 levou a uma maior perda óssea aos 14 dias de lesão periapical e que isso ocorre via modulação da formação e atividade dos osteoclastos. Portanto, o NLRP12 inibe a osteoclastogênese e a atividade dos osteoclastos durante a fase inicial da lesão periapical, retardando o desenvolvimento da doença. / NLRP12 is an intracellular sensor that recongnizes PAMPS and DAMPs during infeccion. It was seen that NLRP12 transcription is down-regulated during osteoclastogenesis, and NLRP12 protect bone via suppression of alternative NF-κB-induced osteoclastogenesis. It has been shown that NLRP12 deficiency in monocytes under RANKL stimuli exhibited more stabilization of NIK and nuclear translocation of RelB, increasing osteoclasts formation in vitro. Therefore, the aim of this study was to evaluate the role of NLRP12 in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and NLRP12 knockout (KO) mice. Samples were collected at 14 and 21 days of the lesion for the analyses. Jaw samples with lesion and control area were subjected to periapical lesions area determination by histological sections; osteoclastogenic markers expression by q-PCR; count of osteoclasts submitted to the enzyme assay for TRAP; evaluation of MMPs activity by Zymography. The expression of T cells markers´ transcription factors was evaluated in lymph nodes by q-PCR. Fourteen days after periapical lesion induction, histological analysis revealed that NLRP12KO mice exhibited higher area of periapical lesion compared to WT group, which was associated with up-regulated mRNA expression of Trap, Cathepsin K and MMP-9 in the jaw samples. Moreover, the number of multinuclear TRAP-stained cells was significantly higher in the NLRP12KO with lesion group when compared to it control, whereas in the WT the number between the lesion and control groups were similar. Still, NLRP12KO showed higher MMP-9 and -2 gelatinolytic activity than it control, unlike WT mice that showed no difference between control and lesion group. In this period, NLRP12KO mice lymph nodes showed more RORγt expression than WT mice and less T-bet expression. At 21 days, WT and NLRP12KO presented periapical lesions of similar sizes. In addition, NLRP12KO group with lesion showed a significant increase in Trap expression when compared with their control, but the increase in Trap e Cathepsin K was similar in both groups. Additionally, there was an increase of multinuclear TRAP-stained cells in both lesion groups when compared with their respective controls; however, there was also no difference between WT and NLRP12KO mice. MMP-9 and -2 activity was similar between WT and NLRP12KO and with their respective controls at 21 days. Our results suggest that NLRP12 deficiency led to increased bone loss at 14 days of periapical lesion and it occurs due to increased osteoclasts formation and activity. Therefore, NLRP12 inhibits osteoclastogenesis and osteoclasts activity during the early stages of periapical lesion, slowing the development of the disease.

O receptor NLRP12 é um receptor intracelular que está envolvido no reconhecimento de PAMPs e DAMPs durante uma infecção. Foi visto que durante a osteoclastogênese, há uma diminuição da transcrição de NLRP12, e que este receptor inibe a reabsorção óssea através da supressão da via alternativa de NF-κB, exercendo um papel protetor sobre o tecido ósseo. Além disso, foi observado que a deficiência de NLRP12 em monócitos sob o estímulo de RANKL levou a maior estabilização de NIK e maior translocação de RelB para o núcleo, aumentando a formação dos osteoclastos in vitro. Portanto, o objetivo deste estudo foi avaliar o papel de NLRP12 no desenvolvimento e progressão da lesão periapical induzida em camundongos. Para isso, foi induzida a lesão periapical dos primeiros molares inferiores dos camundongos fêmeas C57/Bl6 (WT) e camundongos deficientes para o receptor NLRP12 (Nlrp12-/-). Após 14 e 21 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos; expressão gênica de marcadores osteoclastogênicos por qPCR; contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); avaliação enzimática das MMPs por Zimografia. Os linfonodos cervicais foram submetidos à análise da expressão dos fatores de transcrição T-bet, RORγt, GATA-3 e Foxp-3 por qPCR. Aos 14 dias, na análise histomorfométrica os animais Nlrp12-/- apresentaram uma maior lesão periapical quando comparados aos animais WT, associado com o aumento da expressão de Trap, Catepsina K e MMP-9 em amostras de hemi-mandíbulas com lesão. Além disso, o número de células TRAP positivas foi significantemente maior em Nlrp12-/- com lesão quando comparado com seu controle, enquanto no grupo WT com e sem lesão foram semelhantes. Assim como foi observado o maior aumento dos osteoclastos presentes no local da lesão dos animais Nlrp12-/-, estes também se mostraram com maior atividade gelatinolítica de MMP-9 e MMP-2 em relação ao seu controle, diferente dos animais WT que não apresentaram diferença entre os grupos controle e com lesão. Ainda nesse período, foi observado nas amostras de linfonodos cervicais que, no grupo Nlrp12-/- houve uma tendência à maior expressão de RORγt, seguidos de menor expressão de T-bet quando comparados com o grupo WT. Aos 21 dias, os animais WT e Nlrp12-/- apresentaram lesões periapicais de tamanhos semelhantes. Além disso, somente o grupo Nlrp12-/- com lesão apresentou um aumento significativo da expressão de Trap em relação ao seu controle e a expressão de Catepsina K foi semelhante em ambos os grupos. Neste período houve um aumento na quantidade de células TRAP positivas em ambos os grupos com lesão quando comparados com seus respectivos controles, entretanto também não houve diferença entre os animais WT e Nlrp12-/-. A atividade de MMP-9 e MMP-2 foram semelhantes entre os animais WT e Nlrp12-/- e entre seus respectivos controles aos 21 dias. Nossos dados sugerem que a deficiência de NLRP12 levou a uma maior perda óssea aos 14 dias de lesão periapical e que isso ocorre via modulação da formação e atividade dos osteoclastos. Portanto, o NLRP12 inibe a osteoclastogênese e a atividade dos osteoclastos durante a fase inicial da lesão periapical, retardando o desenvolvimento da doença. / NLRP12 is an intracellular sensor that recongnizes PAMPS and DAMPs during infeccion. It was seen that NLRP12 transcription is down-regulated during osteoclastogenesis, and NLRP12 protect bone via suppression of alternative NF-κB-induced osteoclastogenesis. It has been shown that NLRP12 deficiency in monocytes under RANKL stimuli exhibited more stabilization of NIK and nuclear translocation of RelB, increasing osteoclasts formation in vitro. Therefore, the aim of this study was to evaluate the role of NLRP12 in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and NLRP12 knockout (KO) mice. Samples were collected at 14 and 21 days of the lesion for the analyses. Jaw samples with lesion and control area were subjected to periapical lesions area determination by histological sections; osteoclastogenic markers expression by q-PCR; count of osteoclasts submitted to the enzyme assay for TRAP; evaluation of MMPs activity by Zymography. The expression of T cells markers´ transcription factors was evaluated in lymph nodes by q-PCR. Fourteen days after periapical lesion induction, histological analysis revealed that NLRP12KO mice exhibited higher area of periapical lesion compared to WT group, which was associated with up-regulated mRNA expression of Trap, Cathepsin K and MMP-9 in the jaw samples. Moreover, the number of multinuclear TRAP-stained cells was significantly higher in the NLRP12KO with lesion group when compared to it control, whereas in the WT the number between the lesion and control groups were similar. Still, NLRP12KO showed higher MMP-9 and -2 gelatinolytic activity than it control, unlike WT mice that showed no difference between control and lesion group. In this period, NLRP12KO mice lymph nodes showed more RORγt expression than WT mice and less T-bet expression. At 21 days, WT and NLRP12KO presented periapical lesions of similar sizes. In addition, NLRP12KO group with lesion showed a significant increase in Trap expression when compared with their control, but the increase in Trap e Cathepsin K was similar in both groups. Additionally, there was an increase of multinuclear TRAP-stained cells in both lesion groups when compared with their respective controls; however, there was also no difference between WT and NLRP12KO mice. MMP-9 and -2 activity was similar between WT and NLRP12KO and with their respective controls at 21 days. Our results suggest that NLRP12 deficiency led to increased bone loss at 14 days of periapical lesion and it occurs due to increased osteoclasts formation and activity. Therefore, NLRP12 inhibits osteoclastogenesis and osteoclasts activity during the early stages of periapical lesion, slowing the development of the disease.

Les maladies inflammatoires chroniques sont associées à un maintien de la réponse immunitaire et notamment la présence de cellules T CD4+ mémoires. Les maladies inflammatoires chroniques de l’intestin ont pour conséquence une perte osseuse due à une augmentation du nombre et de l'activité des ostéoclastes, les cellules responsables de la résorption osseuse. Les cellules CD4+ ont été décrites comme participant à la différenciation des ostéoclastes mais la nature exacte de ces cellules reste encore indéterminée. Des études in vivo et in vitro chez la souris et chez l’Homme nous ont permis de décrire une population inflammatoire Th17 TNFα CD4+ présente dans la moelle osseuse participant à la différenciation des ostéoclastes. Ces cellules Th17 participent au recrutement et à la différenciation des monocytes en ostéoclastes en agissant sur les cellules stromales mésenchymateuses. De plus, les maladies inflammatoires chroniques de l’intestin sont associées à une augmentation de la prolifération et de la différenciation des cellules souches hématopoïétiques en cellules myéloïdes. Suite à un stress chimique ou mécanique, les ostéoclastes participent à la mobilisation des cellules souches hématopoïétiques depuis la moelle osseuse. Nous avons montré qu’au cours des maladies inflammatoires de l’intestin, l’augmentation de l’activité des ostéoclastes participe à la prolifération et la différenciation des cellules souches en cellules myéloïdes. Ainsi, les ostéoclastes sont devenus une nouvelle cible thérapeutique intéressante dans la lutte des maladies inflammatoires chroniques de l’intestin / Chronic inflammatory diseases are associated to maintain of memory response and the presence of memory CD4+ T cells. Inflammatory bowel diseases are associated with bone loss due to an increase of the number and activity of osteoclasts, the bone-resorbing cells. CD4+ T cells participated to osteoclasts differentiation, but their nature is still undetermined. In vivo and in vivo studies in mice and human allow us to describe an inflammatory Th17 TNFα CD4+ T cells in the bone marrow that participated to osteoclast differentiation and link inflammation and bone loss. Th17 T cells induce the recruitment and the differentiation of monocytes into osteoclasts acting on mesenchymal stromal cells. Moreover, inflammatory bowel diseases are associated in an increase of hematopoietic stem cell (HSC) mobilization. In stress condition, osteoclasts act on HSC niches and induce their mobilization. We have shown that in inflammatory bowel disease, increase osteoclast activity is involved in the proliferation and differentiation of stem cells into myeloid cells. These myeloid cells are responsible for the intestinal inflammation. Thus, osteoclasts have become an exciting new therapeutic target in the fight of inflammatory bowel diseases

O biofilme bacteriano é o agente etiológico primário no desenvolvimento da resposta inflamatória observada na doença periodontal. Os receptores do tipo NOD (NLRs) são proteínas citosólicas que reconhecem componentes microbianos presentes no citoplasma liberados por bactérias invasoras. Sabendo que bactérias periodontopatogênicas têm a capacidade de invadir e colonizar diversas células do tecido periodontal, o presente projeto tem o objetivo de estudar a participação dos receptores do tipo NOD (NOD1 e NOD2) no reconhecimento das bactérias Porphyromonas gingivalis, na modulação da resposta imune e no processo de reabsorção óssea. Camundongos isogênicos machos da linhagem C57BL/6 (WT) e camundongos deficientes para o receptor NOD1 (NOD1-/-) e receptor NOD2 (NOD2-/-) foram infectados com Phorphyromonas gingivalis utilizando um modelo experimental de doença periodontal. O grau de reabsorção óssea foi avaliado por análise morfométrica macroscópica e histológica da maxila, além da quantificação do número de osteoclastos na crista óssea alveolar. O grau de inflamação local foi realizado por contagem do número total de bactérias orais, quantificação de neutrófilos no tecido gengival (MPO) e avaliação dos mediadores inflamatórios por ELISA. Foi também avaliada a expressão de marcadores osteogênicos e osteoclastogênicos no tecido gengival pela técnica de qPCR. Animais NOD1-/- e NOD2-/- apresentaram menor delta de reabsorção óssea quando comparados aos animais WT. Animais NOD2-/- infectados apresentaram debilitado controle bacteriano quando comparados aos animais WT infectados. Animais NOD1-/- e NOD2-/- infectados apresentaram baixa expressão de Cxcl1 e MPO quando comparados aos animais WT infectados. Além disso, foi observado que animais NOD2-/- infectados apresentaram reduzida produção de TNF-α e elevada produção de IL-10 quando comparados aos animais WT infectados. Não foi detectado diferença estatística na expressão de fatores osteogênicos, Runx2 e osteocalcina, entre animais WT, NOD1-/- e NOD2-/- infectados. Embora não houve diferença no número de células TRAP positivas presentes na crista óssea alveolar entre os grupos no tempo avaliado, animais WT infectados apresentaram elevada expressão gênica de RANKL/OPG quando comparados aos animais NOD2-/- infectados. Além disso, a expressão de marcadores de atividades dos osteoclastos, catepsina k e matrix metaloproteinase-9, foi significantemente baixa nos animais NOD1-/- e NOD2-/- infectados quando comparados aos animais WT infectados. Esses resultados permitem sugerir que independente das variáveis observadas, verificamos que a ausência dos dois receptores impede a rápida progressão da reabsorção óssea alveolar observada na periodontite, portanto os receptores NOD1 e NOD2 contribuem para progressão da reabsorção óssea no modelo experimental de periodontite. / The bacterial biofilm has been identified as an etiological agent in the pathogenesis of periodontal disease. The NOD-like receptors (NLRs) are cytoplasmic proteins that sense microbial by products released by invasive bacteria. Since periodontopathogenic bacteria are able to invade and colonize some periodontal tissue cells, the purpose of this study is to determine the role of NOD1 and NOD2 receptors in the recognition of invasive periodontopathogenic bacteria such as Porphyromonas gingivalis, modulating the immune response and bone resorption. Isogenic strain C57BL/6 males (WT), NOD1 (NOD1-/-) and NOD2 (NOD2-/-) knockout mice were infected with Porphyromonas gingivalis in an experimental model of periodontal disease. Alveolar bone resorption was evaluated by macroscopic and histological morphometric analysis, quantification of osteoclasts numbers in bone crest alveolar. Imune inflammatory response was evaluated by, bacterial load, neutrophils quantification and inflammatory mediators levels by ELISA. We also evaluated the osteogenic and osteoclastogenic markers expression in gingival tissue by Real time PCR techniques. NOD1-/- and NOD2-/- animals showed lower bone resorption when compared to WT animals. NOD2-/- infected animals expressed higher bacterial load compared to WT infected ones. NOD1-/- and NOD2-/- infected animals presented lower Cxcl1 and MPO levels compared to WT infected animals. In addition, NOD2-/- infected animals presented lower level of TNF-α and higher level of IL-10 when compared to WT infected animals. There was no significant difference in the osteogenic factors expression, Runx2 and osteocalcin, when compared NOD1-/- and NOD2-/- infected animals to WT infected ones. Although there was no difference in TRAP-positive cells number evaluated in the alveolar bone crest among the studied groups, WT infected animals showed elevated ratio RANKL/OPG when compared to NOD2-/- infected animals. Moreover, the expression of osteoclasts activity markers, cathepsin k and matrix metalloproteinase-9, was significantly lower in NOD1-/- and NOD2-/- infected animals compared to WT infected ones. These results suggest that NOD1 and NOD2 receptors contribute to progression of bone resorption in experimental model of periodontitis, since the lack of NOD like receptors impair the bone resorption.

O biofilme bacteriano é o agente etiológico primário no desenvolvimento da resposta inflamatória observada na doença periodontal. Os receptores do tipo NOD (NLRs) são proteínas citosólicas que reconhecem componentes microbianos presentes no citoplasma liberados por bactérias invasoras. Sabendo que bactérias periodontopatogênicas têm a capacidade de invadir e colonizar diversas células do tecido periodontal, o presente projeto tem o objetivo de estudar a participação dos receptores do tipo NOD (NOD1 e NOD2) no reconhecimento das bactérias Porphyromonas gingivalis, na modulação da resposta imune e no processo de reabsorção óssea. Camundongos isogênicos machos da linhagem C57BL/6 (WT) e camundongos deficientes para o receptor NOD1 (NOD1-/-) e receptor NOD2 (NOD2-/-) foram infectados com Phorphyromonas gingivalis utilizando um modelo experimental de doença periodontal. O grau de reabsorção óssea foi avaliado por análise morfométrica macroscópica e histológica da maxila, além da quantificação do número de osteoclastos na crista óssea alveolar. O grau de inflamação local foi realizado por contagem do número total de bactérias orais, quantificação de neutrófilos no tecido gengival (MPO) e avaliação dos mediadores inflamatórios por ELISA. Foi também avaliada a expressão de marcadores osteogênicos e osteoclastogênicos no tecido gengival pela técnica de qPCR. Animais NOD1-/- e NOD2-/- apresentaram menor delta de reabsorção óssea quando comparados aos animais WT. Animais NOD2-/- infectados apresentaram debilitado controle bacteriano quando comparados aos animais WT infectados. Animais NOD1-/- e NOD2-/- infectados apresentaram baixa expressão de Cxcl1 e MPO quando comparados aos animais WT infectados. Além disso, foi observado que animais NOD2-/- infectados apresentaram reduzida produção de TNF-α e elevada produção de IL-10 quando comparados aos animais WT infectados. Não foi detectado diferença estatística na expressão de fatores osteogênicos, Runx2 e osteocalcina, entre animais WT, NOD1-/- e NOD2-/- infectados. Embora não houve diferença no número de células TRAP positivas presentes na crista óssea alveolar entre os grupos no tempo avaliado, animais WT infectados apresentaram elevada expressão gênica de RANKL/OPG quando comparados aos animais NOD2-/- infectados. Além disso, a expressão de marcadores de atividades dos osteoclastos, catepsina k e matrix metaloproteinase-9, foi significantemente baixa nos animais NOD1-/- e NOD2-/- infectados quando comparados aos animais WT infectados. Esses resultados permitem sugerir que independente das variáveis observadas, verificamos que a ausência dos dois receptores impede a rápida progressão da reabsorção óssea alveolar observada na periodontite, portanto os receptores NOD1 e NOD2 contribuem para progressão da reabsorção óssea no modelo experimental de periodontite. / The bacterial biofilm has been identified as an etiological agent in the pathogenesis of periodontal disease. The NOD-like receptors (NLRs) are cytoplasmic proteins that sense microbial by products released by invasive bacteria. Since periodontopathogenic bacteria are able to invade and colonize some periodontal tissue cells, the purpose of this study is to determine the role of NOD1 and NOD2 receptors in the recognition of invasive periodontopathogenic bacteria such as Porphyromonas gingivalis, modulating the immune response and bone resorption. Isogenic strain C57BL/6 males (WT), NOD1 (NOD1-/-) and NOD2 (NOD2-/-) knockout mice were infected with Porphyromonas gingivalis in an experimental model of periodontal disease. Alveolar bone resorption was evaluated by macroscopic and histological morphometric analysis, quantification of osteoclasts numbers in bone crest alveolar. Imune inflammatory response was evaluated by, bacterial load, neutrophils quantification and inflammatory mediators levels by ELISA. We also evaluated the osteogenic and osteoclastogenic markers expression in gingival tissue by Real time PCR techniques. NOD1-/- and NOD2-/- animals showed lower bone resorption when compared to WT animals. NOD2-/- infected animals expressed higher bacterial load compared to WT infected ones. NOD1-/- and NOD2-/- infected animals presented lower Cxcl1 and MPO levels compared to WT infected animals. In addition, NOD2-/- infected animals presented lower level of TNF-α and higher level of IL-10 when compared to WT infected animals. There was no significant difference in the osteogenic factors expression, Runx2 and osteocalcin, when compared NOD1-/- and NOD2-/- infected animals to WT infected ones. Although there was no difference in TRAP-positive cells number evaluated in the alveolar bone crest among the studied groups, WT infected animals showed elevated ratio RANKL/OPG when compared to NOD2-/- infected animals. Moreover, the expression of osteoclasts activity markers, cathepsin k and matrix metalloproteinase-9, was significantly lower in NOD1-/- and NOD2-/- infected animals compared to WT infected ones. These results suggest that NOD1 and NOD2 receptors contribute to progression of bone resorption in experimental model of periodontitis, since the lack of NOD like receptors impair the bone resorption.

Knochengewebe besitzt die einzigartige Fähigkeit sich nach einem Bruch komplett zu regenerieren. Dennoch zeigen 10-15% der Patienten einen gestörten Heilungsverlauf. Das Immunsystem spielt eine entscheidende Rolle in der Frakturheilung. Im Rahmen der hier präsentierten Doktorarbeit wurde der Einfluss der CD4+ regulatorischen T-Zellen (Treg) auf die Knochenheilung untersucht. In einem Maus-Osteotomie-Modell wurde die zeitliche und räumliche Verteilung ausgewählter Immun- und Knochenzellen im osteotomierten Knochen untersucht. Dabei konnte gezeigt werden, dass Immunzellen im gesamten Heilungsverlauf in der Frakturzone zu finden waren und oft eine direkte Kolokalisation mit den Knochenzellen aufwiesen. Diese Ergebnisse zeigen deutlich die starke Interaktion beider Systeme. Ein adaptiver Transfer muriner Treg vor Setzen der Osteotomie diente als immunmodulatorischer Ansatz zur Verbesserung des Frakturheilungsprozesses. Tiere mit einem unerfahrenen Immunsystem (SPF-Haltung) zeigten eine verbesserte Heilung nach Treg-Transfer. Mäuse mit einem erfahrenen Immunsystem (semi-sterile Haltung) zeigten einen kontroversen Heilungserfolg: eine Hälfte der Mäuse heilte signifikant besser und die andere Hälfte signifikant schlechter. Die Schlechtheiler zeigten eine höhere Ratio von CD8+ Effektoren zu Treg im Vergleich zu den Gutheilern. In einer darauffolgenden Proof-of-concept-Studie konnte gezeigt werden, dass eine prä-OP definierte Ratio von CD8+ Effektoren zu Treg mit dem Heilungserfolg nach Osteotomie korrelierte. Im Rahmen dieser Doktorarbeit konnte ein potentiell positiver Einfluss von CD4+ Treg auf den Frakturheilungsprozess bestätigt werden. Dennoch wurde auch der enorme Einfluss des prä-OP Immunstatus auf den Heilungserfolg deutlich. Für die Klinik ist es also im Rahmen einer Immuntherapie umso wichtiger Patienten-basierte Therapieformen zu entwickeln, bei denen der individuelle Immunstatus eines jeden Patienten vor Anwendung der Therapie berücksichtigt wird. / Bone tissue possesses the remarkable capacity to fully regenerate after injury. However, in 10-15% of patients, unsuccessful bone repair is still a present problem. Components of the adaptive immune system play an indispensable role in bone regeneration. The here presented PhD thesis focused on the interaction of CD4+ regulatory T cells (Treg) during fracture healing. In a murine osteotomy model, the spatiotemporal distribution of immune and bone cells was analyzed within the healing bone. Cells of the immune system were detectable throughout the whole healing cascade in the injured area und showed often a direct co-localization with bone cells. These results highlight the interconnectivity of immune and bone cells during regeneration. By adoptive transfer of murine CD4+ Treg prior to osteotomy, an immunomodulatory approach to improve bone healing was conducted. Mice possessing an unexperienced immune system (SPF housing) showed a consistent improved healing outcome after adoptive Treg transfer. However, mice with a more experienced immune system (semi-sterile housing) receiving an adoptive Treg transfer demonstrated a controversial healing outcome: half of the mice showed a significantly improved and the other half a significantly poorer healing outcome. In the mice with a poorer healing outcome, a higher ratio of CD8+ effector T cells and Treg was observed. In a following proof of concept study, a pre-osteotomy defined ratio of CD8+ effector T cells and Treg could predict the healing outcome after adoptive Treg transfer and osteotomy. A potential positive impact of Treg in bone repair was confirmed in this study. However, the tremendous impact of the environment and thereby of the immune status prior to immunomo-dulation was also clearly demonstrated. Hence, for the clinic, it is even more important to develop and to apply patient based immunomodulatory treatment approaches considering the individual immune status of each patient prior to treatment.