Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies directed against ubiquitous self-antigens, many of which are nuclear autoantigens like dsDNA and chromatin (Pisetsky, 2016), and by elevated type I interferons (IFN) (Hooks et al., 1979; Weckerle et al., 2011), a family of pro-inflammatory cytokines that have antiviral activity (Pestka et al., 2004). Microarray analysis of peripheral blood mononuclear cells (PBMC) from SLE patients discovered the increased expression of IFN-responsive genes that was named the IFN Signature (Baechler et al., 2003a; Bennett et al., 2003b; Crow et al., 2003). Genome wide association studies indicate a clear genetic component in lupus pathogenesis (Chung et al., 2011; SLEGEN et al., 2008) and murine models of SLE confirm genetic drivers of the disease (Morel, 2010; Morel et al., 2000). However, the concordance of SLE in monozygotic twins is only 30-40% (Connolly and Hakonarson, 2012), while the inc / Microbiology and Immunology

Rheumatoid arthritis (RA) is a chronic disease characterized by cycles of inflammation and resolution. Previously, it was believed that the resolution of inflammation is simply dissipation of pro-inflammatory signals, although current research indicates that resolution is an active process. Acute inflammation follows defined phases of induction, inflammation and resolution, and resolution occurs by an active process that requires COX-2 activity. This study aims to address whether this paradigm extends to a recognized model of chronic inflammation. We demonstrated in murine collageninduced arthritis that chronic inflammation follows the same sequential course. While there is the normal production of pro-inflammatory cytokines during inflammation and anti-inflammatory mediators such as 15-deoxyΔ12,14PGJ2 (15d-PGJ2) during resolution, interestingly there is sustained production of both COX-2 and the presumably proinflammatory PGE2 during both phases. Blocking COX-2 activity and therefore production of PGE2 during the resolution phase perpetuated instead of attenuated inflammation. Repletion with PGE2 analogs restored homeostasis, and this function is mediated by the pro-resolving lipoxygenase metabolite, lipoxin A4 (LXA4), which is a potent stop signal. Thus, the study provided in vivo evidence for a natural, endogenous link between the cyclooxygenase-lipoxygenase pathways and showed that PGE2 serves as a feedback inhibitor essential for limiting chronic inflammation in autoimmune arthritis. These findings may explain the enigma regarding why COX-2 inhibitors are palliative rather than curative in humans because blocking resolution may mitigate the benefit of preventing induction. / Microbiology and Immunology

Developmental 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure can permanently alter immune system ontogeny, resulting in the dysregulation of a number of vital immune pathways. We hypothesized that developmental exposure to TCDD may also impair the establishment of self-tolerance, resulting in an increased risk of autoimmunity. For example, we observed that a single prenatal TCDD exposure given to non-autoimmune-prone high affinity aryl hydrocarbon receptor (AhR) C57BL/6 mice resulted in an immune complex-mediated autoimmune disease during the adult stage. Further using a similar TCDD exposure protocol, autoimmune-prone low affinity AhR SNF1 mice exhibited acceleration and exacerbation of lupus-like nephritis in adulthood. Examination of these mice showed that perinatal TCDD exposure adversely affected both primary immune organs of the adaptive immune system. In the thymic compartment, prenatal TCDD affected thymocyte cellularity, differentiation and maturation as well as central tolerance as indicated by high levels of autoreactive Vβ TCR T cells in the periphery. Prenatal TCDD also altered bone marrow B lymphopoiesis and B cell maturation and differentiation in the spleen. Functionally, these B cell changes resulted in high serum autoantibodies titers to dsDNA, ssDNA and cardiolipin suggesting a loss in central B cell tolerance. The functional assessment of T cells, via cytokine production showed that prenatal TCDD mice altered Th1/Th2 levels. As a result, significant changes were detected in the kidney characterized by increased immune complex deposition in the glomeruli, lymphocytic infiltration and general pathologic changes. This would suggest that multiple immune pathways are affected by prenatal TCDD and work either independently or synergistically to display immune-mediated disease during aging. Importantly, this study has also shown that the sex of an individual appears to influence both the type of immune pathways affected by TCDD as well as the progression and severity of the autoimmunity. In summary, these studies clearly demonstrate that postnatal immune system impairment due to prenatal TCDD exposure is not limited to immunosuppression but also can include inappropriate immune activation manifested as a hypersensitivity that can lead to the onset of autoimmune disease. / Ph. D.

Perturbing gut microbiota early in life can lead to the development of autoimmunity. We are just beginning to unravel how early immune programming by microbiota may have long-term effects on noncommunicable diseases. In this thesis, we lay groundwork for programming of the immune system by commensal bacteria early in life through our studies on the induction of early endogenous neonatal IgA, and we evaluate Limosilactobacillus reuteri's characteristics as an inducer. Garnering attention for use a probiotic, L. reuteri has many proven health promoting benefits, such as IgA induction, but emerging evidence also links specific strains to autoimmune disease. "Super-induction" of neonatal IgA can be achieved through cross-fostering immunocompetent pups on immunocompromised dams. We found that this phenomenon was categorically due to transferal of microbes from dam to offspring. By comparing strain CF48-3A to the non-gastric-related organism L. oris, we discovered that L. reuteri is a microorganism that can enhance early neonatal IgA induction. Further investigations revealed that the ability to induce neonatal IgA is not ubiquitous in all L. reuteri strains, as ATCC PTA 6475 did not significantly elevate IgA. We discovered that 6475 has the antigenic ability to stimulate B cell differentiation and IgA production, but it is suppressed by a mechanism related to differences in surface architecture of this strain. L. reuteri strains also vary in their potency of aryl hydrocarbon receptor (AhR) stimulation. In mice, activation of AhR during gestation by a potent prototypical ligand, TCDD, leads to development of autoimmunity offspring. We found that TCDD exacerbated autoimmunity in adult mice using a strain of mice with similar AhR affinity to humans. Further investigations can clarify whether differential AhR ligand expression between L. reuteri strains contributes to the relationship between L. reuteri and autoimmunity. Overall, we conclude that differences between strains of L. reuteri have profoundly different immunological consequences that contribute to our understanding of the linkage between strains and autoimmunity. / Doctor of Philosophy / Differences in microbes transferred to infants through maternal routes shapes the early development of the immune system. In general, transferred microbes are healthy for the infant, and studies suggest that disruption of healthy microbes in the infant gut is linked to long-term health consequences, like autoimmune diseases. We found that a particular difference in maternally transferred microbes increases the early appearance of immunoglobulin A (IgA, a gut-related antibody) in neonatal mice, which is an antibody important for protecting against gut-related infections. We were able to link this early IgA production to a probiotic species Limosilactobacillus reuteri. Within the species classification as L. reuteri, several genetically different strains are health-promoting and broadly marketed over-the-counter for use in probiotic supplements for infants, children, and pregnant and nursing mothers. Emerging scientific evidence also points to a potential connection between other L. reuteri strains and autoimmune disease. Secreted products of genetically different L. reuteri strains have been discovered to activate aryl hydrocarbon receptor (AhR) with various potency. We used a prototypical AhR ligand and found exacerbation of autoimmune disease in adult mice. Thus, we have concluded that different strains of L. reuteri have broadly different effects on immune system development, and strain variability may explain the different effects on autoimmunity that have been observed.

Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that acetylates several proteins that are involved in the immune response. HDAC6 inhibition has been shown in various models to decrease inflammation by altering various proteins involved in the dysregulation of B and T cell responses. In our current studies we sought to determine if HDAC6 inhibition would decrease disease in lupus-prone mice using two murine mouse models of SLE: MRL/lpr mice and NZB/W F1 mice. Both mouse models were fed a rodent diet formulated with the selective HDAC6 inhibitor ACY-738 (N-hydroxy-2-(1-phenylcycloproylamino) pyrimidine-5-carboxamide). NZBW mice received 18 weeks of treatment starting at 16-weeks-of-age and had an average of 57.3 +/- 14.6 ng/mL of ACY-738 in the plasma. MRL/lpr mice received 7 weeks of treatment starting at 11-weeks-of-age and had an average of 78.5 +/- 17.3 ng/mL of ACY-738 in the plasma. Controls received either dexamethasone 5x a week or were left untreated. As the mice aged, body weight, urine protein, and blood sera was collected weekly. Spleen cells were isolated following euthanasia for flow cytometry and kidneys were also collected for histological analyses. We found that in both mouse models that mice treated with ACY-738 had reduced splenic weight and IgG immunoglobulin isotypes. MRL/lpr mice that were treated with ACY-738 had a reduction in the number of IL-17+, ROR-gamma-t TH17 cells. NZBW/ F1 mice that received ACY-738 treatment also had a reduction in the TH17 cells and we observed a significant reduction in kidney pathology. Selective HDAC6 targeting may warrant future investigations as a potential therapeutic target for the treatment of SLE. / Master of Science

No / Combinatorial antibody libraries were constructed from the spleen of a patient with concomitant systemic lupus erythematosus and idiopathic thrombocytopenia. Following selection of the libraries with DNA, a panel of 15 anti-DNA Fabs was isolated. Sequence analysis of these antibodies coupled with measurements of their affinities for ss- and dsDNA were used to investigate the role of somatic mutation in affinity maturation of the anti-DNA response. Examination of the germline genes used by these Fabs supports previous studies that suggest there is no restriction of the gene usage in the anti-DNA response. However, data are presented indicating that VH3 genes and the A27 V¿ paired with the J¿1 may be over-expressed in the anti-DNA repertoire. Analysis of the role of somatic mutation in increasing affinity for DNA indicates that affinity maturation has occurred and suggests that the CDR1 and CDR2 of the heavy chain are of importance in this process.

L’hépatite auto-immune (HAI) est une maladie auto-immune multifactorielle du foie de pathogenèse inconnue. Cette maladie est caractérisée par une perte de la tolérance immunologique envers des antigènes du soi hépatiques entraînant une destruction progressive du parenchyme hépatique en absence de traitements. La prednisone avec l’azathioprine est le traitement de première ligne pour les patients atteints d’HAI. Cette combinaison d’immunosuppresseurs permet de réduire l’inflammation du foie et permet d’induire une rémission chez environ 80 à 90% des patients. Bien que la majorité des patients atteints d’HAI répondent bien à long terme aux traitements disponibles, 10 à 20% de ceux-ci ne répondront pas aux traitements, développeront une cirrhose et pourront nécessiter éventuellement d’une greffe hépatique. Puisqu’à l’heure actuelle aucun test clinique, biologique ou histologique n’est capable de prédire s’il y aura réponse aux traitements et rémission chez les patients atteints d’HAI, il est important d'identifier des biomarqueurs capables de prédire la réponse au traitement et l'activité de la maladie chez ces patients. L’hypothèse de ce projet est que la rémission clinique et une réponse favorable au traitement chez les patients HAI sont influencées par des médiateurs immunologiques (cellules et/ou cytokines) qui, lorsqu’identifiés, pourraient être utilisés pour évaluer la réponse au traitement. Le but de ce projet est donc d’identifier un ou plusieurs biomarqueurs de l'activité de la maladie et de la réponse au traitement chez les patients HAI. Selon les résultats obtenus, il est possible de suggérer que l’IL-16 pourrait influencer l’activité de l’HAI en affectant à la fois les voies pro-inflammatoires et anti-inflammatoires. Les résultats obtenus suggèrent aussi que l’IL-10, l’IL-16 et certaines sous-populations de lymphocytes T CD4 effecteurs et régulateurs pourraient être des biomarqueurs potentiels de l'activité de la maladie et de la réponse au traitement chez les patients HAI. Cependant, des recherches supplémentaires sont nécessaires pour valider l'utilité de ces biomarqueurs dans la prédiction de la réponse au traitement et de l'activité de la maladie chez les patients atteints d’HAI. / Autoimmune hepatitis (AIH) is a multifactorial autoimmune liver disease of unknown pathogenesis characterized by a loss of immunological tolerance towards hepatic self-antigens leading to progressive destruction of the liver parenchyma in the absence of treatment. Prednisone with azathioprine is the first-line treatment for AIH patients. This combination of immunosuppressants helps reduce liver inflammation and induces remission in approximately 80 to 90% of patients. Although most AIH patients respond to available treatments, 10 to 20% will not respond to treatment and will develop cirrhosis and may eventually require a liver transplant. Currently, no clinical, biological, or histological parameters can predict the initial response to treatment or long-term remission of patients with AIH. Therefore, there is a crying need for reliable biomarkers capable of predicting response to treatment and disease activity in these patients. The hypothesis of this project is that clinical remission and a favorable response to treatment in AIH patients are influenced by immunological mediators (cells and/or cytokines) which, when identified, could be used to evaluate the response to treatment. The aim of this project is therefore to identify biomarker(s) of disease activity and treatment response in AIH patients. According to our results, we believe that IL-16 could influence AIH activity by affecting both proinflammatory and anti-inflammatory pathways. Our results also suggest that IL-10, IL-16 and subpopulations of effector and regulatory CD4 T cells could be putative biomarkers of disease activity and response to treatment in AIH patients. However, further research is needed to validate the usefulness of these biomarkers in predicting treatment response and disease activity in AIH patients.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by abnormalities in multiple components of the immune system resulting in progressive damage to multiple organs. Current treatments for SLE are often intensive and result in side effects and the potential for continued flares and progression of disease. Histone deacetylase (HDAC) enzymes control multiple cellular functions by removing acetyl groups from lysine residues in various proteins. HDAC inhibitors have been investigated as a potential treatment for SLE with promising results, however selective HDAC6 inhibition (HDAC6i) has become a leading candidate for pharmacologic inhibition to reduce the potential for side effects. We hypothesize that HDAC6i will decrease SLE disease by targeting substrates of HDAC6 in multiple components of immunity and organ systems. NZB/W mice were treated with ACY-738 or ACY-1083, followed by evaluation of multiple disease parameters and mechanisms involved in disease pathogenesis within the kidney, bone marrow, and spleen. Within the kidney, HDAC6i decreased glomerular pathology scores, proteinuria, and IgG and C3 deposition. Within glomerular cells, HDAC6i increased alpha-tubulin acetylation and decreased nuclear NF-κB. Within the spleen, there was a dose-dependent decrease in the frequency of Th17 cells and a mild decrease in the frequency of Treg cells. Concurrently, there were decreased levels of IL-12/IL-23 and minimal decreases in TGF-β in the serum. Within the bone marrow, B cell development through Hardy fractions exhibited accelerated progression through later stages as NZB/W mice aged. This accelerated progression may allow B cells to bypass important regulatory checkpoints in maintaining immune tolerance and contribute to autoimmunity. Treatment with an HDAC6i corrected the aberrant B cell development in the bone marrow and RNAseq analysis unveiled six genes (Cebpb, Ccr9, Spib, Nfil3, Lgals1, and Pou2af1) that may play a role in the aforementioned abnormalities. Overall, these findings show that HDAC6i decreased disease in NZB/W mice by targeting multiple components of the immune response, including glomerular cells, T cell subsets in the spleen, and bone marrow B cells. In conclusion, selective HDAC6i is an excellent candidate for pharmacologic therapy for SLE because it targets multiple immune abnormalities involved in SLE pathogenesis while remaining selective and safe. / Ph. D. / Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by multiple abnormalities in the immune system resulting in progressive immune-mediated damage to multiple organs. Current treatment regimens are often intensive, result in side effects, and may only provide temporary relief of disease. Histone deacetylase (HDAC) inhibition is currently being investigated as a new treatment modality for SLE with aims for improved efficacy and decreased potential for unwanted side effects. HDAC enzymes remove acetyl groups from multiple proteins (substrates) and subsequently regulate their function. HDAC6 is a specific HDAC enzyme that is of particular interest and are the subject of the following studies. These studies hypothesize that HDAC6 inhibition will decrease SLE by targeting multiple protein targets involved in the immune-mediated pathway of disease initiation and progression. NZB/W mice were utilized as a model of the human disease, and were treated by HDAC6 inhibitors during various stages of disease progression. Long-term treatment initiated early in disease decreases disease as evidenced by decreased renal pathology scores, immune complex deposition in the kidneys, decreased T cell subtypes in the spleen, and decreased inflammatory cytokines. HDAC6 inhibition corrects abnormal B cell development within the bone marrow of NZB/W mice, which is otherwise altered during disease progression. Furthermore, HDAC6 inhibition altered gene expression within the bone marrow, and deep sequencing analysis revealed multiple genes that may be involved in the pathway of disease progression. Overall, HDAC6 inhibition targets multiple pathways involved in SLE disease initiation and progression in various organs including the bone marrow, spleen, and kidneys. Because SLE is a disease that is multi-factorial and effects multiple organs, it would be ideal that a potential drug therapy also targets multiple targets and organ systems while remaining safe to use. Based on these studies, HDAC6 inhibitors are excellent candidates for the treatment of SLE.

T cells chronically stimulated by their antigen often become dysfunctional and lose effector functions and proliferative capacity. This state of unresponsiveness is referred as T cell exhaustion. In order to investigate this, we developed a laboratory model, which allowed us to stimulate chronically in vivo a monoclonal population of CD4+ T cells. This model is based on the adoptive transfer of TCR transgenic CD4+ T cells specific for the male mHAg into male recipient mice. We found that systemic exposure to the male antigen modified deeply anti-male TcR-transgenic CD4+ T cells, plunging them into a state of functional unresponsiveness. Microarray analysis revealed that, in comparison with naive T cells, transferred T cells displayed a gene expression profile very similar to that of virus-specific exhausted CD8+ T cells. Moreover, like exhausted CD8+ T cells, exhausted CD4+ T cells lost their capacity to secrete IFN-ã as well as to proliferate in response to antigen stimulation, and T cell unresponsiveness was controlled by the engagement of programmed death receptor 1 (PD-1) present at the surface of T cells. <p>MicroRNAs are key molecules in shaping T cell function. In order to explore the possibility that chronic antigenic stimulation could shape the pool of microRNAs in exhausted anti-male CD4+ T cells that would account for specific changes in protein synthesis, we compared by microarray analysis the specific expression of microRNAs in naive CD4+ T cells and exhausted CD4+ T cells. Ninety five of them were found differentially expressed, among which, microRNA-155 (miR-155) displayed one of the highest changes. To identify the importance of miR-155 in T cell exhaustion, we analyzed miR-155-deficient CD4+ T cells after chronic exposure to systemic antigen. We found that, chronically-stimulated miR-155-/- CD4+ T cells were retained in a deeper state of unresponsiveness than miR-155+/+ CD4+ T cells. Furthermore, inhibition of PD-1/PD-L1 interaction did not promote antigen-dependent expansion of miR-155-deficient CD4+ T cells, nor did it stimulate T cell inflammation of several organs, contrary to what was observed in mice that received miR-155-sufficient CD4+ T cells. Thus, our observations demonstrated that miR-155 deficiency played a dominant role over PD-1-mediated inhibition of T cells and that miR-155 was required for restoring function in exhausted CD4+ T cells.<p>Next, we explored the mechanism by which exhausted miR-155-/- CD4+ T cells were kept in a deeper unresponsiveness state than miR-155+/+ counterparts. By comparative microarray analysis of gene expression between exhausted miR-155+/+ CD4+ T cells and miR-155-/- CD4+ T cells, heme oxygenase 1 (HO-1) was identified as a specific target of miR-155. Finally, inhibition of HO-1 activity restored the capacity of exhausted miR-155-/- CD4+ T cells to promote autoimmune inflammation in adoptively-transferred recipients. <p>Taken together, our study identified miR-155-mediated regulation of protein expression as a critical factor for restoring function in exhausted CD4+ T cells. Our results also present regulation of HO-1 expression in T cells as one of the mechanisms by which miR-155 promote T cell-driven inflammation. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Human endogenous retroviruses are the remnants of ancient retroviral infections present within our genome. These molecular fossils show similarities with present day exogenous retroviruses but act as typical Mendelian elements that are passed vertically between generations. Despite being repeatedly linked to a number of autoimmune diseases and disorders, no conclusive proof has been identified. Rheumatoid arthritis (RA) is one such disease which has been associated with an increase in HERV expression, compared to controls. In order to elucidate a clear role for HERVs in RA pathogenesis, autoantigens implicated in disease pathogenesis were scanned for sequence homology to retroviral genes. Such epitopes would induce antibodies cross reactive with host proteins, resulting in disease. Short peptides mimicking these regions were synthesised and the prevalence of anti-HERV antibodies was determined in RA patients and disease controls. Additionally, a novel real-time Polymerase Chain Reaction (PCR) assay was developed to accurately quantify levels of HERV-K (HML-2) gag expression, relative to normalised levels of housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) activity in RA patients compared to disease controls with CD4+ lymphocytes harbouring the highest activity. The real-time assay was also used to determine whether factors within the synovium could modulate HERVs, resulting in their upregulation. Exogenous viral protein expression and pro-inflammatory cytokines were shown to exert a significant modulatory effect over HERV-K (HML-2) transcription. From this data, it is clear that RA patients have increased levels of HERV-K (HML-2) gag activity compared to controls. Despite this it is likely that factors within the synovium such as exogenous viral expression and pro-inflammatory cytokines also influence HERV-K (HML-2) transcription possibly contributing to a role of bystander activation, i.e. being influenced by external factors, rather than actively contributing to disease processes. The exact role of HERVs in RA pathology remains elusive; however this research proposes several mechanisms by which HERV-K (HML-2) may contribute to disease.