| Abstract |
Autoimmune diseases develop upon aberrant activation of lymphocytes mainly due
to failure of self-tolerance mechanisms. Both innate and adaptive immune system
over-activation contribute to autoimmunity and tissue inflammation. Rheumatoid
arthritis (RA) is considered as an autoimmune disease against post-translationally
modified proteins, characterized by uncontrolled inflammation. Genetic data have
revealed CD4+ lymphocytes and dendritic cells (DCs) as important contributors to the
breach of immunologic tolerance and development of autoimmunity in RA patients.
Currently biologic therapies (bDMARDs) applied in clinical practice, mainly target the
inflammatory process, such as anti-tumor necrosis factor α (TNF-α)- agents,
Interleukin 6 receptor (IL-6R) and JAK-STATs inhibitors, or target the adaptive immune
response via B-cell depletion (anti- CD20 antibodies) and T-cell co-stimulation
molecule blockade (CTLA4-Ig). Nevertheless, data from registries and cohort studies
have shown that in clinical practice, 50-60% of RA patients starting a bDMARD will
stop treatment due to inefficacy or toxicity in the long-term, while clinical response,
remission or low disease activity, is unpredictable and is achieved by 20-40% of
patients. Development of predictive markers of response for everyday clinical
practice will be an important step in optimizing the treatment of autoimmune
diseases.
DCs drive the immune responses through their crucial role in the antigen presentation
process. The ability of DCs to modulate T-cell responses has made them an interesting
target of study for the immunotherapy of autoimmune diseases. Until now, human
DC-based therapeutic strategies have mainly relied on ex vivo generated DCs
differentiated from peripheral blood (PB) monocytes and have demonstrated limited
efficacy in clinical trials. Thus, identifying alternative therapeutic strategies that aim
at manipulating human DCs towards an immunoregulatory phenotype may yield
improved treatments.
Strategies to manipulate DCs to exploit their immunoregulatory potential are
currently under investigation. Cytotoxic T lymphocyte antigen 4 (CTLA4) is a negative costimulatory molecule expressed on the T cell membrane and its significant
immunoregulatory role in the context of autoimmune diseases is confirmed through
the clinical benefit upon CTLA4-Ig treatment (abatacept) in patients with RA. Several
studies support that CTLA4 delivers reverse signals on DCs, thus reducing their
inflammatory function and enhancing their tolerogenic phenotype. Nevertheless, the
molecular mechanisms that are involved in the induction of the tolerogenic
phenotype of CTLA4-mediated effects on DCs, have not been extensively studied.
In this study we aimed to identify biomarkers that predict clinical responses to
etanercept and abatacept, based on either demographic and clinical characteristics or
a detailed immunological profiling of PB of RA patients, respectively. We assessed the
levels of pathogenic and regulatory cell populations, as well as the sera transcriptome
of abatacept-treated RA patients at baseline, on 3rd and 6th month after treatment
initiation in order identify a signature of cell populations and sera proteins, that are
associated with clinical responses to abatacept. Furthermore, we used CTLA4-Ig as a
tool to generate in vitro tolerogenic DCs (tolDCs) and we investigated the intracellular
pathways that are implicated in the induction of the tolerogenic phenotype in CTLA4-
treated DCs. To address this, human monocytes from healthy donors were isolated
and differentiated into DCs. DCs were cultured with CTLA4-Ig and subjected to
genome-wide transcriptomics. The anti-inflammatory function of DCs was further
assessed.
In the observational study, we showed that approximately half of the patients starting
etanercept discontinued within the first year of therapy. We observed that treatment
inefficacy, which was the most frequent cause of therapy disruption, was associated
with female gender, obesity and higher baseline swollen joint count. Moreover, we
highlighted that the groups of patients that improved disease activity were
overrepresented by males, patients had shorter disease duration and better
functional status. Next, focusing on the immunological studies, we found a strong
association of high levels of T helper 1 cells (Th1), myeloid- derived suppressor cells
(MDSCs) and DCs with a better response to abatacept treatment at 6 months.
Moreover, proteomics analysis revealed 10 amongst 303 proteins in peripheral serum
to have differential expression according to clinical responses at 6 months. Interestingly, a composite index based on the above described cellular and protein
markers showed a high discriminative ability to predict response to abatacept.
Furthermore, we revealed that CTLA4-Ig-treated DCs demonstrated significantly
upregulated transcriptional levels of anti-inflammatory gene IL10. However, the
expression of inflammatory cytokines IL6 and TNFα were decreased, accompanied
with reduced T cell proliferation in co-culture experiments, highlighting the
immunosuppressive properties of CTLA4-Ig-treated DCs. Interestingly, transcriptomic
analysis revealed 1270 differentially expressed genes in CTLA4-mediated tolDCs, the
majority of them participated in metabolic processes, specifically in OXPHOS pathway
and mitochondrial function. Notably, we confirmed that tolDCs had lower basal
OXPHOS and decreased ATP production via Seahorse assays. Furthermore, expression
of phosphorylated mammalian target of rapamycin (mTOR) and AKT1, central
regulators of metabolism, were increased in CTLA4-mediated tolDCs. Mechanistically,
we showed that mTOR controlled the expression of the transcription factor Sp1, which
enhanced the expression of anti-inflammatory gene IL10, thus contributing to the
regulatory function of CTLA4-mediated tolDCs.
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