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Theses Year : 2014

Pathophysiology of hereditary recurrent fever syndromes: cellular and molecular approaches



Hereditary recurrent fevers (HRFs) are a group of rare monogenic autoinflammatory disorders characterized by sterile self-directed inflammation, pathogenic autoantibodies or activated T lymphocytes. Clinical signs include recurrent episodes of fever, accompanied by systemic inflammation (high plasma levels of acute phase reactants) associated with localized inflammation mainly in serosal membranes. The major long-term complication in HRFs is amyloidosis mainly in kidneys, which can progress to renal failure. Despite the discovery, in several HRFs, of molecular defects in various innate immunity genes (e.g. NLRP3, NLRP12 or MEFV), the cellular consequences of these sequence variations are unknown. The pathogenicity of several identified sequence variations is even controversial, and the disease remains genetically unexplained in the majority (70‐80%) of patients. On a fundamental level, although a number of easily transfectable cell lines exist, they do not represent physiologically relevant study models, as the patients’ cells that express disease-causing mutations are mainly of myelomonocytic origin (monocytes & neutrophils) and so far particularly difficult to transfect. The pathophysiological role of the encoded proteins and the signaling pathways linking the molecular defects to increased IL-1β secretion (hallmark of HRFs) are still poorly understood During this thesis, we aimed to develop a disease-relevant cellular model based on human primary cells in order to decipher the functional consequences of the identified HRF mutations and to characterize the molecular and signaling networks to which these proteins belong. In parallel, we aimed to identify new genes involved in HRFs. Following substantial efforts, we first developed a disease-specific cellular model, using human primary monocyte-derived macrophages, which express the HRF-related genes as well as the molecular cascades believed to be involved in the pathophysiology of HRFs. We set up the conditions to express recombinant wild-type NLRP3 (also known as cryopyrin) or pyrin (encoded by MEFV) as well as the most frequent disease-causing mutations of NLRP3 or MEFV in monocyte-derived macrophages, and tested their functional consequences on different signaling pathways. NLRP3 is down-regulated during in-vitro differentiation of human monocytes to macrophages. In macrophages, NLRP3 co-localizes with the centrosome marker γ-tubulin indicating that it may interact with components of the cytoskeleton. Expression of recombinant NLRP3 in human monocyte-derived macrophages resulted in lower expression of the D303N and R260W mutants associated with cryopyrin-associated periodic syndromes (or CAPS, a subgroup of HRFs) suggesting that either they are more prone to degradation or that they may be secreted. Expression of either wild-type or mutated pyrin carrying the most common disease-causing mutations (M694V or V726A involved in familial Mediterranean fever or FMF) was shown to induce cell death and oligomerization/speck formation of the intracellular adaptor protein ASC. Interestingly, ASC oligomers were found in the cytoplasm and near the cell membrane, suggesting that ASC may be secreted. Somewhat unexpectedly, we were indeed able to detect ASC in the supernatants of the transfected monocyte-derived macrophages. Expression of pyrin-M694V was shown to activate the endoplasmic reticulum (ER) stress signaling pathway and to increase production of the pro-inflammatory cytokines IL-1β, IL-8 and TNF-α. Ex-vivo studies on monocytes isolated from HRF patients resulted in increased expression of ER stress-related genes (GRP78, spliced XBP1 and DDIT3/CHOP) and pro-inflammatory cytokines (IL-1B, IL-8, TNFA). Overall, these results demonstrate that ER stress can be a disease-contributing mechanism in FMF. We also showed that plasma levels of IL-18 are elevated in crisis-free FMF patients. In the same line, we found that the levels of this pro-inflammatory cytokine were extremely high in a FMF patient who developed chronic myelomonocytic leukemia leading to an uncontrolled and fatal inflammatory syndrome, thereby unveiling the interplay between two different disorders involving the same target cells. In order to identify new genes involved in HRFs, whole-exome sequencing combined with homozygosity mapping was performed on the genomic DNA of a consanguineous patient with a clinical picture of HRF. Analysis of the exome data led to the identification of a homozygous missense variation in TBC1D23 (c.1030C>T; p.R344C). This evolutionarily conserved gene, which is expressed in blood monocytes and tissue macrophages, plays an important role in the spatiotemporal inhibition of innate immunity signaling in response to microbial pathogens. Using human peripheral blood mononuclear cells transfected with an expression plasmid encoding TBC1D23, we showed that this sequence variation diminishes the ability of TBC1D23 to inhibit LPS-induced cytokine production, thereby suggesting that the R344C missense variation in TBC1D23 is responsible for the disease. In conclusion, we anticipate that the set-up of this disease-relevant model based on transfected human macrophages should represent a starting point for future studies designed to expand our knowledge on the molecular and cellular bases of HRFs, which may pave the way for improved/new therapeutic strategies in these diseases.
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tel-03934824 , version 1 (11-01-2023)


  • HAL Id : tel-03934824 , version 1


Fawaz Awad. Pathophysiology of hereditary recurrent fever syndromes: cellular and molecular approaches. Human genetics. Université Pierre et Marie Curie, 2014. English. ⟨NNT : ⟩. ⟨tel-03934824⟩
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