Photoaging and skin cancer: Is the inflammasome the missing link?

Photoaging and epithelial skin tumorigenesis are complex processes triggered mainly by UV radiation from chronic sun exposure. This leads to DNA damage and reactive oxygen species (ROS) production, which initiate an inflammatory response that alters cell structure and function. Changes in cell homeostasis and ROS production activate intracellular multiprotein platforms called inflammasomes. Inflammasomes nucleate around cytoplasmic receptors mainly of the NLR (nucleotide-binding domain and leucine-rich repeat) family and regulate caspase-1-dependant secretion of pro-inflammatory interleukin (IL)1β and IL18 cytokines, and an inflammatory form of death named pyroptosis. NLRP1 inflammasomes have taken centre stage in skin biology, as mutations in NLRP1 underlie the genetic etiology of dermatological diseases and increase the susceptibility to skin cancer. Targeting inflammasome(s) might be an important approach to improve skin inflammation, photoaging and reduce the risk of epithelial skin tumorigenesis. In this context, we discuss the potential implication of NLRP1 and NLRP3 inflammasomes

Bases moléculaires et cellulaires des maladies autoinflammatoires systémiques, y compris la maladie de Still

Les maladies auto-inflammatoires systémiques (SAIDs) sont des maladies rares caractérisées par des attaques fébriles récurrentes et une inflammation systémique stérile. Leur étiologie est expliquée par des mutations dans des gènes induisant une dérégulation du système immunitaire. Les travaux présentés dans cette thèse visaient à (i) étudier les conséquences fonctionnelles d’un variant NLRP3 identifié dans deux familles distinctes, (ii) identifier les bases moléculaires de l’urticaire inflammatoire chronique et (iii) étudier les bases moléculaires et cellulaires de la maladie de Still de l’adulte (MSA). Nous avons identifié un variant NLRP3 (c.1322C>T, p.A441V) présent à l’état hétérozygote dans une famille française multi-générationnelle et chez un cas sporadique, présentant deux phénotypes NLRP3-AID différents. Des tests fonctionnels in vitro ont montré que la protéine NLRP3 mutée induit une augmentation significative de la formation de specks ASC et de la sécrétion d’IL1β, deux résultantes de l’activation de l’inflammation. Afin d’identifier chez deux cas sporadiques, les bases moléculaires de l’urticaire inflammatoire chronique associé à une inflammation systémique, des analyses de séquençage NGS ont été effectuées. Un variant NLRP3 présent à l’état de mosaïque somatique a été identifié chez les deux patients. Les études fonctionnelles in vitro du variant ont démontré un effet gain de fonction sur l’activation de l’inflammasome NLRP3. Le profil de cytokines plasmatiques chez les patients avec une MSA a révélé des taux d’IL18 très élevés. Des premiers résultats suggèrent que les monocytes peuvent jouer un rôle important dans la pathogenèse de la maladie.Systemic autoinflammatory diseases (SAIDs) are rare disorders characterized by recurrent febrile attacks and systemic sterile inflammation. Mutations in genes that cause dysregulation of the innate immune system underlie the etiology of several SAIDs. The work presented in this thesis aimed at (i) Studying the functional consequences of an identified NLRP3 variant in two apparently unrelated NLRP3-AID families (ii) identifying the molecular bases of chronic inflammatory urticaria and (iii) studying the molecular and cellular bases of AOSD We identified a heterozygous missense NLRP3 variant (c.1322C>T, p.A441V) in a multigenerational French family and in a sporadic case presenting with two different NLRP3-AID phenotypes. In vitro functional assays showed that the mutated NLRP3 protein is associated with significantly higher ASC speck formation and IL1β secretion, two major readouts of inflammasome activation. In order to identify the molecular bases of histamine-resistant chronic urticarial skin rash associated with systemic inflammation in two sporadic cases, next generation sequencing was performed. A somatic mosaic NLRP3 variant was identified in both patients. Despite the late-disease onset, the mutations were widely distributed in different patients’ cells. In vitro functional studies of the identified variants clearly demonstrated a gain-of-function effect on NLRP3-inflammasome activation. Plasma cytokine profile in AOSD patients revealed significantly elevated plasma IL18 levels as compared to healthy controls. Primary results from cytokine profile in AOSD patients’ monocytes and macrophages suggested that monocytes may play an important role in disease pathogenesi

Photoaging and skin cancer: Is the inflammasome the missing link?

International audiencePhotoaging and epithelial skin tumorigenesis are complex processes triggered mainly by UV radiation from chronic sun exposure. This leads to DNA damage and reactive oxygen species (ROS) production, which initiate an inflammatory response that alters cell structure and function. Changes in cell homeostasis and ROS production activate intracellular multiprotein platforms called inflammasomes. Inflammasomes nucleate around cytoplasmic receptors mainly of the NLR (nucleotide-binding domain and leucine-rich repeat) family and regulate caspase-1-dependant secretion of pro-inflammatory interleukin (IL)1β and IL18 cytokines, and an inflammatory form of death named pyroptosis. NLRP1 inflammasomes have taken centre stage in skin biology, as mutations in NLRP1 underlie the genetic etiology of dermatological diseases and increase the susceptibility to skin cancer. Targeting inflammasome(s) might be an important approach to improve skin inflammation, photoaging and reduce the risk of epithelial skin tumorigenesis. In this context, we discuss the potential implication of NLRP1 and NLRP3 inflammasome

Inflammasome biology, molecular pathology and therapeutic implications

International audienceInflammasomes are intracellular multiprotein signaling complexes, mainly present in myeloid cells. They commonly assemble around a cytoplasmic receptor of the nucleotide-binding leucine-rich repeat containing receptor (NLR) family, although other cytoplasmic receptors like pyrin have been shown to form inflammasomes. The nucleation of the multiprotein scaffolding platform occurs upon detection of a microbial, a danger or a homeostasis pattern by the receptor that will, most commonly, associate with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) through homotypic domain interactions resulting in recruitment of procaspase-1. This will lead to the autoproteolytic activation of caspase-1, which regulates the secretion of proinflammatory IL1β and IL18 cytokines and pyroptosis, a caspase-1-mediated form of cell death. Pyroptosis occurs through cleavage of Gasdermin D, a membrane pore forming protein. Recently, non-canonical inflammasomes have been described, which directly sense intracellular pathogens through caspase-4 and -5 in humans, leading to pyroptosis. Inflammasomes are important in host defense; however, a deregulated activity is associated with a number of inflammatory, immune and metabolic disorders. Furthermore, mutations in inflammasome receptor coding genes are causal for an increasing number of rare autoinflammatory diseases. Biotherapies targeting the products of inflammasome activation as well as molecules that directly or indirectly inhibit inflammasome nucleation and activation are promising therapeutic areas. This review discusses recent advances in inflammasome biology, the molecular pathology of several inflammasomes, and current therapeutic approaches in autoinflammatory diseases and in selected common multifactorial inflammasome-mediated disorders

Expression of SAA1, SAA2 and SAA4 genes in human primary monocytes and monocyte-derived macrophages

International audienceCirculating serum amyloid A (SAA) is increased in various inflammatory conditions. The human SAA protein family comprises the acute phase SAA1/SAA2, known to activate a large set of innate and adaptive immune cells, and the constitutive SAA4. The liver synthesis of SAA1/SAA2 is well-established but there is still an open debate on extrahepatic SAA expression especially in macrophages. We aimed to investigate the ability of human primary monocytes and monocyte-derived macrophages to express SAA1, SAA2 and SAA4 at both the transcriptional and protein levels, as previous studies almost exclusively dealt with monocytic cell lines. Monocytes and derived macrophages from healthy donors were stimulated under various conditions. In parallel with SAA, pro-inflammatory IL1A, IL1B and IL6 cytokine expression was assessed. While LPS alone was non-effective, a combined LPS/dexamethasone treatment induced SAA1 and to a lesser extent SAA2 transcription in human monocytes and macrophages. In contrast, as expected, pro-inflammatory cytokine expression was strongly induced following stimulation with LPS, an effect which was dampened in the presence of dexamethasone. Furthermore, in monocytes polarized towards a pro-inflammatory M1 phenotype, SAA expression in response to LPS/dexamethasone was potentiated; a result mainly seen for SAA1. However, a major discrepancy was observed between SAA mRNA and intracellular protein levels under the experimental conditions used. Our results demonstrate that human monocytes and macrophages can express SAA genes, mainly SAA1 in response to an inflammatory environment. While SAA is considered as a member of a large cytokine network, its expression in the monocytes-macrophages in response to LPS-dexamethasone is strikingly different from that observed for classic pro-inflammatory cytokines. As monocytes-macrophages are major players in chronic inflammatory diseases, it may be hypothesized that SAA production from macrophages may contribute to the local inflammatory microenvironment, especially when macrophages are compactly organized in granulomas as in sarcoidosis

Expression des gènes SAA par les monocytes et macrophages humains

National audienceLes taux des protéines serum amyloid A (SAA) sont augmentés par diverses conditions inflammatoires. Chez l’Homme, la famille de protéines SAA comporte les protéines de phase aigüe SAA1/SAA2, connues pour activer de nombreuses cellules immunitaires, et la protéine constitutive SAA4. La synthèse hépatique des protéines SAA1/SAA2 est bien établie mais une expression extra-hépatique est encore débattue, particulièrement dans les macrophages. De plus, des auteurs ont mis en évidence la présence d’ARNm SAA dans des cellules spumeuses de plaques d’athéromes. Nous avons étudié la capacité des monocytes et macrophages dérivés de monocytes humains à exprimer SAA1, SAA2 et SAA4, au niveau transcriptionnel et protéique, tandis que les études précédentes ont principalement investigué des lignées monocytaires. Des monocytes et macrophages dérivés de monocytes de donneurs sains ont été traités par différentes conditions. L’expression des cytokines pro-inflammatoires IL1A, IL1B et IL6 a été étudiée en parallèle de l’expression des gènes SAA. Nous avons mis en évidence l’expression du gène SAA1, et de SAA2 à une moindre mesure, lorsque les cellules sont simultanément traitées par un agent pro-inflammatoire et un agent anti-inflammatoire. L’agent pro-inflammatoire seul n’avait pas d’effet sur l’expression des gènes SAA, bien qu’il induise, comme attendu, l’expression des cytokines pro-inflammatoires, effet amoindri par l’ajout du stimulus anti-inflammatoire. De plus, la polarisation des monocytes en phénotype pro-inflammatoire M1 potentialisait l’expression des gènes SAA, principalement SAA1. Nous avons cependant observé un écart entre les taux d’ARNm SAA et les taux intracellulaires de protéines. Nos résultats montrent que les monocytes et macrophages humains peuvent exprimer les gènes SAA, principalement SAA1, dans un environnement inflammatoire. Les monocytes et macrophages étant des acteurs majeurs de maladies inflammatoires, il est possible d’émettre l’hypothèse que la production de SAA par les monocytes et macrophages peut contribuer à la persistance d’un microenvironnement local inflammatoire et à la formation des plaques d’athéromes

Expression des gènes SAA par les monocytes et macrophages humains

National audienceLes taux des protéines serum amyloid A (SAA) sont augmentés par diverses conditions inflammatoires. Chez l’Homme, la famille de protéines SAA comporte les protéines de phase aigüe SAA1/SAA2, connues pour activer de nombreuses cellules immunitaires, et la protéine constitutive SAA4. La synthèse hépatique des protéines SAA1/SAA2 est bien établie mais une expression extra-hépatique est encore débattue, particulièrement dans les macrophages. De plus, des auteurs ont mis en évidence la présence d’ARNm SAA dans des cellules spumeuses de plaques d’athéromes. Nous avons étudié la capacité des monocytes et macrophages dérivés de monocytes humains à exprimer SAA1, SAA2 et SAA4, au niveau transcriptionnel et protéique, tandis que les études précédentes ont principalement investigué des lignées monocytaires. Des monocytes et macrophages dérivés de monocytes de donneurs sains ont été traités par différentes conditions. L’expression des cytokines pro-inflammatoires IL1A, IL1B et IL6 a été étudiée en parallèle de l’expression des gènes SAA. Nous avons mis en évidence l’expression du gène SAA1, et de SAA2 à une moindre mesure, lorsque les cellules sont simultanément traitées par un agent pro-inflammatoire et un agent anti-inflammatoire. L’agent pro-inflammatoire seul n’avait pas d’effet sur l’expression des gènes SAA, bien qu’il induise, comme attendu, l’expression des cytokines pro-inflammatoires, effet amoindri par l’ajout du stimulus anti-inflammatoire. De plus, la polarisation des monocytes en phénotype pro-inflammatoire M1 potentialisait l’expression des gènes SAA, principalement SAA1. Nous avons cependant observé un écart entre les taux d’ARNm SAA et les taux intracellulaires de protéines. Nos résultats montrent que les monocytes et macrophages humains peuvent exprimer les gènes SAA, principalement SAA1, dans un environnement inflammatoire. Les monocytes et macrophages étant des acteurs majeurs de maladies inflammatoires, il est possible d’émettre l’hypothèse que la production de SAA par les monocytes et macrophages peut contribuer à la persistance d’un microenvironnement local inflammatoire et à la formation des plaques d’athéromes

Mosaic variants in TNFRSF1A : an emerging cause of tumour necrosis factor receptor-associated periodic syndrome

International audienceObjective To identify the molecular basis of a systemic autoinflammatory disorder (SAID) evocative of TNF receptor-associated periodic syndrome (TRAPS). Methods (i) Deep next generation sequencing (NGS) through a SAID gene panel; (ii) variant allele distribution in peripheral blood subpopulations; (iii) in silico analyses of mosaic variants using TNF receptor superfamily 1A (TNFRSF1A) crystal structure; (iv) review of the very rare TNFRSF1A mosaic variants reported previously. Results In a 36-year-old man suffering from recurrent fever for 12 years, high-depth NGS revealed a TNFRSF1A mosaic variant, c.176G>A p.(Cys59Tyr), which Sanger sequencing failed to detect. This mosaic variant displayed a variant allele fraction of 14% in whole blood; it affects both myeloid and lymphoid lineages. p.(Cys59Tyr), a recurrent germline pathogenic variant, affects a crucial cysteine located in the first cysteine-rich domain (CRD1) and involved in a disulphide bridge. Introduction of a tyrosine at this position is expected to disrupt the CRD1 structure. Review of the three previously reported TNFRSF1A mosaic variants revealed that they are all located in a small region of CRD2 and that germinal cells can be affected. Conclusion This study expands the localization of TNFRSF1A mosaic variants to the CRD1 domain. Noticeably, residues involved in germline TNFRSF1A mutational hot spots can also be involved in post-zygotic mutational events. Including our study, only four patients have been thus far reported with TNFRSF1A mosaicism, highlighting the need for a high-depth NGS-based approach to avoid the misdiagnosis of TRAPS. Genetic counselling has to consider the potential occurrence of TNFRSF1A mosaic variants in germinal cells

A critical region of A20 unveiled by missense TNFAIP3 variations that lead to autoinflammation

International audienceA20 haploinsufficiency (HA20) is an autoinflammatory disease caused by heterozygous loss-of-function variations in TNFAIP3 , the gene encoding the A20 protein. Diagnosis of HA20 is challenging due to its heterogeneous clinical presentation and the lack of pathognomonic symptoms. While the pathogenic effect of TNFAIP3 truncating variations is clearly established, that of missense variations is difficult to determine. Herein, we identified a novel TNFAIP3 variation, p.(Leu236Pro), located in the A20 ovarian tumor (OTU) domain and demonstrated its pathogenicity. In the patients’ primary cells, we observed reduced A20 levels. Protein destabilization was predicted in silico for A20_Leu236Pro and enhanced proteasomal degradation was confirmed in vitro through a flow cytometry-based functional assay. By applying this approach to the study of another missense variant, A20_Leu275Pro, for which no functional characterization has been performed to date, we showed that this variant also undergoes enhanced proteasomal degradation. Moreover, we showed a disrupted ability of A20_Leu236Pro to inhibit the NF-κB pathway and to deubiquitinate its substrate TRAF6. Structural modeling revealed that two residues involved in OTU pathogenic missense variations (i.e. Glu192Lys and Cys243Tyr) establish common interactions with Leu236. Interpretation of newly identified missense variations is challenging, requiring, as illustrated here, functional demonstration of their pathogenicity. Together with functional studies, in silico structure analysis is a valuable approach that allowed us (i) to provide a mechanistic explanation for the haploinsufficiency resulting from missense variations and (ii) to unveil a region within the OTU domain critical for A20 function