Regulatory T-cell dysfunctions are associated with increase in tumor necrosis factor α in autoimmune hemolytic anemia and participate in Th17 polarization

Warm autoimmune hemolytic anemia (wAIHA) is a rare acquired autoimmune disease mediated by antibodies targeting red blood cells. The involvement of CD4 T-helper cells has been scarcely explored, with most findings extrapolated from animal models. Here, we performed quantification of both effector T lymphocytes (Teff) and regulatory T cells (Treg), associated with functional and transcriptomic analyses of Treg in human wAIHA. We observed a shift of Teff toward a Th17 polarization concordant with an increase in serum interleukin-17 concentration that correlates with red blood cell destruction parameters, namely lactate dehydrogenase and bilirubin levels. A decrease in circulating Treg, notably effector Treg, associated with a functional deficiency, as represented by their decrease capability to inhibit Teff proliferation, were also observed. Treg deficiency was associated with a reduced expression of Foxp3, the master transcription factor known to maintain the Treg phenotype stability and suppressive functions. Transcriptomic profiling of Treg revealed activation of the tumor necrosis facto (TNF)-α pathway, which was linked to increased serum TNF-α concentrations that were twice as high as in controls. Treg transcriptomic profiling also suggested that post-translational mechanisms possibly accounted for Foxp3 downregulation and Treg dysfunctions. Since TNF-α participates in the rupture of immune tolerance during wAIHA, its inhibition could be of interest. To this end, the effects of fostamatinib, a SYK inhibitor, were investigated in vitro, and we showed that besides the inhibition of erythrocyte phagocytosis by monocytes, fostamatinib is also able to dampen TNF-α production, thus appearing as a promising multitargeting therapy in wAIHA (clinicaltrials gov. Identifier: NCT02158195)

HuMoSC, une nouvelle thérapie pour le traitement des maladies dysimmunitaires et fibrosantes

Our team has developed an experimental method to generate immunosuppressive cells of monocyte origin called Human Monocyte-derived Suppressor Cells (HuMoSC). Although their immunosuppressive properties have already been shown, no study had yet evaluated their interest in tissue remodeling.The objective of this thesis was to evaluate the therapeutic potential of HuMoSCs and their supernatant for the treatment of tissue remodeling. On the other hand, we sought to identify the mechanisms of action involved in the functions of the supernatant of HuMoSC. The effect of HuMoSCs was first evaluated in giant cell arteritis using an ex vivo temporal artery culture model, and then in an in vitro and in vivo fibrosis model, similar to that described in Systemic Scleroderma.Our results showed that HuMoSC supernatant decreased the expression of matrix proteins and pro-fibrosing factors as well as cell proliferation.In conclusion, this thesis work has highlighted the anti-remodeling properties of HuMoSC supernatant thus representing a new hope in the treatment of fibrosing diseases.Notre équipe a développé une méthode expérimentale pour générer des cellules immunosuppressives d’origine monocytaire baptisées les Human Monocyte-derived Suppressor Cells (HuMoSC). Si leurs propriétés immunosuppressives ont déjà été montrées, aucune étude n’avait encore évalué leurs intérêts sur le remodelage tissulaire.L'objectif de cette thèse était d’une part, d'évaluer le potentiel thérapeutique des HuMoSC et de leur surnageant pour le traitement du remodelage tissulaire. D’autre part, nous avons cherché à identifier les mécanismes d’action impliqués dans les fonctions du surnageant. L’effet des HuMoSC a d’abord été évalué dans l'artérite à cellules géantes en utilisant un modèle de culture d’artères temporales ex vivo, puis dans un modèle de fibrose in vitro et in vivo, similaire à celle décrite dans la Sclérodermie Systémique.Nos résultats ont montré que le surnageant de HuMoSC diminuait l’expression des protéines matricielles et des facteurs pro-fibrosants ainsi que la prolifération cellulaire.En conclusion, ce travail de thèse a mis en avant les propriétés anti-remodelage du surnageant de HuMoSC, représentant ainsi un nouvel espoir dans le traitement des maladies fibrosantes

HuMoSC, a new therapy for the treatment of auto-immune diseases and fibrosis

Notre équipe a développé une méthode expérimentale pour générer des cellules immunosuppressives d’origine monocytaire baptisées les Human Monocyte-derived Suppressor Cells (HuMoSC). Si leurs propriétés immunosuppressives ont déjà été montrées, aucune étude n’avait encore évalué leurs intérêts sur le remodelage tissulaire.L'objectif de cette thèse était d’une part, d'évaluer le potentiel thérapeutique des HuMoSC et de leur surnageant pour le traitement du remodelage tissulaire. D’autre part, nous avons cherché à identifier les mécanismes d’action impliqués dans les fonctions du surnageant. L’effet des HuMoSC a d’abord été évalué dans l'artérite à cellules géantes en utilisant un modèle de culture d’artères temporales ex vivo, puis dans un modèle de fibrose in vitro et in vivo, similaire à celle décrite dans la Sclérodermie Systémique.Nos résultats ont montré que le surnageant de HuMoSC diminuait l’expression des protéines matricielles et des facteurs pro-fibrosants ainsi que la prolifération cellulaire.En conclusion, ce travail de thèse a mis en avant les propriétés anti-remodelage du surnageant de HuMoSC, représentant ainsi un nouvel espoir dans le traitement des maladies fibrosantes.Our team has developed an experimental method to generate immunosuppressive cells of monocyte origin called Human Monocyte-derived Suppressor Cells (HuMoSC). Although their immunosuppressive properties have already been shown, no study had yet evaluated their interest in tissue remodeling.The objective of this thesis was to evaluate the therapeutic potential of HuMoSCs and their supernatant for the treatment of tissue remodeling. On the other hand, we sought to identify the mechanisms of action involved in the functions of the supernatant of HuMoSC. The effect of HuMoSCs was first evaluated in giant cell arteritis using an ex vivo temporal artery culture model, and then in an in vitro and in vivo fibrosis model, similar to that described in Systemic Scleroderma.Our results showed that HuMoSC supernatant decreased the expression of matrix proteins and pro-fibrosing factors as well as cell proliferation.In conclusion, this thesis work has highlighted the anti-remodeling properties of HuMoSC supernatant thus representing a new hope in the treatment of fibrosing diseases

New Insights into the Pathogenesis of Giant Cell Arteritis: Mechanisms Involved in Maintaining Vascular Inflammation

The giant cell arteritis (GCA) pathophysiology is complex and multifactorial, involving a predisposing genetic background, the role of immune aging and the activation of vascular dendritic cells by an unknown trigger. Once activated, dendritic cells recruit CD4 T cells and induce their activation, proliferation and polarization into Th1 and Th17, which produce interferon-gamma (IFN-γ) and interleukin-17 (IL-17), respectively. IFN-γ triggers the production of chemokines by vascular smooth muscle cells, which leads to the recruitment of additional CD4 and CD8 T cells and also monocytes that differentiate into macrophages. Recent data have shown that IL-17, IFN-γ and GM-CSF induce the differentiation of macrophage subpopulations, which play a role in the destruction of the arterial wall, in neoangiogenesis or intimal hyperplasia. Under the influence of different mediators, mainly endothelin-1 and PDGF, vascular smooth muscle cells migrate to the intima, proliferate and change their phenotype to become myofibroblasts that further proliferate and produce extracellular matrix proteins, increasing the vascular stenosis. In addition, several defects in the immune regulatory mechanisms probably contribute to chronic vascular inflammation in GCA: a defect in the PD-1/PD-L1 pathway, a quantitative and qualitative Treg deficiency, the implication of resident cells, the role of GM-CSF and IL-6, the implication of the NOTCH pathway and the role of mucosal-associated invariant T cells and tissue-resident memory T cells

Forensic age estimation at the University Center of Legal Medicine Lausanne-Geneva: a retrospective study over 12 years.

With the undeniable increase in asylum requests from unaccompanied alleged minors, age estimation of living individuals has become an essential part of the routine work in European forensic centers. This study aims to review the forensic age estimations performed in our center since 2010, to evaluate the state-of-the-art of this practice in Switzerland with the evolution of the methodology according to upcoming recommendations. Our institute's expert reports performed between 2010 and 2022 were retrospectively analyzed. We gathered the following parameters: demographic data, morphological characteristics, alleged age compared with the assessed minimum age, sexual maturation, dental and bone age. When available, we collected personal and family history, medical history, records of torture-related/self-inflicted injuries, and information about eating habits that might affect skeletal development. Data collection amounted to 656 cases. Forensic age estimations ordered by the Swiss Secretariat for Migration (SEM) represented 76.4% of cases, with 23.6% of them ordered by the Court/Public Prosecutor. Most alleged minors were male (94.5%) and came from Afghanistan (53.4%). Adjunction of CT scans of the sternoclavicular joints was necessary in 86.4% of cases. Only 25.2% of our reports concluded on most probable minority, with 55.6% of definite majors; in 19.2% of our cases, minority could not be excluded. This study aspires to further broaden our expertise regarding forensic age estimations. Given the increasing migratory flows, we can expect a notable increase in the frequency of these requests. Consequently, this study aims to promote a multidisciplinary approach and the international standardization of the methodology of these estimations

Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

International audienceHuman secretory immunoglobulins (SIg) A1 and SIgA2 guide mucosal responses toward tolerance or inflammation, notably through reverse-transcytosis, the apical-to-basal transport of IgA2 immune complexes via M cells of gut Peyer's patches. As such, the maintenance of a diverse gut microbiota requires broad affinity IgA and glycan-glycan interaction. Here, we asked whether IgA1 and IgA2-microbiota interactions might be involved in dysbiosis induction during inflammatory bowel diseases. Using stool HPLC-purified IgA, we show that reverse-transcytosis is abrogated in ulcerative colitis (UC) while it is extended to IgA1 in Crohn's disease (CD). 16S RNA sequencing of IgA-bound microbiota in CD and UC showed distinct IgA1- and IgA2-associated microbiota; the IgA1+ fraction of CD microbiota was notably enriched in beneficial commensals. These features were associated with increased IgA anti-glycan reactivity in CD and an opposite loss of reactivity in UC. Our results highlight previously unknown pathogenic properties of IgA in IBD that could support dysbiosis

Human monocyte-derived suppressive cells (HuMoSC) for cell therapy in giant cell arteritis

This article is part of the Research Topic "Novel Therapeutic Options in Large Vessel Vasculitis".International audienceIntroduction The pathogenesis of Giant Cell Arteritis (GCA) relies on vascular inflammation and vascular remodeling, the latter being poorly controlled by current treatments. Methods This study aimed to evaluate the effect of a novel cell therapy, Human Monocyte-derived Suppressor Cells (HuMoSC), on inflammation and vascular remodeling to improve GCA treatment. Fragments of temporal arteries (TAs) from GCA patients were cultured alone or in the presence of HuMoSCs or their supernatant. After five days, mRNA expression was measured in the TAs and proteins were measured in culture supernatant. The proliferation and migration capacity of vascular smooth muscle cells (VSMCs) were also analyzed with or without HuMoSC supernatant. Results Transcripts of genes implicated in vascular inflammation ( CCL2 , CCR2 , CXCR3 , HLADR ), vascular remodeling ( PDGF , PDGFR ), angiogenesis (VEGF) and extracellular matrix composition ( COL1A1 , COL3A1 and FN1 ) were decreased in arteries treated with HuMoSCs or their supernatant. Likewise, concentrations of collagen-1 and VEGF were lower in the supernatants of TAs cultivated with HuMoSCs. In the presence of PDGF, the proliferation and migration of VSMCs were both decreased after treatment with HuMoSC supernatant. Study of the PDGF pathway suggests that HuMoSCs act through inhibition of mTOR activity. Finally, we show that HuMoSCs could be recruited in the arterial wall through the implication of CCR5 and its ligands. Conclusion Altogether, our results suggest that HuMoSCs or their supernatant could be useful to decrease vascular in flammation and remodeling in GCA, the latter being an unmet need in GCA treatment

Neointimal myofibroblasts contribute to maintaining Th1/Tc1 and Th17/Tc17 inflammation in giant cell arteritis

International audienceVascular smooth muscle cells (VSMCs) have been shown to play a role in the pathogenesis of giant cell arteritis (GCA) through their capacity to produce chemokines recruiting T cells and monocytes in the arterial wall and their ability to migrate and proliferate in the neointima where they acquire a myofibroblast (MF) phenotype, leading to vascular stenosis. This study aimed to investigate if MFs could also impact T-cell polarization. Confocal microscopy was used to analyze fresh fragments of temporal artery biopsies (TABs). Healthy TAB sections were cultured to obtain MFs, which were then treated or not with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) and analyzed by immunofluorescence and RT-PCR. After peripheral blood mononuclear cells and MFs were co-cultured for seven days, T-cell polarization was analyzed by flow cytometry. In the neointima of GCA arteries, we observed a phenotypic heterogeneity among VSMCs that was consistent with a MF phenotype (α-SMA + CD90 + desmin + MYH11 +) with a high level of STAT1 phosphorylation. Co-culture experiments showed that MFs sustain Th1/Tc1 and Th17/Tc17 polarizations. The increased Th1 and Tc1 polarization was further enhanced following the stimulation of MFs with IFN-γ and TNF-α, which induced STAT1 phosphorylation in MFs. These findings correlated with increases in the production of IL-1β, IL-6, IL-12 and IL-23 by MFs. Our study showed that MFs play an additional role in the pathogenesis of GCA through their ability to maintain Th17/Tc17 and Th1/Tc1 polarizations, the latter being further enhanced in case of stimulation of MF with IFN-γ and TNF-α

Mucosal-associated invariant T cells in giant cell arteritis

International audienceThis study aimed to assess the implication of mucosal-associated invariant T (MAIT) cells in GCA. Blood samples were obtained from 34 GCA patients (before and after 3 months of treatment with glucocorticoids (GC) alone) and compared with 20 controls aged >50 years. MAIT cells, defined by a CD3(+)CD4(-)TCRγδ(-)TCRVα7.2(+)CD161(+) phenotype, were analyzed by flow cytometry. After sorting, we assessed the ability of MAIT cells to proliferate and produce cytokines after stimulation with anti CD3/CD28 microbeads or IL-12 and IL-18. MAIT were stained in temporal artery biopsies (TAB) by confocal microscopy. MAIT cells were found in the arterial wall of positive TABs but was absent in negative TAB. MAIT frequency among total αβ-T cells was similar in the blood of patients and controls (0.52 vs. 0.57%; P = 0.43) and not modified after GC treatment (P = 0.82). Expression of IFN-γ was increased in MAIT cells from GCA patients compared to controls (44.49 vs. 32.9%; P = 0.029), and not modified after 3 months of GC therapy (P = 0.82). When they were stimulated with IL-12 and IL-18, MAIT from GCA patients produced very high levels of IFN-γ and displayed a stronger proliferation compared with MAIT from controls (proliferation index 3.39 vs. 1.4; P = 0.032). In GCA, the functional characteristics of MAIT cells are modified toward a pro-inflammatory phenotype and a stronger proliferation capability in response to IL-12 and IL-18, suggesting that MAIT might play a role in GCA pathogenesis. Our results support the use of treatments targeting IL-12/IL-18 to inhibit the IFN-γ pathway in GCA

Improvement of Treg immune response after treatment with tocilizumab in giant cell arteritis

International audienceOBJECTIVES: To study the percentage, suppressive function and plasticity of Treg in giant cell arteritis (GCA), and the effects of glucocorticoids and tocilizumab. METHODS: Blood samples were obtained from 40 controls and 43 GCA patients at baseline and after treatment with glucocorticoids + IV tocilizumab (n = 20) or glucocorticoids (n = 23). Treg percentage and phenotype were assessed by flow cytometry. Suppressive function of Treg was assessed by measuring their ability to inhibit effector T-cell (Teff) proliferation and polarisation into Th1 and Th17 cells. RESULTS: Treg (CD4(+)CD25(high)FoxP3(+)) frequency in total CD4(+) T cells was decreased in active GCA patients when compared to controls (2.5% vs. 4.7%, P < 0.001) and increased after treatment with tocilizumab but worsened after treatment with glucocorticoids alone. Treg lacking exon 2 of FoxP3 were increased in GCA patients when compared to controls (23% vs. 10% of total Treg, P = 0.0096) and normalised after treatment with tocilizumab + glucocorticoids but not glucocorticoids alone. In GCA patients, Treg were unable to control Teff proliferation and induced ˜50% increase in the amount of IL-17(+) Teff, which was improved after in vitro blockade of the IL-6 pathway by tocilizumab. CONCLUSION: This study reports quantitative and functional disruptions in the regulatory immune response of GCA patients and demonstrates that, unlike glucocorticoids, tocilizumab improves Treg immune response