Genetic Depletion or Hyperresponsiveness of Natural Killer Cells Do Not Affect Atherosclerosis Development.

RATIONALE: Chronic inflammation is central in the development of atherosclerosis. Both innate and adaptive immunities are involved. Although several studies have evaluated the functions of natural killer (NK) cells in experimental animal models of atherosclerosis, it is not yet clear whether NK cells behave as protective or proatherogenic effectors. One of the main caveats of previous studies was the lack of specificity in targeting loss or gain of function of NK cells. OBJECTIVES: We used 2 selective genetic approaches to investigate the role of NK cells in atherosclerosis: (1) Ncr1iCre/+R26lsl-DTA/+ mice in which NK cells were depleted and (2) Noé mice in which NK cells are hyperresponsive. METHODS AND RESULTS: No difference in atherosclerotic lesion size was found in Ldlr-/- (low-density lipoprotein receptor null) mice transplanted with bone marrow (BM) cells from Ncr1iCreR26Rlsl-DTA , Noé, or wild-type mice. Also, no difference was observed in plaque composition in terms of collagen content, macrophage infiltration, or the immune profile, although Noé chimera had more IFN (interferon)-γ-producing NK cells, compared with wild-type mice. Then, we investigated the NK-cell selectivity of anti-asialoganglioside M1 antiserum, which was previously used to conclude the proatherogenicity of NK cells. Anti-asialoganglioside M1 treatment decreased atherosclerosis in both Ldlr-/- mice transplanted with Ncr1iCreR26Rlsl-DTA or wild-type bone marrow, indicating that its antiatherogenic effects are unrelated to NK-cell depletion, but to CD8+ T and NKT cells. Finally, to determine whether NK cells could contribute to the disease in conditions of pathological NK-cell overactivation, we treated irradiated Ldlr-/- mice reconstituted with either wild-type or Ncr1iCreR26Rlsl-DTA bone marrow with the viral mimic polyinosinic:polycytidylic acid and found a significant reduction of plaque size in NK-cell-deficient chimeric mice. CONCLUSIONS: Our findings, using state-of-the-art mouse models, demonstrate that NK cells have no direct effect on the natural development of hypercholesterolemia-induced atherosclerosis, but may play a role when an additional systemic NK-cell overactivation occurs

Discovery of a novel cytokine signature for the diagnosis of autism spectrum disorder in young Arab children in Qatar

BackgroundAutism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by impaired social interaction and communication and the occurrence of stereotyped and repetitive behaviors. Several studies have reported altered cytokine profiles in ASD and hence may serve as potential diagnostic biomarkers of the disorder. This study aims to identify diagnostic biomarkers for ASD in a well-defined study cohort in Qatar.MethodsWe measured the protein levels of 45 cytokines in the plasma samples of age- and gender-matched children (2–4 years) with ASD (n = 100) and controls (n = 60) using a Luminex multiplex assay. We compared the differences in the levels of these cytokines between the two study groups and then fitted the significantly altered cytokines into a logistic regression model to examine their diagnostic potential for ASD.ResultsWe found elevated levels of IFN-γ, FGF-2, IL-1RA, and IL-13 and reduced levels of eotaxin, HGF, IL-1 alpha, IL-22, IL-9, MCP-1, SCF, SDF-1 alpha, VEGFA, and IP-10 in the plasma of children with ASD compared to controls. Furthermore, we observed that elevated levels of IFN-γ (odds ratio (OR) = 1.823; 95% (confidence interval) CI = 1.206, 2.755; p = 0.004) and FGF-2 (OR = 2.528; 95% CI = 1.457, 4.385; p < 0.001) were significantly associated with increased odds of ASD, whereas reduced levels of eotaxin (OR = 0.350; 95% CI = 0.160, 0.765; p = 0.008) and HGF (OR = 0.220; 95% CI = 0.070, 0.696; p = 0.010) were significantly associated with lower odds of ASD relative to controls. The combination of these four cytokines revealed an area under the curve (ROC-AUC) of 0.829 (95% CI = 0.767, 0.891; p < 0.001), which demonstrates the diagnostic accuracy of the four-cytokine signature.ConclusionsOur results identified a panel of cytokines that could discriminate between children with ASD and controls in Qatar. In addition, our findings support the predominance of a Th1 immune phenotype in ASD children and emphasize the need to validate these results in larger populations

Validation of plasma protein glycation and oxidation biomarkers for the diagnosis of autism

Autism Spectrum Disorder (ASD) is a common neurodevelopmental disorder in children. It is currently diagnosed by behaviour-based assessments made by observation and interview. In 2018 we reported a discovery study of a blood biomarker diagnostic test for ASD based on a combination of four plasma protein glycation and oxidation adducts. The test had 88% accuracy in children 5–12 years old. Herein, we present an international multicenter clinical validation study (N = 478) with application of similar biomarkers to a wider age range of 1.5–12 years old children. Three hundred and eleven children with ASD (247 male, 64 female; age 5.2 ± 3.0 years) and 167 children with typical development (94 male, 73 female; 4.9 ± 2.4 years) were recruited for this study at Sidra Medicine and Hamad Medical Corporation hospitals, Qatar, and Hospital Regional Universitario de Málaga, Spain. For subjects 5–12 years old, the diagnostic algorithm with features, advanced glycation endproducts (AGEs)—Nε-carboxymethyl-lysine (CML), Nω-carboxymethylarginine (CMA) and 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and oxidative damage marker, o,o’-dityrosine (DT), age and gender had accuracy 83% (CI 79 – 89%), sensitivity 94% (CI 90–98%), specificity 67% (CI 57–76%) and area-under-the-curve of receiver operating characteristic plot (AUROC) 0.87 (CI 0.84–0.90). Inclusion of additional plasma protein glycation and oxidation adducts increased the specificity to 74%. An algorithm with 12 plasma protein glycation and oxidation adduct features was optimum for children of 1.5–12 years old: accuracy 74% (CI 70–79%), sensitivity 75% (CI 63–87%), specificity 74% (CI 58–90%) and AUROC 0.79 (CI 0.74–0.84). We conclude that ASD diagnosis may be supported using an algorithm with features of plasma protein CML, CMA, 3DG-H and DT in 5–12 years-old children, and an algorithm with additional features applicable for ASD screening in younger children. ASD severity, as assessed by ADOS-2 score, correlated positively with plasma protein glycation adducts derived from methylglyoxal, hydroimidazolone MG-H1 and Nε(1-carboxyethyl)lysine (CEL). The successful validation herein may indicate that the algorithm modifiable features are mechanistic risk markers linking ASD to increased lipid peroxidation, neuronal plasticity and proteotoxic stress.</p

Selective EGF-Receptor Inhibition in CD4+ T Cells Induces Anergy and Limits Atherosclerosis.

BACKGROUND: Several epidermal growth factor receptor (EGFR) inhibitors have been successfully developed for the treatment of cancer, limiting tumor growth and metastasis. EGFR is also expressed by leukocytes, but little is known about its role in the modulation of the immune response. OBJECTIVES: The aim of this study was to determine whether EGFR expressed on CD4+ T cells is functional and to address the consequences of EGFR inhibition in atherosclerosis, a T cell-mediated vascular chronic inflammatory disease. METHODS: The authors used EGFR tyrosine kinase inhibitors (AG-1478, erlotinib) and chimeric Ldlr-/-Cd4-Cre/Egfrlox/lox mouse with a specific deletion of EGFR in CD4+ T cells. RESULTS: Mouse CD4+ T cells expressed EGFR, and the EGFR tyrosine kinase inhibitor AG-1478 blocked in vitro T cell proliferation and Th1/Th2 cytokine production. In vivo, treatment of Ldlr-/- mice with the EGFR inhibitor erlotinib induced T cell anergy, reduced T cell infiltration within atherosclerotic lesions, and protected against atherosclerosis development and progression. Selective deletion of EGFR in CD4+ T cells resulted in decreased T cell proliferation and activation both in vitro and in vivo, as well as reduced interferon-γ, interleukin-4, and interleukin-2 production. Atherosclerotic lesion size was reduced by 2-fold in irradiated Ldlr-/- mice reconstituted with bone marrow from Cd4-Cre/Egfrlox/lox mice, compared to Cd4-Cre/Egfr+/+ chimeric mice, after 4, 6, and 12 weeks of high-fat diet, associated with marked reduction in T cell infiltration in atherosclerotic plaques. Human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both in vitro and in vivo. CONCLUSIONS: EGFR blockade induced T cell anergy in vitro and in vivo and reduced atherosclerosis development. Targeting EGFR may be a novel strategy to combat atherosclerosis

Dissection du rôle des cellules NK dans l'athérosclérose en utilisant des approches génétiques sélectives

Chronic inflammation is central in the development of atherosclerosis. Both innate and adaptive immunity are involved in this process. Although several studies have evaluated the functions of NK cells in experimental animal models of atherosclerosis, it is not yet clear whether NK cells behave as protective or pro-atherogenic effectors. One of the main caveats of previous studies was the lack of specificity in targeting loss- or gain-of-function of NK cells. Here, we used two selective genetic approaches to investigate the role of NK cells in atherosclerosis: 1) Ncr1iCre/+R26lslDTA/+ mice in which NK cells were depleted, 2) Noé mice in which NK cells are hyperresponsive. No difference in atherosclerotic lesion size was found in Ldlr-/- mice transplanted with bone marrow cells from Ncr1iCreR26Rlsl−DTA, Noé or WT mice. Also, no difference was observed in plaque composition in terms of collagen content, macrophage infiltration or the immune profile in blood and spleen, although Noé chimera had more IFN-y-producing NK cells in comparison with WT mice. Then, we investigated the NK cell selectivity of anti-asialo GM1 anti-serum, which was previously used to conclude to the pro-atherogenicity of NK cells. Anti-asialo GM1 treatment decreased atherosclerosis in both Ldlr-/- mice transplanted with Ncr1iCreR26Rlsl−DTA or WT BM, indicating that its anti-atherogenic effects are unrelated to NK cell depletion. Finally, to determine whether NK cells could contribute to the disease in conditions of pathological NK cell overactivation, we treated irradiated Ldlr-/- mice reconstituted with either WT or Ncr1iCreR26Rlsl−DTA BM with the viral mimic Poly(I:C) and found a significant reduction of plaque size in NK-cell deficient chimeric mice. Our findings, using state-of-the-art mouse models, clearly demonstrate that NK cells have no direct effect on the natural development of hypercholesterolemia-induced atherosclerosis, but may play a role when an additional systemic NK cell overactivation occurs.L'inflammation chronique en réponse à l'accumulation de lipoprotéines dans la paroi artérielle est centrale dans le développement de l'athérosclérose. L’immunité innée et adaptée sont impliquées dans ce processus. Les cellules Natural Killer (NK), un des éléments clés de l'immunité innée, ont été identifiées dans les lésions athérosclérotiques humaines et murines. Bien que plusieurs études aient cherché à évaluer le rôle des cellules NK dans des modèles animaux expérimentaux d'athérosclérose, les résultats restent contradictoires, certaines rapportant des effets pro-athérogéniques, d’autres anti-athérogéniques. L'une des principales limites de ces études est le manque de spécificité dans le ciblage de la perte ou du gain de fonction des cellules NK. Nous avons utilisé deux approches génétiques sélectives pour étudier le rôle des cellules NK dans l'athérosclérose: 1) des souris Ncr1iCre/+R26lslDTA/+ dans lesquelles les cellules NK ont été déplétées 2) des souris Noé, dont les cellules NK sont hyper-réactives. Les cellules de la moelle osseuse (BM) de ces souris ont été utilisées pour reconstituer le système hématopoïétique de souris Ldlr -/- irradiées. Après une période de récupération de 4 semaines, les souris ont été mises sous un régime riche en matières grasses (HFD) pendant 8 semaines. L'analyse morphométrique de la taille des lésions ‘athérosclérose dans le sinus aortique et l'aorte thoracique n'a montré aucune différence statistiquement significative entre les 3 groupes. De plus, aucune différence n'a été observée dans la composition de la plaque en termes de teneur en collagène, d'infiltration de macrophages ou de profil immunitaire dans le sang et la rate des souris Ncr1iCreR26lsl-DTA, Noé ou contrôles. Nous avons ensuite étudié la sélectivité de des anticorps anti-asialo-GM1 dans la déplétion des cellules NK, qui avaient été utilisés précédemment pour démontrer le rôle pro-athérogène des cellules NK. Nous avons confirmé les effets non spécifiques de cet anticorps, qui déplète non seulement les cellules NK, mais aussi les lymphocytes NKT et CD8+. Enfin, pour déterminer si l'activation des cellules NK par un stimulus externe pouvait avoir des effets sur l’athérosclérose, nous avons traité les souris chimériques (souris Ldlr -/- irradiées reconstituées soit avec les cellules de moelle contrôle ou déficiente en cellules NK) avec du poly IC (un mimétique viral) pendant 8 semaines de HFD. Nous avons trouvé une réduction significative de la taille des lésions au niveau du sinus aortique et de l'aorte thoracique dans les souris déficientes en cellules NK. Nos résultats, à partir de modèles de souris spécifiques, contredisent les études antérieures et démontrent clairement que chez les souris hypercholestérolémiques, les cellules NK n'ont aucun effet direct sur l'athérosclérose, sauf si elles sont pré-stimulée, comme par exemple dans un contexte d’infection virale ou de présence de tumeurs

DataSheet_1_In search of immune cellular sources of abnormal cytokines in the blood in autism spectrum disorder: A systematic review of case-control studies.zip

Abnormal cytokine levels in circulating blood have been repeatedly reported in autism; however, the underlying cause remains unclear. This systematic review aimed to investigate cytokine levels in peripheral blood compartments and identify their potential immune cellular sources in subjects with autism through comparison with controls. We conducted an electronic database search (PubMed, Scopus, ProQuest Central, Ovid, SAGE Journals, and Wiley Online Library) from inception (no time limits) to July 9, 2020, and identified 75 relevant articles. Our qualitative data synthesis focused on results consistently described in at least three independent studies, and we reported the results according to the PRISMA protocol. We found that compared with controls, in subjects with autism, cytokines IL-6, IL-17, TNF-α, and IL-1β increased in the plasma and serum. We also identified monocytes, neutrophils, and CD4+ T cells as potential sources of these elevated cytokines in autism. Cytokines IFN-γ, TGF-β, RANTES, and IL-8 were increased in the plasma/serum of subjects with autism, and IFN-γ was likely produced by CD4+ T cells and natural killer (NK) cells, although conflicting evidence is present for IFN-γ and TGF-β. Other cytokines—IL-13, IL-10, IL-5, and IL-4—were found to be unaltered in the plasma/serum and post-stimulated blood immune cells in autistic individuals as compared with controls. The frequencies of T cells, monocytes, B cells, and NK cells were unchanged in subjects with autism as opposed to controls, suggesting that abnormal cytokines were unlikely due to altered cell numbers but might be due to altered functioning of these cells in autism. Our results support existing studies of abnormal cytokines in autism and provide comprehensive evidence of potential cellular sources of these altered cytokines in the context of autism.Systematic Review Registrationhttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020205224, identifier [CRD42020205224].</p

DISSECTION OF THE ROLE OF NATURAL KILLER CELLS IN ATHEROSCLEROSIS USING SELECTIVE GENETIC APPROACHES

85th Congress of the European-Atherosclerosis-Society (EAS), Prague, CZECH REPUBLIC, APR 23-26, 2017International audienc

Genetic Depletion or Hyperresponsiveness of Natural Killer Cells Do Not Affect Atherosclerosis Development.

RATIONALE: Chronic inflammation is central in the development of atherosclerosis. Both innate and adaptive immunities are involved. Although several studies have evaluated the functions of natural killer (NK) cells in experimental animal models of atherosclerosis, it is not yet clear whether NK cells behave as protective or proatherogenic effectors. One of the main caveats of previous studies was the lack of specificity in targeting loss or gain of function of NK cells. OBJECTIVES: We used 2 selective genetic approaches to investigate the role of NK cells in atherosclerosis: (1) Ncr1iCre/+R26lsl-DTA/+ mice in which NK cells were depleted and (2) Noé mice in which NK cells are hyperresponsive. METHODS AND RESULTS: No difference in atherosclerotic lesion size was found in Ldlr-/- (low-density lipoprotein receptor null) mice transplanted with bone marrow (BM) cells from Ncr1iCreR26Rlsl-DTA , Noé, or wild-type mice. Also, no difference was observed in plaque composition in terms of collagen content, macrophage infiltration, or the immune profile, although Noé chimera had more IFN (interferon)-γ-producing NK cells, compared with wild-type mice. Then, we investigated the NK-cell selectivity of anti-asialoganglioside M1 antiserum, which was previously used to conclude the proatherogenicity of NK cells. Anti-asialoganglioside M1 treatment decreased atherosclerosis in both Ldlr-/- mice transplanted with Ncr1iCreR26Rlsl-DTA or wild-type bone marrow, indicating that its antiatherogenic effects are unrelated to NK-cell depletion, but to CD8+ T and NKT cells. Finally, to determine whether NK cells could contribute to the disease in conditions of pathological NK-cell overactivation, we treated irradiated Ldlr-/- mice reconstituted with either wild-type or Ncr1iCreR26Rlsl-DTA bone marrow with the viral mimic polyinosinic:polycytidylic acid and found a significant reduction of plaque size in NK-cell-deficient chimeric mice. CONCLUSIONS: Our findings, using state-of-the-art mouse models, demonstrate that NK cells have no direct effect on the natural development of hypercholesterolemia-induced atherosclerosis, but may play a role when an additional systemic NK-cell overactivation occurs

Table_1_Discovery of a novel cytokine signature for the diagnosis of autism spectrum disorder in young Arab children in Qatar.docx

BackgroundAutism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by impaired social interaction and communication and the occurrence of stereotyped and repetitive behaviors. Several studies have reported altered cytokine profiles in ASD and hence may serve as potential diagnostic biomarkers of the disorder. This study aims to identify diagnostic biomarkers for ASD in a well-defined study cohort in Qatar.MethodsWe measured the protein levels of 45 cytokines in the plasma samples of age- and gender-matched children (2–4 years) with ASD (n = 100) and controls (n = 60) using a Luminex multiplex assay. We compared the differences in the levels of these cytokines between the two study groups and then fitted the significantly altered cytokines into a logistic regression model to examine their diagnostic potential for ASD.ResultsWe found elevated levels of IFN-γ, FGF-2, IL-1RA, and IL-13 and reduced levels of eotaxin, HGF, IL-1 alpha, IL-22, IL-9, MCP-1, SCF, SDF-1 alpha, VEGFA, and IP-10 in the plasma of children with ASD compared to controls. Furthermore, we observed that elevated levels of IFN-γ (odds ratio (OR) = 1.823; 95% (confidence interval) CI = 1.206, 2.755; p = 0.004) and FGF-2 (OR = 2.528; 95% CI = 1.457, 4.385; p ConclusionsOur results identified a panel of cytokines that could discriminate between children with ASD and controls in Qatar. In addition, our findings support the predominance of a Th1 immune phenotype in ASD children and emphasize the need to validate these results in larger populations.</p