Bifidobacterium longum and Chlorella sorokiniana Combination Modulates IFN-γ, IL-10, and SOCS3 in Rotavirus-Infected Cells.

Rotavirus is the main cause of acute diarrhea in children up to five years of age. In this regard, probiotics are commonly used to treat or prevent gastroenteritis including viral infections. The anti-rotavirus effect of Bifidobacterium longum and Chlorella sorokiniana, by reducing viral infectivity and improving IFN-type I response, has been previously reported. The present study aimed to study the effect of B. longum and/or C. sorokiniana on modulating the antiviral cellular immune response mediated by IFN-γ, IL-10, SOCS3, STAT1, and STAT2 genes in rotavirus-infected cells. To determine the mRNA relative expression of these genes, HT-29 cells were treated with B. longum and C. sorokiniana alone or in combination, followed by rotavirus infection. In addition, infected cells were treated with B. longum and/or C. sorokiniana. Cellular RNA was purified, used for cDNA synthesis, and amplified by qPCR. Our results demonstrated that the combination of B. longum and C. sorokiniana stimulates the antiviral cellular immune response by upregulating IFN-γ and may block pro-inflammatory cytokines by upregulating IL-10 and SOCS3. The results of our study indicated that B. longum, C. sorokiniana, or their combination improve antiviral cellular immune response and might modulate pro-inflammatory responses.

Évaluation du statut de phosphorylation de STAT1 par cytométrie en flux / Evaluation of STAT1 Phosphorylation Status by Flow Cytometry

INTRODUCTION: Une demande d'introduction d'une nouvelle analyse au Répertoire québécois et système de mesure des procédures de biologie médicale (ci-après nommé Répertoire) a été transmise à l'Institut national d'excellence en santé et en services sociaux (INESSS) à travers le mécanisme d'évaluation des nouvelles analyses de biologie médicale. Le mandat vise à évaluer la pertinence d'introduire le statut de phosphorylation de la protéine STAT1 par cytométrie en flux au Répertoire. MÉTHODOLOGIE: L'évaluation a été réalisée selon l'approche basée sur l'appréciation globale de la valeur que l'Institut préconise dans son Énoncé de principes et fondements éthiques, et qui stipule qu'une intervention apporte de la valeur dans la mesure où son usage ou sa mise en place contribue à la triple finalité du système de santé et de services sociaux dans le contexte québécois. La démarche d'évaluation comprend une revue de la documentation scientifique, une recherche de la littérature grise et une consultation ad hoc menée auprès de cliniciens spécialisés en immunologie-allergologie pédiatrique et adulte. Une revue de la littérature économique a été réalisée concernant l'efficience du statut de phosphorylation de STAT1 (pSTAT1). Une analyse d'impact budgétaire considérant les coûts liés à l'ajout au Répertoire de ce test a également été réalisée. Les coûts ont été projetés sur un horizon de trois ans selon la perspective du système de soins de santé québécois. L'ensemble des données scientifiques, contextuelles et expérientielles a été interprété et apprécié à l'aide d'une approche basée sur l'appréciation globale de la valeur définie selon cinq dimensions : socioculturelle, populationnelle, clinique, organisationnelle et économique. Les constats issus de cette démarche évaluative ont servi à guider les discussions et le processus d'analyse de la Direction de l'évaluation des médicaments et des technologies à des fins de remboursement. CONTEXTE DE L'ÉVALUATION: Les erreurs innées de l'immunité (EII), précédemment appelées déficits immunitaires primaires (DIP), sont un groupe de maladies génétiques qui se manifestent entre autres par une susceptibilité accrue aux infections récurrentes, atypiques, sévères ou chroniques, aux réactions allergiques, inflammatoires ou auto-immunes, aux insuffisances médullaires et à certains cancers. Ces maladies sont monogéniques et causées par des variants germinaux pathogènes qui entraînent la perte ou le gain de fonction d'un des gènes impliqués dans les EII. Plusieurs protéines sont impliquées dans les EII, telles que celles de la voie de signalisation JAK/STAT. La famille des STAT comprend sept membres : STAT1, 2, 3, 4, 5A, 5B et 6. Une perte de fonction de STAT1 peut être responsable d'une susceptibilité accrue aux infections mycobactériennes. Les mutations activatrices de STAT1 provoquent un défaut de déphosphorylation qui se manifeste cliniquement par de la candidose mucocutanée chronique et des manifestations auto-immunes systémiques. DIMENSION SOCIOCULTURELLE: Plusieurs des sociétés savantes consultées s'étant positionnées sur le sujet recommandent le test du statut de pSTAT1 dans le diagnostic de certaines EII. Les cliniciens consultés sont aussi de cet avis. L'ajout éventuel d'un test fonctionnel comme celui de pSTAT1 est en concordance avec la Politique québécoise pour les maladies rares, Pour une meilleure reconnaissance et prise en charge des personnes atteintes de maladies rares, qui a été publiée en 2023. Cette politique vise à « optimiser l'accès à des soins et à des services de santé de qualité, sécuritaires, équitables, inclusifs et adaptés aux besoins particuliers des patients atteints de maladies rares, et culturellement sensibles ¼. Dimension populationnelle: Les trois types de dysfonctions associés à la phosphorylation de la protéine STAT1 (le gain ou la perte de fonction et la présence d'autoanticorps anti-IFN-γ neutralisants) peuvent être détectés par le test de cytométrie en flux (CMF). Le traitement des dysfonctions de STAT1 inclut principalement des combinaisons d'antifongiques ou d'antibiotiques souvent à long terme, des greffes de cellules souches hématopoïétiques (GCSH) et des inhibiteurs de la voie JAK/STAT. L'ajout du test de pSTAT1 au Répertoire contribuerait à la prise en charge rapide des patients et aiderait au choix de traitement, limitant ainsi l'errance médicale de ces patients. De plus, il permettrait de promouvoir la disponibilité du test et de standardiser les résultats obtenus. DIMENSION CLINIQUE: Afin de documenter la validité et l'utilité clinique du test, huit articles ont été retenus, soit six documents sur les pertes et les gains de fonction de STAT1 et deux articles sur la présence d'autoanticorps anti-IFN-γ. Les données montrent que l'évaluation du statut de pSTAT1 contribue à détecter les défauts de la boucle d'amplification IL-12/IFN-γ, à valider les anomalies fonctionnelles de STAT1 et à identifier la présence d'autoanticorps neutralisants anti-IFN-γ chez les patients cliniquement suspectés de certaines EII. Il sert également à évaluer la fonctionnalité des variants de signification incertaine (VSI) obtenus après des analyses génétiques et aide à cibler les investigations génétiques. Enfin, certaines données montrent que le test pSTAT1 améliore la prise en charge des patients, notamment en ce qui concerne le recours à l'antibioprophylaxie, à l'immunomodulation ou à la GCSH. DIMENSIONS ORGANISATIONNELLE ET ÉCONOMIQUE: Le test de pSTAT1 est offert par la grappe Montréal-CHU Sainte-Justine depuis 2014. Il a été effectué entre 2014 et 2023 sous un code générique qui regroupe tous les tests de CMF associés à une valeur pondérée inférieure à celle estimée du test pSTAT1. Le laboratoire demandeur mentionne avoir la capacité d'effectuer la totalité des analyses pour couvrir les besoins de toute la province et de faire face à une augmentation de la volumétrie, le cas échéant. Selon les cliniciens consultés, la réalisation du test localement apporterait un gain de temps et un soutien pour l'interprétation des résultats. L'accès à d'autres tests comme celui du dosage de l'IL-12 devrait demeurer disponible via les envois hors Québec pour couvrir des besoins exceptionnels. Une réponse sera obtenue en 24 à 36 heures; ce délai est jugé adéquat par les cliniciens consultés. L'efficience de l'analyse proposée ne peut être évaluée justement compte tenu du fait que les issues cliniques et les coûts ne peuvent être adéquatement mesurés ou approximés. Toutefois, l'introduction de l'analyse proposée au Répertoire devrait engendrer des économies d'environ 132 000 $ au cours des trois premières années. Bien que le coût de cette analyse ne puisse être justifié par des écrits scientifiques ou une évaluation robuste, aucun signal n'indique que son utilisation, spécifique au contexte de certaines EII, pourrait constituer une utilisation non responsable des ressources. Le risque économique de son introduction est considéré comme très faible. CONCLUSIONS: À la lumière des constats formulés ci-dessus, l'INESSS recommande l'introduction de l'analyse portant sur l'évaluation du statut de phosphorylation de STAT1 par cytométrie en flux au Répertoire, étant donné que: cette analyse est recommandée par les sociétés savantes et les cliniciens spécialisés dans le diagnostic de certaines erreurs innées de l'immunité; la littérature scientifique montre que le test permet la détection des dysfonctions de STAT1 et la vérification de la fonctionnalité des VSI. Il aide à la prise en charge adéquate des patients, afin de diminuer l'errance diagnostique souvent observée avec les maladies rares et d'offrir un traitement approprié rapidement; l'analyse est disponible depuis plus de dix années dans le réseau québécois de la santé et des services sociaux; le recours à l'analyse dans le contexte des EII constituerait une utilisation responsable des ressources; e risque économique de l'inscription de l'analyse au Répertoire est considéré comme étant très faible. Le ou les laboratoires désignés devront satisfaire aux exigences de la norme ISO 15189.

INTRODUCTION: The Institut national d'excellence en santé et en services sociaux (INESSS) has received a request to introduce a new assay to the Répertoire québécois et système de mesure des procédures de biologie médicale (hereinafter referred to as the Répertoire), through the evaluation mechanism for new medical biology assays. The mandate is to evaluate the relevance of introducing the phosphorylation status of the STAT1 protein by flow cytometry into the Répertoire. METHODOLOGY: The evaluation was conducted according to the overall value assessment approach advocated by the Institute in its Statement of Ethical Principles and Foundations, which stipulates that an intervention brings value insofar as its use or implementation contributes to the triple aim of the health and social services system in Quebec. The evaluation process included a review of the scientific literature, a grey literature search and an ad hoc consultation with clinicians specializing in pediatric and adult immunologyallergology. A review of the economic literature was conducted on the efficiency of STAT1 phosphorylation status (pSTAT1). A budget impact analysis was also conducted, considering the costs associated with adding this test to the Répertoire. Costs were projected over a three-year horizon from the perspective of the Quebec healthcare system. All scientific, contextual, and experiential data were interpreted and assessed using an approach based on an overall value assessment defined according to five dimensions: sociocultural, population-based, clinical, organizational, and economic. The findings of this evaluative approach were used to guide the discussions and analysis process of the Direction de l'évaluation des médicaments et des technologies à fins de remboursement. EVALUATION CONTEXT: Inborn errors of immunity (IEIs), previously known as primary immune deficiencies (PIDs), are a group of genetic diseases manifested by, among other things, increased susceptibility to recurrent, atypical, severe, or chronic infections, allergic, inflammatory, or autoimmune reactions, bone marrow failure and certain cancers. These diseases are monogenic and caused by pathogenic germline variants that result in the loss or gain of function of one of the genes involved in IEIs. Several proteins are involved in IEIs, such as those of the JAK/STAT signaling pathway. The STAT family comprises seven members: STAT1, 2, 3, 4, 5A, 5B and 6. Loss of STAT1 function may be responsible for increased susceptibility to mycobacterial infections. Activating mutations in STAT1 cause a dephosphorylation defect that manifests clinically as chronic mucocutaneous candidiasis and systemic autoimmune manifestations. Socio-Cultural Dimension: Several of the learned societies consulted on this subject recommend testing for pSTAT1 status in the diagnosis of certain IEIs. The clinicians consulted also share this opinion. The possible addition of a functional test such as pSTAT1 is in line with the Politique québécoise pour les maladies rares, Pour une meilleure reconnaissance et prise en charge des personnes atteintes de maladies rares, published in 2023. This policy aims to "optimize access to quality health care and services that are safe, equitable, inclusive, and adapted to the specific needs of culturally sensitive patients with rare diseases." Population Dimension: The three types of dysfunctions associated with phosphorylation of the STAT1 protein (gain or loss of function and the presence of neutralizing anti-IFN-γ autoantibodies) can be detected by flow cytometry testing. Treatment of STAT1 dysfunction mainly includes often long-term combinations of antifungals or antibiotics, hematopoietic stem cell transplantation (HSCT), and inhibitors of the JAK/STAT pathway. The addition of the pSTAT1 test to the Répertoire would contribute to the rapid management of patients and help in the choice of treatment, thus limiting medical wandering for these patients. It would also promote the availability of the test and standardize the results obtained. Clinical Dimension: In order to document the validity and clinical utility of the test, eight papers were selected: six papers on loss and gain of function of STAT1 and two papers on the presence of antiIFN-γ autoantibodies. The data show that evaluation of pSTAT1 status helps to detect defects in the IL-12/IFN-γ amplification loop, validate STAT1 functional abnormalities and identify the presence of neutralizing anti-IFN-γ autoantibodies in patients clinically suspected of certain IEIs. It is also used to assess the functionality of variants of uncertain significance (VUSs) obtained after genetic analysis and helps target genetic investigations. Finally, there is some evidence that pSTAT1 improves patient management, particularly regarding the use of antibiotic prophylaxis, immunomodulation, or HSCT. Organizational and Economic Dimensions: The pSTAT1 test has been offered by the Montreal-CHU Sainte-Justine laboratories since 2014. It was performed between 2014 and 2023 under a generic code that groups together all flow cytometry tests associated with a weighted value lower than that estimated for the pSTAT1 test. The applicant laboratory states that it has the capacity to perform all the tests to cover the needs of the entire province, and to cope with any increase in volume. According to the clinicians consulted, performing the test locally would save time and provide support in interpreting results. Access to other tests, such as the IL-12 assay, should remain available via shipments outside Quebec to cover exceptional needs. A response will be obtained within 24 to 36 hours; this timeframe is deemed adequate by the clinicians consulted. The efficiency of the proposed analysis cannot be accurately evaluated, as clinical outcomes and costs cannot be adequately measured or approximated. However, the introduction of the proposed analysis to the Répertoire is expected to generate savings of approximately $132,000 over the first three years. Although the cost of this analysis cannot be justified by scientific literature or robust evaluation, there are no indications that its use, specific to the context of certain IEIs, might constitute an unaccountable use of resources. The economic risk of its introduction is considered very low. CONCLUSIONS: Considering the above findings, the INESSS recommends the introduction of the analysis of STAT1 phosphorylation status by flow cytometry to the Répertoire, given that: This analysis is recommended by learned societies and clinicians specializing in the diagnosis of certain innate immune errors; the scientific literature shows that the test enables detection of STAT1 dysfunction and verification of VUS functionality. It helps to ensure that patients are properly managed, so as to reduce the diagnostic wandering often observed with rare diseases, and to offer appropriate treatment rapidly; analysis has been available for over ten years in the Quebec health and social services network; the use of analysis in the context of IEIs would be a responsible use of resources; the economic risk of listing the analysis in the Répertoire is considered to be very low. The designated laboratory(ies) must meet ISO 15189 requirements.

Fatal COVID-19 Infection in Two Children with STAT1 Gain-of-Function.

While SARS-CoV-2 infection causes a mild disease in most children, SARS-CoV-2 infection may be lethal in a few of them. In the defense against SARS-CoV-2, type I interferons are key players, and several studies have identified a defective or neutralized interferon response as the cause of overwhelming viral infection. However, inappropriate, untimely, or excessive interferon production may also be detrimental to the host. Here, we describe two patients with STAT1 gain-of-function (GOF), a known type I interferonopathy, who died of COVID-19. Whole-exome sequencing and interferon-gamma-activated sequence (GAS) and interferon-sensitive responsive element (ISRE) reporter assay were performed to identify and characterize STAT1 variants. Patient 1 developed hemophagocytic lymphohistiocytosis (HLH) in the context of COVID-19 infection and died in less than a week at the age of 4 years. Patient 2 developed a high fever, cough, and hypoxemia and succumbed to COVID-19 pneumonia at the age of 5 years. Two heterozygous missense variants, p.E563Q and p.K344E, in STAT1 were identified. Functional validation by reporter assay and immunoblot confirmed that both variants are gain-of-function (GOF). GOF variants transiently expressing cells exhibited enhanced upregulation of downstream genes, including ISG15, MX1, and OAS1, in response to IFN-α stimulation. A catastrophic course with HLH or acute respiratory failure is thought to be associated with inappropriate immunoregulatory mechanisms to handle SARS-CoV-2 in STAT1 GOF. While most patients with inborn errors of immunity who developed COVID-19 seem to handle it well, these cases suggest that patients with STAT1-GOF might be at risk of developing fatal complications due to SARS-CoV-2.

Butyrate facilitates immune clearance of colorectal cancer cells by suppressing STAT1-mediated PD-L1 expression.

OBJECTIVE: Immunotherapy has been proven to improve the prognosis of patients with advanced malignancy but has shown limited efficacy in patients with Colorectal Cancer (CRC). Increasing evidence suggests that butyrate, a bacterial metabolite, enhances the efficacy of cancer therapies by modulating immune responses. Here, the effect and the mechanism of butyrate on anti-PD-L1 therapy were investigated in CRC. METHODS: The expression of PD-L1 and STAT1, and the lysine acetylation of STAT1 in CRC cells were observed after treatment with butyrate (2, 5, and 10 mM) for 24h or butyrate (5 mM) for 8, 16, and 24h. Site-directed mutations of STAT1 (K410R or K413R) were introduced to determine the role of STAT1 acetylation in modulating PD-L1 expression. The effect of butyrate on the cytotoxicity of CD8+ T-cells against CRC cells with or without PD-L1 overexpression was explored in vitro and in vivo. RESULTS: Butyrate could suppress IFN-γ-induced PD-L1 up-regulation in CRC cells in a dose- and time-dependent way. Butyrate promoted the lysine acetylation of STAT1 to reduce STAT1 expression. Non-acetylated mutant STAT1 not only ameliorated butyrate-induced suppression of lysine acetylation and nuclear translocation of STAT1 but also blocked the effect of butyrate on PD-L1. Butyrate attenuated the IFN-γ-induced impairment of CD8+ T-cell cytotoxicity against CRC cells. Meanwhile, butyrate suppressed CRC tumor growth by enhancing CD8+ T-cell infiltration. However, directly overexpressing PD-L1 in CRC cells could abolish the effect of butyrate. CONCLUSION: Butyrate strengthens the immune response to CRC cells by suppressing PD-L1 expression via acetylation of STAT1.

Modulation of STAT-1, STAT-3, and STAT-6 activities in THP-1 derived macrophages infected with two Trypanosoma cruzi strains.

Trypanosoma cruzi is the causative protozoan of Chagas' Disease, a neglected tropical disease that affects 6-7 million people worldwide. Interaction of the parasite with the host immune system is a key factor in disease progression and chronic symptoms. Although the human immune system is capable of controlling the disease, the parasite has numerous evasion mechanisms that aim to maintain intracellular persistence and survival. Due to the pronounced genetic variability of T. cruzi, co-infections or mixed infections with more than one parasite strain have been reported in the literature. The intermodulation in such cases is unclear. This study aimed to evaluate the co-infection of T. cruzi strains G and CL compared to their individual infections in human macrophages derived from THP-1 cells activated by classical or alternative pathways. Flow cytometry analysis demonstrated that trypomastigotes were more infective than extracellular amastigotes (EAs) and that strain G could infect more macrophages than strain CL. Classically activated macrophages showed lower number of infected cells and IL-4-stimulated cells displayed increased CL-infected macrophages. However, co-infection was a rare event. CL EAs decreased the production of reactive oxygen species (ROS), whereas G trypomastigotes displayed increased ROS detection in classically activated cells. Co-infection did not affect ROS production. Monoinfection by strain G or CL mainly induced an anti-inflammatory cytokine profile by decreasing inflammatory cytokines (IFN-γ, TNF-α, IL-1ß) and/or increasing IL-4, IL-10, and TGF-ß. Co-infection led to a predominant inflammatory milieu, with reduced IL-10 and TGF-ß, and/or promotion of IFN-γ and IL-1ß release. Infection by strain G reduced activation of intracellular signal transducer and activator of transcription (STAT) factors. In EAs, monoinfections impaired STAT-1 activity and promoted phosphorylation of STAT-3, both changes may prolong cell survival. Coinfected macrophages displayed pronounced activation of all STATs examined. These activations likely promoted parasite persistence and survival of infected cells. The collective results demonstrate that although macrophages respond to both strains, T. cruzi can modulate the intracellular environment, inducing different responses depending on the strain, parasite infective form, and co-infection or monoinfection. The modulation influences parasite persistence and survival of infected cells.

Gain-of-function STAT1 mutation and visceral leishmaniasis.

Gain-of-function mutations in the STAT1 gene have been initially associated with chronic mucocutaneous candidiasis. However, further research has shown that STAT1 GOF variants may increase susceptibility to infection by other intracellular pathogens. This report describes the first case of disseminated leishmaniasis associated with a STAT1 GOF mutation in a pediatric patient who did not have chronic mucocutaneous candidiasis. The patient was a four-year-old boy presenting with fever, severe asthenia, hepatosplenomegaly, pancytopenia, and liver failure. Bone marrow aspirate revealed hemophagocytosis and Leishmania parasites. Treatment consisted primarily of liposomal amphotericin B, as per the Hemophagocytic Lymphohistiocytosis 2004 protocol. After eight weeks of treatment, the patient did not improve and was submitted to diagnostic splenectomy. Activated macrophages and nodular spleen necrosis secondary to the visceral leishmaniasis were detected. Unfortunately, the patient died in the second week after splenectomy due to overwhelming systemic infection. DNA sequencing revealed a pathogenic (p. R274Q) GOF mutation in STAT1.

STAT1/IFIT2 signaling pathway is involved in PD-L1-mediated epithelial-to-mesenchymal transition in human esophageal cancer.

BACKGROUND: We have previously reported significant change of epithelial to mesenchymal transition (EMT) phenotype of Eca-109 cells upon PD-L1 operation, and the cytoplasmic domain of PD-L1 played an essential role in promoting EMT of esophageal cancer cells. However, the underlying mechanism of how PD-L1 regulated EMT in esophageal cancer remained unclear. METHODS: The overexpression and knockdown expression models of PD-L1 and IFIT2 were established by using lenti-virus transfection and RNAi method. Western blotting, qRT-PCR, CCK8 assay, transwell assay and wound healing assay were chosen to investigate their impact on the cells. The expression levels of IFIT2 and EMT markers in esophageal cancer tissues were examined by immunohistochemical staining. The rescue experiments were further applied to investigate the role of STAT1/IFIT2 signal pathway in the PD-L1-mediated EMT. Luciferase reporter assays were performed to examine the IFIT2 promoter activities upon knockdown expression of PD-L1 to identify the putative targeted region of IFIT2 promoter. RESULTS: The STAT1/IFIT2 signal pathway was activated when PD-L1 was knockdown in human esophageal cancer cells. Decreased IFIT2 expression significantly increased the cellular abilities of viability, invasion and migration by using RNAi method in human esophageal cancer cells. Decreased IFIT2 expression in esophageal cancer tissues significantly correlated with EMT status, and could be used as an independent prognostic predictor for the patients. Rescue experiments in PD-L1 knockdown cells further confirmed that STAT1/IFIT2 pathway was involved in the PD-L1 mediated EMT of esophageal cancer cells. Moreover, the luciferase reporter assay also confirmed that in esophageal cancer cells, the promoter region of IFIT2 (-3K~-1K) remains more active in PD-L1 knockdown expression cells compared with controls. CONCLUSION: Our present work reveals a novel mechanism of how PD-L1 regulates EMT of cancer cells, namely STAT1/IFIT2 signal pathway is required in PD-L1 mediated EMT in human esophageal cancer.

STAT1 Is Required for Decreasing Accumulation of Granulocytic Cells via IL-17 during Initial Steps of Colitis-Associated Cancer.

Signal transducer and activator of transcription 1 (STAT1) acts as a tumor suppressor molecule in colitis-associated colorectal cancer (CAC), particularly during the very early stages, modulating immune responses and controlling mechanisms such as apoptosis and cell proliferation. Previously, using an experimental model of CAC, we reported increased intestinal cell proliferation and faster tumor development, which were consistent with more signs of disease and damage, and reduced survival in STAT1-/- mice, compared with WT counterparts. However, the mechanisms through which STAT1 might prevent colorectal cancer progression preceded by chronic inflammation are still unclear. Here, we demonstrate that increased tumorigenicity related to STAT1 deficiency could be suppressed by IL-17 neutralization. The blockade of IL-17 in STAT1-/- mice reduced the accumulation of CD11b+Ly6ClowLy6G+ cells resembling granulocytic myeloid-derived suppressor cells (MDSCs) in both spleen and circulation. Additionally, IL-17 blockade reduced the recruitment of neutrophils into intestinal tissue, the expression and production of inflammatory cytokines, and the expression of intestinal STAT3. In addition, the anti-IL-17 treatment also reduced the expression of Arginase-1 and inducible nitric oxide synthase (iNOS) in the colon, both associated with the main suppressive activity of MDSCs. Thus, a lack of STAT1 signaling induces a significant change in the colonic microenvironment that supports inflammation and tumor formation. Anti-IL-17 treatment throughout the initial stages of CAC related to STAT1 deficiency abrogates the tumor formation possibly caused by myeloid cells.

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis, inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models.

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer's disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aß1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.

In cardiac fibroblasts, interferon-beta attenuates differentiation, collagen synthesis, and TGF-ß1-induced collagen gel contraction.

Cardiac fibroblasts (CF) play a key role in the homeostasis of the extracellular matrix in cardiac tissue and are newly recognized as inflammatory supporter cells. Besides, CF-to-Cardiac myofibroblast differentiation is commanded by TGF-b, through SMAD signaling pathways, and these last cells are strongly implicated in cardiac fibrosis. In the heart IFN-ß is produced by CF; however, the role of IFN-ß, STAT proteins, and STAT-homo or heterodimers in the regulation of CF function with or without a fibrotic environment is unknown. CF were isolated from hearts of adult rats, and by western blot analysis we studied STAT1, STAT2, and STAT3 phosphorylation and through specific siRNA against these proteins we analyzed their role in CF functions such as differentiation (α-SMA expression); and pro-collagen type-I synthesis and secretion expression levels; collagen gels contraction and CF migration. In cultured adult rats CF, IFN-ß increases phosphorylation of STAT1, STAT2, and STAT3. Both STAT1 and STAT2 were involved in decreasing α-SMA and CF migration induced by TGF-ß1. Also, IFN-ß through STAT1 regulated pro-collagen type-I protein expression levels, and collagen gels contraction induced by TGF-ß1. STAT3 was not involved in any effects of IFN-ß studied. In conclusion, IFN-ß through STAT1 and STAT2 shows antifibrotic effects on CF TGF-ß1-treated, whereas STAT3 did not participate in such effect.

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis, inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer's disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aβ1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.

Immune Monitoring of Patients With Primary Immune Regulation Disorders Unravels Higher Frequencies of Follicular T Cells With Different Profiles That Associate With Alterations in B Cell Subsets.

Primary immune regulation disorders lead to autoimmunity, allergy and inflammatory conditions due to defects in the immune homeostasis affecting different T, B and NK cell subsets. To improve our understanding of these conditions, in this work we analyzed the T and B cell compartments of 15 PID patients with dysregulation, including 3 patients with STAT1 GOF mutation, 7 patients with CVID with dysregulation, 3 patients with mutations in CTLA4, 1 patient with CD25 mutation and 1 patient with STAT5b mutation and compared them with healthy donors and with CVID patients without dysregulation. CD4+ and CD8+ T cells from the patients exhibited a significant decreased frequency of naïve and regulatory T cells with increased frequencies of activated cells, central memory CD4+ T cells, effector memory CD8+ T cells and terminal effector CD8+ T cells. Patients also exhibited a significantly increased frequency of circulating CD4+ follicular helper T cells, with altered frequencies of cTfh cell subsets. Such cTfh cells were skewed toward cTfh1 cells in STAT1 GOF, CTLA4, and CVID patients, while the STAT5b deficient patient presented a skew toward cTfh17 cells. These alterations confirmed the existence of an imbalance in the cTfh1/cTfh17 ratio in these diseases. In addition, we unraveled a marked dysregulation in the B cell compartment, characterized by a prevalence of transitional and naïve B cells in STAT1 GOF and CVID patients, and of switched-memory B cells and plasmablast cells in the STAT5b deficient patient. Moreover, we observed a significant positive correlation between the frequencies cTfh17 cells and switched-memory B cells and between the frequency of switched-memory B cells and the serum IgG. Therefore, primary immunodeficiencies with dysregulation are characterized by a skew toward an activated/memory phenotype within the CD4+ and CD8+ T cell compartment, accompanied by abnormal frequencies of Tregs, cTfh, and their cTfh1 and cTfh17 subsets that likely impact on B cell help for antibody production, which likely contributes to their autoimmune and inflammatory conditions. Therefore, assessment of these alterations by flow cytometry constitutes a simple and straightforward manner to improve diagnosis of these complex clinical entities that may impact early diagnosis and patients' treatment. Also, our findings unravel phenotypic alterations that might be associated, at least in part, with some of the clinical manifestations observed in these patients.

Nova mutação no gene STAT1 associada com candidíase mucocutânea crônica / New STAT1 gene mutation associated with chronic mucocutaneous candidiasis

Mutações no gene STAT1 (signal transducer and activator of transcription 1) têm sido identificadas como responsáveis pela maioria dos casos sindrômicos da candidíase mucocutânea crônica com herança autossômica dominante (AD). Nesse artigo, descrevemos uma menina de 7 anos que apresentou candidíase da mucosa oral e unhas, além de infecção disseminada da pele e couro cabeludo por Microspora gipseum. Recentemente, a paciente foi diagnosticada e tratada de meningite por Cryptococcus neoformans. Na família não existem outros casos de candidíase. A avaliação imunológica incluiu a detecção de subpopulações de linfócitos (CD3, CD4, CD8, CD20 e células NK), assim como a dosagem de IgG, IgA, IgM e IgE, subclasses de IgG e autoanticorpos. Excluindo-se discreta diminuição de CD3, CD4, CD8, NK e leve aumento de IgG1, os demais exames estiveram dentro da normalidade. O sequenciamento do exoma detectou uma rara mutação em heterozigose no exon 14 do domínio de ligação do DNA (DNA-binding domain) do gene STAT1, ocasionando um provável ganho de função (GOF) responsável pela doença (Gly384Asp). Essa variação foi também identificada pelo sequenciamento de Sanger, não estando reportada nos bancos de dados públicos e apresentando elevado potencial de dano (índice CADD=32). Será interessante contarmos com informações clínicas e estudos com outros pacientes para conhecermos mais essa mutação patológica. Além da apresentação do caso, discutiremos as formas de tratamento existentes.

STAT1 (signal transducer and activator of transcription 1) gene mutations have been identified as responsible for most syndromic cases of chronic mucocutaneous candidiasis with autosomal dominant (AD) inheritance. In this article, we described a 7-year-old girl who presented with candidiasis of the oral mucosa and nails, as well as disseminated infection of the skin and scalp caused by Microsporum gypseum. Recently, the patient was diagnosed and treated for Cryptococcus neoformans meningitis. There are no other cases of candidiasis in the family. The immunological evaluation consisted of detection of subpopulations of lymphocytes (CD3, CD4, CD8, CD20, and NK cells), as well as measurement of IgG, IgA, IgM, and IgE, IgG subclasses, and autoantibodies. Excluding a slight decrease in CD3, CD4, CD8, NK and a minimal increase in IgG1, the others were within normal limits. Exome sequencing detected a rare heterozygous variation in exon 14 of the DNA-binding domain of the STAT1 gene, causing a probable gain of function (GOF) responsible for the disease (Gly384Asp). This variation was also identified by Sanger sequencing, but it was not reported in public databases and had a high potential for damage (Combined Annotation-Dependent Depletion [CADD] score = 32). Having clinical information and conducting studies of other patients will be helpful to learn more about this pathological mutation. In addition to the presentation of the case, we will discuss the existing forms of treatment.

Interferon-gamma signaling is downregulated in peripheral blood mononuclear cells from asthma patients / A sinalização de interferon-gama é diminuída em células mononucleares do sangue periférico de pacientes asmáticos

Introduction: Interferon-gamma (IFN-g) signaling is mediated by crosstalk of receptors, such as IFN-g receptor 1 (IFN-g R1), transcription factors, such as signal transducer and activator of transcription 1 (STAT1) and suppressors of cytokine signaling 1 (SOCS1). Here, we evaluated the role of IFN-g signaling in peripheral blood mononuclear cells (PBMCs) from asthma patients and control individuals. Methods: PBMCs from adult healthy nonasthmatic controls (n = 12; male and female, 18-60 years old) and patients diagnosed with asthma (n = 18; male and female, 18-60 years old) were stimulated with IFN-g (0.25, 0.5 and/or 1.0 ng/mL) and, after 24h, the production of CXC motif chemokine 10 (CXCL10) was evaluated (by enzyme linked immunosorbent assay) as well as the expression of IFN-g R1, STAT1 (both by flow cytometry assay) and SOCS1 (by real-time qPCR assay). Results: CXCL10 production was reduced in a dose-dependent manner in PBMCs from asthma patients stimulated with IFN-g when compared to control individuals. While IFN-g induced an increase in IFN-g R1 expression and phosphorylated STAT1 (pSTAT1) activation in PBMCs from the control group, a reduction in both IFN-g R1 and pSTAT1 was observed in PBMCs from asthma patients. IFN-g increased SOCS1 mRNA expression in PBMCs from asthma patients when compared to IFN-g-stimulated cells from control individuals. Conclusion: Taken together, our results demonstrated that IFN-g signaling is downregulated in asthma patients.

Introdução: A sinalização de interferon-gama (IFN-g) é mediada por receptores, como o receptor 1 de IFN-gama (IFN-gR1), fatores de transcrição, como o transdutor de sinal e o ativador de transcrição 1 (STAT1) e supressores de sinalização de citocina 1 (SOCS1). Neste trabalho, avaliamos o papel da sinalização de IFN-g em células mononucleares do sangue periférico (PBMCs) de indivíduos com asma e controle. Métodos: Células mononucleares do sangue periférico (PBMCs) de adultos saudáveis e não asmáticos (n = 12, homens e mulheres, 18-60 anos) e pacientes diagnosticados com asma (n = 18, homens e mulheres, 18-60 anos) foram estimuladas com IFN-g (0,25, 0,5 e/ou 1,0 ng/mL) e após 24 horas a produção de CXCL10 foi avaliada por ensaio de imunoabsorção enzimática (ELISA), bem como o receptor 1 de IFN-g (IFN-g R1). Também foram avaliadas as expressões do transdutor de sinal e ativador da transcrição 1 (STAT1) (por citometria de fluxo) e supressor de expressão de sinalização de citocinas 1 (SOCS1) (por ensaio qPCR em tempo real). Resultados: A produção de CXCL10, uma quimiocina induzida por IFNg, foi reduzida de maneira dependente da dose em PBMCs de pacientes com asma estimulados com IFN-g (0,25-1,0 ng/mL) quando comparado ao grupo controle. Enquanto IFN-g induziu um aumento da expressão de IFN-g R1 e ativação da fosforilação de STAT1 (pSTAT1) em PBMCs do grupo controle, uma redução de ambas (IFN-g R1 e pSTAT1) foi observada em PBMCs de pacientes com asma. O IFN-g aumentou as PBMCs de expressão do mRNA de SOCS1 de pacientes com asma quando comparado às células estimuladas por IFN-g do controle. Conclusão: Em conjunto, nossos resultados demonstraram que a sinalização de IFN-g é sub-regulada em pacientes com asma.

MicroRNA-548-3p overexpression inhibits proliferation, migration and invasion in osteoblast-like cells by targeting STAT1 and MAFB.

Osteoporosis is the most common bone disease and a public health issue with increasing prevalence in Mexico. This disease is caused by an imbalance in the bone remodelling process mediated by osteoclast and osteoblast. MicroRNAs have emerged as key players during the differentiation of both types of cells specialized involved in bone metabolism. We found high expression levels of miR-548x-3p in circulating monocytes derived from postmenopausal osteoporotic women. This study aimed to analyse the functional characterization of miR-548x-3p roles in the bone remodelling process. We validated by RT-qPCR, the elevated levels of miR-548x-3p in circulating monocytes derived from osteoporosis women. Through bioinformatics analysis, we identify MAFB and STAT1 as potential target genes for miR-548x-3p. Both genes showed low levels of expression in circulating monocytes derived from osteoporotic women. In addition, we demonstrated the binding of miR-548x-3p to the 3'-UTR of both mRNAs. MiR-548x-3p was overexpressed in osteoblasts-like cell lines decreasing the levels of MAFB and STAT1 mRNA and protein. We found that miR-548x-3p overexpression inhibits the proliferation, migration and invasion of the cell lines evaluated. Our results identified, by the first time, the potential role of miR-548x-3p as a modulator of the bone remodelling process by regulating the expression of MAFB and STAT1.

STAT1-NFκB crosstalk triggered by interferon gamma regulates noradrenaline-induced pineal hormonal production.

Melatonin production by pineal glands is modulated by several immune signals. The nuclear translocation of nuclear factor kappa-B (NFκB) homodimers, lacking transactivation domains, once induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF), inhibits the expression of Aanat gene and the synthesis of noradrenaline (NA)-induced melatonin. Interferon gamma (IFN-γ), on the other hand, increases melatonin synthesis. Furthermore, this cytokine activates the signal transducer as well as the activator of transcription 1 (STAT1) pathway, which was never evaluated as a melatonin synthesis modulator before. Reports demonstrated that IFN-γ might also activate NFκB. The present study evaluated the role of STAT1-NFκB crosstalk triggered by IFN-γ regarding the regulation of NA-induced pineal glands' hormonal production. Moreover, IFN-γ treatment increased NA-induced Aanat transcription, in addition to the synthesis of N-acetylserotonin (NAS) and melatonin. These effects were associated with STAT1 nuclear translocation, confirmed by the co-immunoprecipitation of STAT1 and Aanat promoter. Pharmacological STAT1 enhancement augmented NA-induced Aanat transcription as well as NAS and melatonin production. Additionally, IFN-γ induced the nuclear translocation of RelA-NFκB subunits. The blockade of this pathway prevented IFN-γ effects on the pineal function. The present data show that STAT1 and NFκB crosstalk controls melatonin production through a synergistic mechanism, disclosing a new integrative mechanism regarding pineal hormonal activity control.

Chloroquine-treated dendritic cells require STAT1 signaling for their tolerogenic activity.

MS and EAE are T cell-driven autoimmune diseases of the CNS where IL-17-producing Th17 cells promote damage and are pathogenic. Conversely, tolerogenic DCs induce Treg cells and suppress Th17 cells. Chloroquine (CQ) suppresses EAE through the modulation of DCs by unknown mechanisms. Here, we show that STAT 1 is necessary for CQ-induced tolerogenic DCs (tolDCs) to efficiently suppress EAE. We observed that CQ induces phosphorylation of STAT1 in DCs in vivo and in vitro. Genetic blockage of STAT1 abrogated the suppressive activity of CQ-treated DCs. Opposed to its WT counterparts, CQ-treated STAT1-/- BMDCs were unable to suppress Th17 cells and increased EAE severity. Our findings show that STAT1 is a major signaling pathway in CQ-induced tolDCs and may shed light on new therapeutic avenues for the induction of tolDCs in autoimmune diseases such as MS.