Reconstituição hemato-imunológica após o transplante de células estaminais hematopoiéticas: recuperação da policlonalidade dos linfócitos T e B da funcionalidade das células

RESUMO: O transplante de células estaminais hematopoiéticas (TCE) é um processo terapêutico usado no tratamento de doenças genéticas e malignas de modo a reconstituir uma hematopoiese normal e aumentar respostas antitumorais. O TCE é feito num contexto autólogo (autoTCE) de forma a recuperar a função da medula óssea após a utilização de terapia antitumoral de alta intensidade. Já o TCE alogénico (aloTCE) envolve sempre um condicionamento inicial mieloablativo, cito-redutor e imunossupressor que permite a aceitação do enxerto e simultaneamente, se for o caso, o controlo de doença tumoral. A recuperação pós-transplante envolve uma regeneração total da hematopoiese, desde a recolonização pelas células do enxerto dos nichos estaminais da medula óssea, ao desenvolvimento, maturação e diferenciação efetora das diversas linhagens celulares sanguíneas e imunológicas. A regeneração é progressiva, mas prolongada, e neste período os doentes apresentam-se severamente imunocomprometidos devido à imaturidade funcional e à restrição da diversidade estrutural dos repertórios linfocitários. No aloTCE, o processo é ainda mais dificultado com a profilaxia e/ou as complicações imunológicas derivam da aloreatividade. Este trabalho teve como objetivo fazer uma avaliação da reconstituição hematoimunológica após o autoTCE e aloTCE. Pretendeu-se, com uma análise abrangente a todos os compartimentos celulares periféricos com atividade imunológica, relacionar e integrar a dinâmica dos processos celulares observados, com os fatores clínicos do transplante e os mecanismos regulatórios da ontogenia hematopoiética e da diferenciação imunológica. Começou-se por estabelecer uma análise da consolidação da recuperação mieloide, nos grupos de doentes com autoTCE e aloTCE, com a avaliação da granulopoiese e monopoiese após o arranque hematológico. Nestas séries de doentes, a recuperação e consolidação da granulopoiese para valores no intervalo normal mostrou-se dependente das caraterísticas do enxerto. Alguns transplantes alogénicos usaram enxertos de medula óssea (MO) com uma dosagem significativamente inferior de células CD34+ comparativamente às colheitas de células estaminais mobilizadas para o sangue periférico (PBSCs). Os doentes com enxerto de MO apresentaram uma capacidade menor de recompor uma granulopoiese com contagens normais de neutrófilos aos 3, aos 6 e também aos 12 meses de evolução. A reconstituição da linfopoiese revelou dinâmicas muito diferentes para as populações e subpopulações de células NK, linfócitos B e T CD4+ e CD8+ e a cinética da recuperação e maturação de cada compartimento celular foi normalmente superior nos transplantes autólogos em relação aos alogénicos. A recuperação das células NK nos doentes com enxerto alogénico foi caraterizada por uma expansão inicial das frações celulares imaturas CD56++CD16- e CD56++CD16+. No entanto, estes doentes apresentaram um arresto da maturação do compartimento de células NK. Observou-se uma recuperação lenta das células maturas CD56dimCD16+ e níveis muito baixos da população de células NK CD56-CD16+ terminalmente diferenciadas em todos os pontos de análise. Já os doentes com transplante autólogo não apresentaram uma acumulação inicial tão extensa das células imaturas CD56++, e a meio do período da análise, as células NK maturas CD56dimCD16+ apresentaram contagens no intervalo normal. O processo de recuperação e desenvolvimento do compartimento celular NK após o transplante deverá ser muito provavelmente determinado pelos níveis de citocinas homeostáticas como a IL-15, e também, pelos níveis de células produtoras de citocinas modeladoras do desenvolvimento celular como a IL-2. De facto, neste trabalho, a evolução da recuperação e maturação das células NK foi correlacionada com a dimensão que os compartimentos de células T CD4+ e CD8+ apresentam nas fases iniciais da evolução dos transplantes. Os transplantes com contagens celulares T superiores apresentam uma expansão limitada de células imaturas CD56++ e uma maior capacidade de maturação do compartimento NK. As células T CD8+ e em especial as subpopulações de memória são particularmente sensíveis à IL-15. Os transplantes autólogos apresentam níveis iniciais mais elevados de células T comparativamente ao transplantes alogénicos provavelmente devido à ausência de um condicionamento imunossupressor presente nos segundos transplantes. Desta forma, nos transplantes autólogos e também nos doentes com transplante alogénico que fizeram uma recuperação inicial forte dos compartimentos de células T, a IL-15 disponível deverá ter sido utilizada em larga medida pela população maioritária de células T CD8+. A disponibilidade da apresentação desta citocina às células CD56++ é então limitada o que poderá justificar a expansão moderada destas células observada nestes grupos de doentes. Por outro lado, os níveis mais elevados de células T CD4+ nestes grupos possibilitam uma maior produção de IL-2, que é fundamental para o processo de maturação das células NK. Foi avaliada a diversidade genética dos genes dos recetores de células NK com domínios Ig (KIR) e foi avaliada a expressão genética do repertório de recetores das células NK no pós-transplante. A diversidade e a organização do cluster de genes KIR presente na população portuguesa está relacionada com encontrada na população caucasiana europeia. Nestas populações existe um balanceamento entre os repertórios de genes inibitórios e de ativação que resulta numa maior frequência de indivíduos com um genótipo constituído pelo haplótipos KIR A/B. A distribuição de genes KIR no transplante é determinada pela frequência dos genes KIR na população e o repertório de genes alocado no enxerto alogénico tem vindo a ser relacionado com a evolução clínica em alguns transplantes. O efeito aloreativo derivado da incompatibilidade do repertório KIR alocado e os ligandos do recetor parece ser determinante pelo menos no controlo da doença residual mínima derivada das leucemias mieloides. As interações do sistema de recetores KIR e de ligandos do HLA classe I foram avaliados nos transplantes alogénicos com dadores com diferentes graus de proximidade genética. Observou-se uma distribuição de interações KIR do dador – ligando do recetor independente da proximidade genética do par-dador recetor devida a uma segregação independente dos sistemas genéticos KIR e HLA, mas também em certa medida porque os vários painéis de dadores apresentam frequências genéticas KIR semelhantes. Foi verificada também uma expressão aleatória dos genes KIR, não dependente da presença dos ligandos HLA e no contexto alogénico foi detetado um repertório de transcritos quimérico que vai progressivamente evoluindo para o genótipo do repertório estrutural NK do dador, o que indicia uma regulação estocástica dos genes germinais. A reconstituição do compartimento linfocitário B é marcada por uma expansão de células transicionais imaturas e um atraso no processo de maturação. A análise de fatores como os sjKRECs indicadores da atividade linfopoiética B da medula óssea, a estimativa do histórico de divisões celulares e a diversidade estrutural do repertório da cadeia pesada das imunoglobulinas IGH, sugere que a recuperação celular B deverá ser estabelecida principalmente por uma neogénese celular derivada da medula óssea, enquanto os fatores homeostáticos das células B deverão promover uma sobrevivência celular, mas não proliferações expansivas. Nesta análise, verificou-se ainda que a maturação das células B, ao fim de um ano de evolução está relacionada com os níveis do primeiro semestre de sjKRECs e com a recuperação de células T CD4+. A recuperação das subpopulações T CD4+ e CD8+ é um processo imperfeito que é estabelecido em larga medida pela diferente sensibilidade de diferentes subpopulações celulares aos níveis elevados das citocinas homeostáticas do póstransplante. A recuparação da linfopenia CD4+ é mais condicionada, pois provavelmente exige intermediários celulares inibidos nas condições do póstransplante, pelo que, a reconstituição destas células parece ser feita quase em exclusivo por renovação tímica. A deficiente recuparação deste compartimento celular, que apresenta funções reguladoras centrais é condicionadora da maturação de outros compartimentos linfocitários incluindo o B e o NK. Quanto ao compartimento T CD8+ apresenta-se expandido com um repertório estrutural limitado e sinais de anergia e senescência. Estes indicadores sugerem que estas células, em particular as subpopulações T CD8+ maturas de memória, são alvo de proliferações sistémicas derivadas dos níveis elevados de citocinas homeostática a que são altamente sensiveis Em conjunto, os dados obtidos neste trabalho, estabelecem os processos de regeneração hematopoiética e reconstituição imunológica, apontando alguns fatores de condicionamento e potenciais marcadores de evolução clínica.ABSTRACT: Hematopoietic stem cell transplantation is a therapy for treatment of genetic diseases and some malignancies by fixing haematopoiesis damages or boost antitumor immune responses. Autologous grafts are used to recover severe bone marrow depleted cancer patients after high-dose chemotherapy. Allogeneic transplantation uses a myeloablative conditioning that is immunosuppressive allowing engraftment and, if necessary, provides tumour control. Post-transplant recovery includes a complete regeneration of hematopoiesis with recolonization by graft of hematopoietic stem cell niches and a progressive ontogeny settlement for all blood cell lineages. This is however; a time-delayed process on which patients’ immune status is severely compromised due to immaturity and skewing of strutural diversity of lymphocytes repertoires. Progress of allogeneic transplant patients is even more unsettled due to alloreactivity prophylaxis and related immune complications. This study aims to evaluate the hemato-immunological reconstitution after autologous and allogeneic stem cell transplantation. It was established an analysis extended to several peripheral cellular immunological effectors, in order to correlate the recovery dynamics with transplant related factors and regulatory mechanisms of cell ontogeny and immunological differentiation. It is well established that the engraftment kinetics is highly conditioned by the graft stem cell dose. In this analysis, we focused our interest in the assessment of factors affecting the consolidation of myelopoiesis after transplantation by evaluating the midterm recovery of granulopoiesis and monopoiesis. Some of the transplants of the allogeneic series used a bone marrow (BM) graft with a significantly lower CD34+ cell dosage when compared with peripheral blood mobilized stem cell grafts (PBSCs). During the time-line of the analysis a higher proportion of patients with a BM transplant showed neutrophils absolute counts beneath the normal range. Consequently, the graft type and stem cell dosage seem to be a factor affecting not the initial engraftment kinetics but also the mid-term consolidation of the myeloid recovery. Each distinct lymphocyte cell type and subtype presented a specific reconstitution dynamic and the overall recovery kinetics of cellular levels and even maturation rate was found higher in autologous transplantation than in the allogeneic settings. Allogeneic transplant patients showed an early expansion of immature CD56++CD16- and CD56++CD16+ NK cell subsets. However, the maturation of NK cell compartment was found severely compromised. These patients presented a slow recovery of mature cytotoxic CD56dimCD16+ NK cell counts and levels of terminally differentiated CD56- CD16+ cell were depleted at all time points of the analysis. The NK cell compartment of autologous transplants showed a different recovery and maturation rate. These patients showed median immature CD56++ cell levels in normal range since the earliest evaluation at 3 months and also presented a better maturation rate. By the second semester after transplant, mature CD56dimCD16+ cell counts reached the lower boundary of normal range and also terminally differentiated CD56-CD16+ cells gone normal in the analysis time line. The reconstitution of the NK cell compartment in the different transplant settings is likely driven by a different exposure of NK cell subsets to homeostatic cytokines such as IL-15 and others such as IL-2. Furthermore, according to the data here described, it is a process highly conditioned by post-transplant CD4+ T cell levels and to a lesser extend with levels of CD8+ T cells. In a low T cell posttransplant condition, as seen in allo-HSCT patients, a large ratio of circulating NK cells were the immature CD56++. Auto-HSCT patients, with higher T cell levels, showed an enhanced NK cell development with no accumulation of immature subsets. It is possible that an effective consumption of homeostatic cytokines by an early dominant CD8+ T cell population determines a limited availability to other sensitive cells such as immature NK cells. Additionally, and while other regulatory mechanisms cannot be excluded, the observed enhancement of NK cells maturation with high levels of CD4+ T cells, is most probably a result of endogenous T cell derived IL-2; that acting through the high-affinity IL-2 receptor of CD56++ NK cells induce their maturation into CD56++ cells with cytoxicity competence. This is a process that is probably severely conditioned by the immunosuppressive regiments of allogeneic transplantation. It was also established the KIR genotypic diversity in Portuguese, the allocation of KIR genes and genetic expression after transplantation. The diversity of KIR gene cluster was evaluated in Portuguese and the genotypic profiles observed were found related to the ones covered in European neighbouring populations with a balance between the activation and inhibitory KIR genes repertoire. These gene frequencies determine the KIR repertoire allocated after stem cell transplantation, which was associated with the allogeneic post-transplant outcome. It has been hypothesized that donor-derived alloreactive NK cells target recipient hematopoietic cells resulting in an antileukemia effect and a lower incidence of graft rejection. However, those effects depend on multiple factors, including disease type, presence of T cells, immunosuppressants and the repertoires of allocated KIR genes and recipient HLA ligands. Although nonuniformly demonstrated, the capture of NK cell alloreactivity is probably shaped by a KIR ligand incompatibility either in the setting of a HLA class I mismatched transplant, or in the presence of a donor KIR loci for which there is no recipient ligand. We evaluated donor-KIR/recipient-ligand interactions in transplants with related donors and unrelated donors found in a local registry or from abroad. A significant fraction of these transplants had all three ligands of inhibitory receptors, and therefore, in theory were not prone to NK mediated alloreactivity. The distribution of KIR alloreactive interactions however, was found independent of the donor-recipient genetic proximity, almost certainly due to different gene segregation and a comparable KIR frequencies in the donor pools. KIR gene expression seems to be established mainly by stochastic regulation of germinal genes and not dependent on HLA ligand presence. Allogeneic transplants showed a chimeric KIR repertoire that was gradually replaced by a donor derived genotype. B cell compartment showed a distinct expansion of immature transitional cells and a maturation arrest. Results from sjKRECs quantifications indicates a recovery of B cell absolute counts mainly by bone marrow neogenesis. Analysis of cell divisions estimates and IGH repertoire diversity suggests that B cell homeostatic factors promote cell survival but not expansive proliferations. Subsequent recovery of mature and memory B cells was found correlated with early sjKRECs high levels and T CD4+ cell recovery. Within this analysis period, CD4+ and CD8+ T cell recovery was found highly deficient. It is modulated by cell sensitivity to post-transplant high levels of homeostatic cytokines and thymic activity. Homeostatic control of CD4+ lymphopenia probably requires a cellular intermediate, not readily available, making recovery almost completely dependent on thymic renewal. T CD8+ cell compartment was found highly disproportional with a skewed repertoire and signs of anergy and senescence. These are signs of systemic cell proliferations made by mature T CD8+ due to high sensitivity to increased homeostatic cytokines levels. Altogether, the data obtained in this study helps to establish the dynamics of hematological regeneration and immunological reconstitution, presenting some efficiency related factors and potential tools for monitoring transplant outcome

Suppression by allogeneic-specific regulatory T cells is dependent on the degree of HLA compatibility

Copyright © 2021 The Authors. This article is distributed under the terms of the CC BY 4.0 Unported license.Regulatory T cell (Treg) infusion for graft-versus-host disease treatment has been increasingly investigated. However, polyclonal Treg may suppress the desired graft-versus-leukemia effect. Although allogeneic-specific (allo-specific) Treg may provide a more-targeted graft-versus-host disease treatment, there is the need to develop easily translatable expansion protocols and to better characterize their specificity and mechanisms of suppression. In this article, we provide a robust protocol for human allo-specific Treg expansion and characterize their phenotype, potency, and specificity of suppression by testing different expansion conditions and suppression assay milieus. We found that higher concentrations of IL-2 during expansion with allogeneic APC yielded allo-specific Treg that were more-potent suppressors and displayed a more activated phenotype. Although responses to the same APC present during expansion were the most suppressed, responses to third-party APC partially matched to the expansion APC were still significantly more suppressed than responses to fully mismatched APC. Furthermore, suppression of responses to the expansion APC was strictly contact dependent, whereas suppression of responses to mismatched APC was partially independent of contact. Finally, distinct subsets in fresh and expanded Treg could be described using multidimensional visualization techniques. We propose that allo-specific Treg are HLA specific and that the mechanisms of suppression elicited depend on their compatibility with the stimulators.This work was supported by Fundação para a Ciência e Tecnologia, Portugal, under the Harvard Medical School–Portugal Program Project Induction of Immune Tolerance in Human Allogeneic Hematopoietic Stem Cell Transplantation (HMSP-ICT/0001/2011) and Gilead Sciences, under Programa Gilead GÉNESE (FPJ001262). J.B. received a Ph.D. grant from Fundação para a Ciência e Tecnologia (PD/BD/105769/2014).info:eu-repo/semantics/publishedVersio

New Insights on Innate Immune Response by Blood Macrophages and Liver Kupffer Cells to Leishmania infantum Parasites

Funding Information: Funding: This study was supported by FCT-Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/28908/2017 and PTDC/CVT-CVT/0228/2020, and by national funds within the scope of Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA, UIDB/00276/2020) and Global Health and Tropical Medicine (GHTM, UID/04413/2020). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.L. infantum is the aetiological agent of zoonotic visceral leishmaniasis (ZVL), a disease that affects humans and dogs. Leishmania parasites are well adapted to aggressive conditions inside the phagolysosome and can control the immune activation of macrophages (MØs). Although MØs are highly active phagocytic cells with the capacity to destroy pathogens, they additionally comprise the host cells for Leishmania infection, replication, and stable establishment in the mammal host. The present study compares, for the first time, the innate immune response to L. infantum infection of two different macrophage lineages: the blood macrophages and the liver macrophages (Kupffer cells, KC). Our findings showed that L. infantum takes advantage of the natural predisposition of blood-MØs to phagocyte pathogens. However, parasites rapidly subvert the mechanisms of MØs immune activation. On the other hand, KCs, which are primed for immune tolerance, are not extensively activated and can overcome the dormancy induced by the parasite, exhibiting a selection of immune mechanisms, such as extracellular trap formation. Altogether, KCs reveal a different pattern of response in contrast with blood-MØs when confronting L. infantum parasites. In addition, KCs response appears to be more efficient in managing parasite infection, thus contributing to the ability of the liver to naturally restrain Leishmania dissemination.publishersversionpublishe

Characterization of the major histocompatibility complex locus association with Behçet’s disease in Iran

© 2015 Xavier et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Introduction: The aim of this study was to characterize the association of human leukocyte antigen (HLA) B alleles and major histocompatibility complex (MHC) single nucleotide polymorphisms (SNPs) with Behçet's disease (BD) in an Iranian dataset. Methods: The association of three SNPs in the MHC region previously identified as the most associated in high-density genotyping studies was tested in a case-control study on 973 BD patients and 825 controls from Iran, and the association of HLA-B alleles was tested in a subset of 681 patients and 414 controls. Results: We found that HLA-B*51 (P = 4.11 × 10(-41), OR [95% CI] = 4.63[3.66-5.85]) and B*15 confer risk for BD (P = 2.83 × 10(-2), OR [95% CI] = 1.75[1.08-2.84]) in Iranian, and in B*51 negative individuals, only the B*15 allele is significantly associated with BD (P = 2.51 × 10(-3), OR [95% CI] = 2.40[1.37-4.20]). rs76546355, formerly known as rs116799036, located between HLA-B and MICA (MHC class I polypeptide-related sequence A), demonstrated the same level of association with BD as HLA-B*51 (P adj = 1.78 × 10(-46), OR [95% CI] = 5.46[4.21-7.09], and P adj = 8.34 × 10(-48), OR [95% CI] = 5.44[4.20-7.05], respectively) in the HLA-B allelotyped subset, while rs2848713 was less associated (P adj = 7.14 × 10(-35), OR [95% CI] = 3.73[2.97-4.69]) and rs9260997 was not associated (P adj = 1.00 × 10(-1)). Additionally, we found that B*51 genotype-phenotype correlations do not survive Bonferroni correction, while carriers of the rs76546355 risk allele predominate in BD cases with genital ulcers, positive pathergy test and positive BD family history (2.31 × 10(-4) ≤ P ≤ 1.59 × 10(-3)). Conclusions: We found that the HLA-B*51 allele and the rs76546355/rs116799036 MHC SNP are independent genetic risk factors for BD in Iranian, and that positivity for the rs76546355/rs116799036 risk allele, but not for B*51, does correlate with specific demographic characteristics or clinical manifestations in BD patients.This work was supported by the Portuguese Fundação para a Ciência e a Tecnologia (grants PTDC/SAU-GMG/098937/2008, PTDC/IIM-GES/5015/2012 and CMUP-ERI/TPE/0028/2013, fellowships SFRH/BD/43895/2008 to JMX, SFRH/BPD/35737/2007 to PA, SFRH/BPD/70008/2010 to IS, a Ciência and an Investigator-FCT contract to SAO), and the Research Committee of the Tehran University of Medical Sciences (grant 132/714).info:eu-repo/semantics/publishedVersio

immune activation of canine hepatic spheroids exposed to leishmania infantum

The application of innovative three-dimensional (3D) spheroids cell culture strategy to Parasitology offers the opportunity to closely explore host–parasite interactions. Here we present a first report on the application of 3D hepatic spheroids to unravel the immune response of canine hepatocytes exposed to Leishmania infantum. The liver, usually considered a major metabolic organ, also performs several important immunological functions and constitutes a target organ for L. infantum infection, the etiological agent of canine leishmaniasis (CanL), and a parasitic disease of major veterinary and public health concern. 3D hepatic spheroids were able to sense and immunologically react to L. infantum parasites, generating an innate immune response by increasing nitric oxide (NO) production and enhancing toll-like receptor (TLR) 2 and interleukin-10 gene expression. The immune response orchestrated by canine hepatocytes also lead to the impairment of several cytochrome P450 (CYP450) with possible implications for liver natural xenobiotic metabolization capacity. The application of meglumine antimoniate (MgA) increased the inflammatory response of 3D hepatic spheroids by inducing the expression of Nucleotide oligomerization domain (NOD)-like receptors 1 and NOD2 and TLR2, TLR4, and TLR9 and enhancing gene expression of tumour necrosis factor α. It is therefore suggested that hepatocytes are key effector cells and can activate and orchestrate the immune response to L. infantum parasites.publishersversionpublishe

The rare DRB1*04:08-DQ8 haplotype is the main HLA class II genetic driver and discriminative factor of Early-onset Type 1 diabetes in the Portuguese population

IntroductionEarly-onset Type 1 diabetes (EOT1D) is considered a disease subtype with distinctive immunological and clinical features. While both Human Leukocyte Antigen (HLA) and non-HLA variants contribute to age at T1D diagnosis, detailed analyses of EOT1D-specific genetic determinants are still lacking. This study scrutinized the involvement of the HLA class II locus in EOT1D genetic control.MethodsWe conducted genetic association and regularized logistic regression analyses to evaluate genotypic, haplotypic and allelic variants in DRB1, DQA1 and DQB1 genes in children with EOT1D (diagnosed at ≤5 years of age; n=97), individuals with later-onset disease (LaOT1D; diagnosed 8-30 years of age; n=96) and nondiabetic control subjects (n=169), in the Portuguese population. ResultsAllelic association analysis of EOT1D and LaOT1D unrelated patients in comparison with controls, revealed that the rare DRB1*04:08 allele is a distinctive EOT1D susceptibility factor (corrected p-value=7.0x10-7). Conversely, the classical T1D risk allele DRB1*04:05 was absent in EOT1D children while was associated with LaOT1D (corrected p-value=1.4x10-2). In corroboration, HLA class II haplotype analysis showed that the rare DRB1*04:08-DQ8 haplotype is specifically associated with EOT1D (corrected p-value=1.4x10-5) and represents the major HLA class II genetic driver and discriminative factor in the development of early onset disease.DiscussionThis study uncovered that EOT1D holds a distinctive spectrum of HLA class II susceptibility loci, which includes risk factors overlapping with LaOT1D and discriminative genetic configurations. These findings warrant replication studies in larger multicentric settings encompassing other ethnicities and may impact target screening strategies and follow-up of young children with high T1D genetic risk as well as personalized therapeutic approaches

Whole blood transcriptional profiling in ankylosing spondylitis identifies novel candidate genes that might contribute to the inflammatory and tissue-destructive disease aspects

Introduction: A number of genetic-association studies have identified genes contributing to ankylosing spondylitis (AS) susceptibility but such approaches provide little information as to the gene activity changes occurring during the disease process. Transcriptional profiling generates a 'snapshot' of the sampled cells' activity and thus can provide insights into the molecular processes driving the disease process. We undertook a whole-genome microarray approach to identify candidate genes associated with AS and validated these gene-expression changes in a larger sample cohort

Genetic variation in PFKFB3 impairs antifungal immunometabolic responses and predisposes to Invasive Pulmonary Aspergillosis

Copyright © 2021 Gonçalves et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Activation of immune cells in response to fungal infection involves the reprogramming of their cellular metabolism to support antimicrobial effector functions. Although metabolic pathways such as glycolysis are known to represent critical regulatory nodes in antifungal immunity, it remains undetermined whether these are differentially regulated at the interindividual level. In this study, we identify a key role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the immunometabolic responses to Aspergillus fumigatus. A genetic association study performed in 439 recipients of allogeneic hematopoietic stem cell transplantation (HSCT) and corresponding donors revealed that the donor, but not recipient, rs646564 variant in the PFKFB3 gene increased the risk of invasive pulmonary aspergillosis (IPA) after transplantation. The risk genotype impaired the expression of PFKFB3 by human macrophages in response to fungal infection, which was correlated with a defective activation of glycolysis and the ensuing antifungal effector functions. In patients with IPA, the risk genotype was associated with lower concentrations of cytokines in the bronchoalveolar lavage fluid samples. Collectively, these findings demonstrate the important contribution of genetic variation in PFKFB3 to the risk of IPA in patients undergoing HSCT and support its inclusion in prognostic tools to predict the risk of fungal infection in this clinical setting. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. Activation of glycolysis is essential for innate immune cells to mount effective antifungal responses. In this study, we report the contribution of genetic variation in the key glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) to the risk of invasive pulmonary aspergillosis (IPA) after allogeneic hematopoietic stem cell transplantation. The PFKFB3 genotype associated with increased risk of infection was correlated with an impairment of the antifungal effector functions of macrophages in vitro and in patients with IPA. This work highlights the clinical relevance of genetic variation in PFKFB3 to the risk of IPA and supports its integration in risk stratification and preemptive measures for patients at high risk of IPA.This work was supported by the Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-SER/29635/2017, PTDC/MED-GEN/28778/2017, UIDB/50026/2020, and UIDP/50026/2020), the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000039), the Institut Mérieux (Mérieux Research Grant 2017), the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017), the European Union’s Horizon 2020 research and innovation program under grant agreement no. 847507, and the “la Caixa” Foundation (ID 100010434) and FCT under the agreement LCF/PR/HR17/52190003. Individual support was provided by FCT (SFRH/BD/136814/2018 to S.M.G., PD/BD/137680/2018 to D.A., CEECIND/04058/2018 to C.C., and CEECIND/03628/2017 to A.C.). M.G.N. was supported by an ERC Advanced Grant and a Spinoza Grant of the Netherlands Organization for Scientific Research.info:eu-repo/semantics/publishedVersio

IL-10 overexpression predisposes to invasive aspergillosis by suppressing antifungal immunity

© 2017 American Academy of Allergy, Asthma & ImmunologyProinflammatory immune responses are critically required for antimicrobial host defenses; however, excessive inflammation has the potential to damage host tissues thereby paradoxically contributing to the progression of infection. A central negative regulator of inflammatory responses is IL-10, an immunosuppressive cytokine with a wide variety of functions across multiple cell types. Although the role of IL-10 during infection appears to vary for different microorganisms, a largely detrimental role has been attributed to this cytokine during fungal disease. Given the variable risk of infection and its outcome among patients with comparable predisposing factors, susceptibility to invasive aspergillosis (IA) is thought to rely largely on genetic predisposition. The initial investigation of genetic variability at the IL10 locus led to the identification of single nucleotide polymorphisms (SNPs) influencing its transcriptional activity; thus, IL-10 may be a reasonable candidate for the genetic regulation of susceptibility to IA in high-risk patients.Supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), the Fundação para a Ciência e Tecnologia (FCT) (contracts IF/00735/2014 to A.C., IF/01390/2014 to E.T., IF/00021/2014 to R.S., and SFRH/BPD/96176/2013 to C.C.), the Conselho de Reitores das Universidades Portuguesas (CRUP), Portugal (Ações Integradas Luso-Alemãs A-43/16), the Deutscher Akademischer Austauschdienst (DAAD) (project-ID 57212690), the Fondo de Investigaciones Sanitarias (Madrid, Spain) (grant #PI12/02688) and the ERA-NET PathoGenoMics (grant #0315900A).info:eu-repo/semantics/publishedVersio

ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines

<p>Abstract</p> <p>Background</p> <p>The T antigen is a tumor-associated structure whose sialylated form (the sialyl-T antigen) involves the altered expression of sialyltransferases and has been related with worse prognosis. Since little or no information is available on this subject, we investigated the regulation of the sialyltransferases, able to sialylate the T antigen, in bladder cancer progression.</p> <p>Methods</p> <p>Matched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: <it>ST3Gal.I</it>, <it>ST3Gal.II </it>and <it>ST3Gal.IV </it>mRNA level by real-time PCR. Sialyl-T antigen was detected by dot blot and flow cytometry using peanut lectin. Sialyltransferase activity was measured against the T antigen in the cell lines.</p> <p>Results</p> <p>In nonmuscle-invasive bladder cancers, <it>ST3Gal.I </it>mRNA levels were significantly higher than corresponding urothelium (p < 0.001) and this increase was twice more pronounced in cancers with tendency for recurrence. In muscle-invasive cancers and matching urothelium, <it>ST3Gal.I </it>mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed <it>ST3Gal.I </it>mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the <it>ST3Gal.I </it>mRNA level, the ST activity (r = 0.99; p = 0.001) and sialyl-T antigen expression, demonstrating that sialylation of T antigen is attributable to ST3Gal.I. The expression of sialyl-T antigens was found in patients' bladder tumors and urothelium, although without a marked relationship with mRNA level. The two <it>ST3Gal.I </it>transcript variants were also equally expressed, independently of cell phenotype or malignancy.</p> <p>Conclusion</p> <p>ST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of <it>ST3Gal.I </it>seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.</p