Microbiota development and mucosal IgA responses during childhood in health and allergic disease

Tesis por compendio[ES] Antecedentes: Los patrones de colonización microbiana alterados durante la infancia pueden ser en parte responsables del aumento de enfermedades alérgicas en los países desarrollados. La microbiota intestinal difiere en composición y diversidad durante los primeros meses de vida en niños que luego desarrollan o no una enfermedad alérgica. Sin embargo, poco se sabe sobre la importancia de las respuestas inmunitarias tempranas de la mucosa a la microbiota intestinal en el desarrollo de alergias infantiles. Además, los estudios con respecto al efecto protector de la microbiota de la leche materna en el riesgo de desarrollar alergias no han sido concluyentes. Aunque la cavidad bucal es el primer lugar de encuentro entre la mayoría de los antígenos exógenos y el sistema inmunológico, no existen datos sobre la influencia de las bacterias orales en el desarrollo de alergias durante la infancia. Objetivos: El objetivo general de esta tesis fue evaluar la composición y diversidad microbiana en muestras orales, intestinales y de leche materna, junto con su interacción con IgA, para estudiar el papel de la colonización microbiana durante edades tempranas de la vida en condiciones de salud y de enfermedad alérgica. Sujetos: Los bebés y las madres incluidas en este estudio forman parte del ensayo aleatorio doble ciego más grande de Suecia, entre 2001 y 2003, donde se evaluaron los posibles efectos preventivos sobre la alergia de Lactobacillus reuteri ATCC 55730 hasta los 2 y 7 años. En esta tesis, utilizamos muestras de heces recogidas a los 1 y 12 meses, y muestras orales de bebés, obtenidas longitudinalmente a los 3, 6, 12, 24 meses y 7 años. Además, analizamos muestras de leche materna, recogidas a un mes después del parto de las madres correspondientes. Métodos: Se utilizaron tecnologías de secuenciación de segunda generación dirigidas al gen 16S rARN, en combinación con citometría de células marcadas por fluorescencia, para abordar las respuestas de IgA de la mucosa hacia las bacterias intestinales y de la leche materna. Además, se utilizó la secuenciación del gen 16S para describir la colonización oral de la microbiota, en muestras de saliva, de niños que desarrollaron alergias o de aquellos que se mantuvieron sanos. Los niveles de carga bacteriana en diferentes hábitats microbianos se obtuvieron mediante la metodología de qPCR y los niveles totales de IgA de las muestras de heces se determinaron mediante inmuno-ensayo ELISA. Resultados y conclusión: La colonización de la cavidad bucal durante la infancia temprana es progresiva, aumenta en complejidad con el tiempo, y varios factores externos parecen influir en gran medida en la maduración de la microbiota oral, ya sea con un impacto a corto o largo plazo. Los cambios tempranos en la composición microbiana oral parecen influir en la maduración inmune y el desarrollo de alergias en la infancia, y la presencia de especies bacterianas específicas puede ser importante para este proceso. Además, las respuestas de IgA alteradas hacia la microbiota intestinal durante la infancia precedieron a las manifestaciones de asma y alergia durante los primeros 7 años de vida, y el consumo de leche materna con una riqueza microbiana reducida en el primer mes de vida puede aumentar el riesgo de desarrollar alergia durante la infancia. Los hallazgos observados en la presente tesis deben confirmarse en cohortes más grandes y la importancia de los factores ambientales postnatales para el desarrollo temprano de la microbiota debe abordarse más a fondo. Las investigaciones futuras deben ir más allá de la caracterización de la composición de la comunidad bacteriana e investigar los mecanismos funcionales entre los microorganismos colonizadores tempranos, la maduración inmunitaria y la alergia, así como el desarrollo del asma durante la infancia.[CA] Antecedents: S'ha proposat que els patrons de colonització microbiana alterats durant la infància podrien ser en part els responsables de l'augment de malalties al·lèrgiques als països desenvolupats. La microbiota intestinal difereix en composició i diversitat durant els primers mesos de vida en els nens que després van desenvolupar una malaltia al·lèrgica. No obstant això, poc es sap sobre la importància de les respostes immunes de la mucosa a la microbiota intestinal en el desenvolupament d'al·lèrgies infantils. A més, les investigacions amb relació a l'efecte protector de la microbiota de la llet materna en el risc de desenvolupar al·lèrgies no han sigut concloents. Encara que la cavitat bucal és el primer lloc de trobada entre la majoria dels gèneres externs i el sistema immunològic, encara no s'ha descobert la influència dels bacteris en el desenvolupament d'una al·lèrgia durant la infància. Objectius: L'objectiu general d'aquesta tesi va ser avaluar la composició microbiana i la diversitat de mostres orals, fecals i llet materns, juntament amb la seva interacció amb IgA, per estudiar el paper del desenvolupament microbià durant el període de la infància primerenca a la salut i la malaltia al·lèrgica. Subjectes: Les mares i xiquets inclosos en aquest estudi formen part d'un estudi aleatori doble-cec a Suècia, entre el 2001 i el 2003, on es van avaluar els possibles efectes preventius de la suplementació amb Lactobacillus ATCC 55730 fins als 2 i 7 anys. En aquesta tesi, s'utilitzaren mostres de bebès arreplegades longitudinalment, obtinguts a 1 i 12 mesos, 3, 6, 12, 24 mesos i 7 anys, respectivament. A més, s'analitzaren les mostres de llet materna, arreplegades a un mes postpart de les corresponents mares. Mètodes: S'han utilitzat tecnologies de seqüenciació de nova generació dirigides al ARNr 16S, en combinació amb la classificació de les cèl·lules activades, per abordar les respostes de la mucosa cap als bacteris intestinals i de la llet materna. A més, s'utilitzà la seqüenciació d'Illumina MiSeq del gen 16S per descriure la colonització microbiana oral, i es van obtenir mostres longitudinals de saliva de menuts que varen desenvolupar al·lèrgies i d'alguns que es van mantenir saludables. Els nivells de càrrega bacteriana en diferents nínxols microbians s'han obtingut mitjançant la metodologia de qPCR i els nivells totals d'IgA de les mostres fecals es determinaren mitjançant l'immunoassaig ELISA. Resultats i conclusions: La colonització de la cavitat bucal durant la primera infància és transitòria, augmenta la seva complexitat amb el temps, i diversos factors externs influeixen en gran mesura el procés de maduració de la microbiota oral, amb un impacte a curt i llarg termini. Els canvis primerencs en la composició microbiana oral pareixen influir en la maduració del sistema immunològic i el desenvolupament d'al·lèrgies a la infància, així com la presència d'espècies bacterianes específiques pot ser important per a aquest progrés. A més, les respostes d'IgA alterades cap a la microbiota intestinal durant la infància precedeixen a les manifestacions relatives a la malaltia asmàtica i al·lèrgiques durant els primers 7 anys de vida. Per altra banda, el consum de llet materna amb una microbiota de riquesa reduïda al primer mes de vida podria augmentar el risc de desenvolupar al·lèrgia durant la infància. Els resultats observats en aquest estudi haurien de confirmar-se en cohorts humanes més grans i la importància dels factors ambientals post natals que influeixen en el desenvolupament de la microbiota primerenca han de ser més estudiats. Les investigacions futures deuen anar més enllà de la caracterització de la composició de la comunitat bacteriana i investigar els mecanismes funcionals entre els microorganismes colonitzadors primerencs, la maduració del sistema immunològic i el desenvolupament de l'al·lèrgia i l'asma durant la in[EN] Background: It has been proposed that altered microbial colonization patterns during infancy may be partly responsible for the increase of allergic diseases in developed countries. The gut microbiota differs in composition and diversity during the first months of life in children who later do or do not develop allergic disease. However, little is known about the significance of early mucosal immune responses to the gut microbiota in childhood allergy development, and the findings regarding the protective effect of breastmilk microbiota in the risk of allergy development have been inconclusive. Furthermore, even though the oral cavity is the first site of encounter between a majority of foreign antigens and the immune system, the influence of oral bacteria on allergy development during childhood has not yet been reported. Objectives: The general aim of this thesis was to assess the microbial composition and diversity of oral, fecal and breastmilk samples, together with its interaction with IgA, in order to study the role of microbial development during early childhood in health and allergic disease. Subjects: The infants and mothers included in this study were part of a larger randomized double-blind trial in Sweden, between 2001 and 2003, where potential allergy preventive effects of Lactobacillus reuteri ATCC 55730 were evaluated until 2 and 7 years of age. In this thesis, we used longitudinally collected stool and oral samples from infants, obtained at 1 and 12 months and 3, 6, 12, 24 months and 7 years of age, respectively. Furthermore, we analyzed breastmilk samples, collected at one month post partum, from the corresponding mothers. Methods: Next-generation sequencing technologies targeting the 16S rRNA gene, in combination with cell activated cell sorting, were used in order to address mucosal IgA responses towards gut and breastmilk bacteria. Furthermore, sequencing of the 16S rRNA gene was used in order to describe oral microbiota colonization, in longitudinally obtained saliva samples, from children developing allergy or staying healthy. Bacterial load levels in different microbial habitats were obtained by qPCR methodology and total IgA levels of stool samples were determined by ELISA immunoassays. Results and conclusion: Colonization of the oral cavity during early childhood is transitional, increasing in complexity with time, and several external factors appear to greatly influence oral microbiota maturation, having either a short or a long-term impact. Early changes in oral microbial composition seem to influence immune maturation and allergy development in childhood, and the presence of specific bacterial species may be important for this progress. Furthermore, altered IgA responses towards the gut microbiota during infancy preceded asthma and allergy manifestations during the first 7 years of life, and consumption of breastmilk with a reduced microbial richness in the first month of life may increase the risk for allergy development during childhood. Findings observed here need to be confirmed in larger cohorts and the importance of postnatal environmental factors for early microbiota development should be addressed further. Future research should go beyond characterization of bacterial community composition and investigate the functional mechanisms between early colonizing microorganisms, immune maturation and allergy and asthma development during childhood.Dzidic, M. (2019). Microbiota development and mucosal IgA responses during childhood in health and allergic disease [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125479TESISCompendi

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

Bugging allergy; role of pre-, pro- and synbiotics in allergy prevention

Large-scale biodiversity loss and complex changes in social behaviors are altering human microbial ecology. This is increasingly implicated in the global rise in inflammatory diseases, most notably the "allergy epidemic" in very early life. Colonization of human ecological niches, particularly the gastrointestinal tract, is critical for normal local and systemic immune development and regulation. Disturbances in composition, diversity and timing of microbial colonization have been associated with increased allergy risk, indicating the importance of strategies to restore a dysbiotic gut microbiota in the primary prevention of allergic diseases, including the administration of probiotics, prebiotics and synbiotics. Here, we summarize and discuss findings of randomized clinical trials that have examined the effects of these microbiome-related strategies on short and long-term allergy preventative effects - including new guidelines from the World Allergy Organization which now recommend probiotics and prebiotics for allergy prevention under certain conditions. The relatively low quality evidence, limited comparative studies and large heterogeneity between studies, have collectively hampered recommendations on specific probiotic strains, specific timing and specific conditions for the most effective preventive management. At the same time the risk of using available products is low. While further research is needed before specific practice guidelines on supplement probiotics and prebiotics, it is equally important that the underlying dietary and lifestyle factors of dysbiosis are addressed at both the individual and societal levels

Allergy development is associated with consumption of breastmilk with a reduced microbial richness in the first month of life

Background Early colonization with a diverse microbiota seems to play a crucial role for appropriate immune maturation during childhood. Breastmilk microbiota is one important source of microbes for the infant, transferred together with maternal IgA antibodies. We previously observed that allergy development during childhood was associated with aberrant IgA responses to the gut microbiota already at 1 month of age, when the IgA antibodies are predominantly maternally derived in breastfed infants. Objective To determine the microbial composition and IgA-coated bacteria in breastmilk in relation to allergy development in children participating in an intervention trial with pre- and post-natal Lactobacillus reuteri supplementation. Methods A combination of flow cytometric cell sorting and 16S rRNA gene sequencing was used to characterize the bacterial recognition patterns by IgA in breastmilk samples collected one month post-partum from 40 mothers whose children did or did not develop allergic and asthmatic symptoms during the first 7 years of age. Results The milk fed to children developing allergic manifestations had significantly lower bacterial richness, when compared to the milk given to children that remained healthy. Probiotic treatment influenced the breastmilk microbiota composition. However, the proportions of IgA-coated bacteria, the total bacterial load and the patterns of IgA-coating were similar in breastmilk between mothers of healthy children and those developing allergies. Conclusion Consumption of breastmilk with a reduced microbial richness in the first month of life may play an important role in allergy development during childhood.Funding Agencies|Spanish Ministry of Economy and Competitiveness [BIO2015-68711-R]; Swedish Research CouncilSwedish Research Council [2016-01698]; Swedish Heart and Lung FoundationSwedish Heart-Lung Foundation [20140321, 20170365]; Cancer and Allergy Foundation; European Research Council (ERC)European Research Council (ERC)Estonian Research Council [639226]</p

Gut Microbiota and Mucosal Immunity in the Neonate

Gut microbiota colonization is a complex, dynamic, and step-wise process that is in constant development during the first years of life. This microbial settlement occurs in parallel with the maturation of the immune system, and alterations during this period, due to environmental and host factors, are considered to be potential determinants of health-outcomes later in life. Given that host–microbe interactions are mediated by the immune system response, it is important to understand the close relationship between immunity and the microbiota during birth, lactation, and early infancy. This work summarizes the evidence to date on early gut microbiota colonization, and how it influences the maturation of the infant immune system and health during the first 1000 days of life. This review will also address the influence of perinatal antibiotic intake and the importance of delivery mode and breastfeeding for an appropriate development of gut immunity.The authors would like to acknowledge the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (ERC Starting Grant, project no. 639226) and the grants AGL2015-707487-P and BIO2015-68711-R from the Spanish Ministerio de Economía, Industria y Competitividad (MINECO). MSR would like to acknowledge the support from the Ayudas para la Contratación de Personal Investigador en Formación (ACIF) program of Generalitat Valenciana with the European Social Fund (ESF).Peer reviewe

Aberrant IgA responses to the gut microbiota during infancy precede asthma and allergy development

Background: Although a reduced gut microbiota diversity and low mucosal total IgA levels in infancy have been associated with allergy development, IgA responses to the gut microbiota have not yet been studied. Objective: We sought to determine the proportions of IgA coating together with the characterization of the dominant bacteria, bound to IgA or not, in infant stool samples in relation to allergy development. Methods: A combination of flow cytometric cell sorting and deep sequencing of the 16S rDNA gene was used to characterize the bacterial recognition patterns by IgA in stool samples collected at 1 and 12 months of age from children staying healthy or having allergic symptoms up to 7 years of age. Results: The children with allergic manifestations, particularly asthma, during childhood had a lower proportion of IgA bound to fecal bacteria at 12months of age compared with healthy children. These alterations cannot be attributed to differences in IgA levels or bacterial load between the 2 groups. Moreover, the bacterial targets of early IgA responses (including coating of the Bacteroides genus), as well as IgA recognition patterns, differed between healthy children and children with allergic manifestations. Altered IgA recognition patterns in children with allergy were observed already at 1 month of age, when the IgA antibodies are predominantly maternally derived in breast-fed children. Conclusion: An aberrant IgAresponsiveness to the gutmicrobiota during infancy precedes asthma and allergy development, possibly indicating an impaired mucosal barrier function in allergic children.Funding Agencies|Swedish Research Council [K2011-56X-21854-01-06]; Swedish Heart-Lung Foundation [20140321]; Ekhaga Foundation [210-53]; Medical Research Council of Southeast Sweden; Olle Engqvist Foundation; Cancer and Allergy Foundation; University Hospital of Linkoping, Sweden; Spanish MINECO [2012-40007]</p

MAMI: a birth cohort focused on maternal-infant microbiota during early life

Early microbial colonization is a relevant aspect in human health. Altered microbial colonization patterns have been linked to an increased risk of non-communicable diseases (NCDs). Advances in understanding host-microbe interactions highlight the pivotal role of maternal microbiota on infant health programming. This birth cohort is aimed to characterize the maternal microbes transferred to neonates during the first 1000 days of life, as well as to identify the potential host and environmental factors, such as gestational age, mode of delivery, maternal/infant diet, and exposure to antibiotics, which affect early microbial colonization.This study is supported through a five-year grant from European Research Council (ERC) - European Union’s Horizon 2020 Framework - with an ERC Starting Grant (ref. 639226). Funding received from this grant supports direct research costs and research assistant salaries. The ERC had no direct role in the design of this study and will not in the collection, analysis or interpreting of data, or manuscript writing

Major alterations in the mononuclear phagocyte landscape associated with COVID-19 severity

10.1073/pnas.2018587118Proceedings of the National Academy of Sciences of the United States of America1186e201858711

SARS‐CoV‐2‐specific humoral and cellular immunity persists through 9 months irrespective of COVID‐19 severity at hospitalisation

Objectives Humoral and cellular immunity to SARS-CoV-2 following COVID-19 will likely contribute to protection from reinfection or severe disease. It is therefore important to characterise the initiation and persistence of adaptive immunity to SARS-CoV-2 amidst the ongoing pandemic. Methods Here, we conducted a longitudinal study on hospitalised moderate and severe COVID-19 patients from the acute phase of disease into convalescence at 5 and 9 months post-symptom onset. Utilising flow cytometry, serological assays as well as B cell and T cell FluoroSpot assays, we assessed the magnitude and specificity of humoral and cellular immune responses during and after human SARS-CoV-2 infection. Results During acute COVID-19, we observed an increase in germinal centre activity, a substantial expansion of antibody-secreting cells and the generation of SARS-CoV-2-neutralising antibodies. Despite gradually decreasing antibody levels, we show persistent, neutralising antibody titres as well as robust specific memory B cell responses and polyfunctional T cell responses at 5 and 9 months after symptom onset in both moderate and severe COVID-19 patients. Conclusion Our findings describe the initiation and, importantly, persistence of cellular and humoral SARS-CoV-2-specific immunological memory in hospitalised COVID-19 patients long after recovery, likely contributing towards protection against reinfection.Medicin