Modulation of B cell antibody production by antigen-specific IL-10 producing regulatory T cells

The ability of B cells to present antigen and produce antibodies has established their absolute requirement in mediating adaptive immune responses. Other key mediators of the adaptive system are regulatory T-cells (Treg) which modulate both T and B cell responses. There are currently three different Treg populations: naturally-occurring regulatory populations (nTreg) designated by the expression of the IL-2 receptor (CD25) and the transcription factor Foxp3. Alternatively there are Tr-1 and Th3 cells which are characterised by the secretion of IL-10 and TGF-β respectively. IL-10 producing T cells have been shown to regulate the adaptive immune system by inducing B cells to secrete IgG4 in a cell-contact dependent manner. The benefit of such non-inflammatory B cell responses is apparent in the hypo-responsive state of patients with allergy or helminth infections such as Onchocerciasis. The present thesis was aimed at analyzing the molecular mechanisms underlying the ability of IL-10 producing T cells to induce the production of the IgG4 by B cells. For these investigations, regulatory T cell clones (Tr-TCC) were generated from human PBMC. The initial results section provides an overview regarding the different aspects of antigen-specific Tr-TCC generation. Antigen-specificity was induced via repeated rounds of stimulation with tetanus toxoid (or Onchocerca Volvulus extract) alone or on combination with dexamethasone and vitamin D3 (DD3). The results show the ability of Tr-TCC to suppress tetanus-specific reactive T cells in a cell-contact independent but IL-10-dependent manner. Generated Tr-TCC were then characterised for their expression of regulatory T cell markers such as CD25 and Foxp3 and compared with isolated nTreg: interestingly the cells had overlapping phenotypes. Using B:T cell co-culture assays, generated Tr-TCC were then shown to preferentially induce B cells to secrete IgG4 and required cell-contact. Experiments also demonstrated that both memory and naïve B cells were required to produce significant levels of IgG4. Mechanistically, molecules like GITR, GITR-L, TGF-β, IL-10 and Foxp3 were shown to play an important role in the induction of this immunoglobulin. It could be shown that blocking GITR molecules selectively prevented IgG4 production as did neutralizing Abs to GITR-L, IL-10 and TGF-β. Furthermore, the prevention of IgG4 induction by anti-GITR Abs was reversed by excess rIL-10 but not rTGF- β indicating a complex relationship. The requirement of Foxp3 in this process was surprising since Tr-1 cells do not constitutively express Foxp3 and an active functional role was not previously reported. To investigate this point further, the levels of Foxp3 were measured during the generation process; here it could be shown that levels of constitutive Foxp3 increased after each round of stimulation and correlated with the ability of T cell lines to induce IgG4 in B cells. The functional requirement of this transcription factor was further shown after silencing Foxp3 in Tr-TCC using specific siRNA since Tr-TCC induced IgG2 instead of IgG4. IgG4 production was also shown using isolated nTreg from healthy untreated donors albeit weaker than Tr-TCC. In further correlation, IgG4 induction by nTreg was also GITR and IL-10 dependant. More interestingly, under the same conditions, isolated CD4+CD25- effector T cells induced the production of IgG2, a result correlating with Foxp3 silenced Tr-TCC. In the final experiments, B cell activation using Toll-like receptor stimuli affected the ability of Tr-TCC to induce IgG4 in B cells. These preliminary findings provide a hypothesis for the mechanism into how the different outcomes of Onchocerca infection occur: hypo-responsive (high IgG4 and low pathology) versus hyper-responsiveness (low IgG4, high IgE and debilitating pathology).Modulation der B-Zell-Antikörper-Produktion von Antigen-spezifischen IL-10 produzierenden regulatorischen T-Zellen Die Fähigkeit von B Zellen Antigene zu präsentieren und Antikörper zu produzieren ist die notwendige Voraussetzung um die Adaptive Immunantwort auszulösen. Andere Schlüssel- Mediatoren des adaptiven Systems sind regulatorische T Zellen (Treg), die sowohl die T- als auch die B-Zell-Reaktionen modulieren. Derzeit gibt es drei verschiedene Treg Populationen. Zum einen gibt es natürliche regulatorische T Zellen (nTreg), die durch ihre Expression des IL-2 Rezeptors (CD25) und des Transkriptionsfaktors Foxp3 charakterisiert sind. Zum anderen gibt es Tr-1 und Th3 Zellen, die durch die Sekretion von IL-10 und TGF-β charakterisiert sind. Es ist festgestellt worden, daß IL-10 produzierende T Zellen das adaptive Immunsystem regulieren, indem sie B Zellen zur Sekretion von IgG4 anregen. Diese Induktion von IgG4 ist Zell Kontakt abhängig. Der Vorteil einer solchen nicht-entzündlichen B Zell-Antwort zeigt sich am hyporesponsiven Zustand von Patienten mit Allergien oder Helminth Infektionen wie Onchozerkose. Das Ziel der vorliegenden Dissertation war die Analyse der molekularen Mechanismen, die IL- 10 produzierende T Zellen dazu befähigen die Produktion von IgG4 durch B Zellen zu induzieren. Für diese Untersuchungen wurden regulatorische T Zell Klone (Tr-TCC) aus Human PBMC generiert. Der erste Ergebnis-Abschnitt dieser Arbeit gibt einen Überblick über die verschiedenen Aspekte von Antigen-spezifischer Tr-TCC-Generierung. Die Antigen-Spezifität wurde durch wiederholte Stimulation mit dem Tetanus-Toxoid (oder Onchocerca Volvulus Extrakt) allein, oder in Kombination mit Dexamethason und Vitamin D3 (DD3) induziert. Die Ergebnisse zeigen, daß die so generierten T Zellen die Fähigkeit haben die Proliferation von Tetanus-spezifischen nicht regulatorischen T Zellen zu unterdrücken. Diese Unterdrückung war Zell-Kontakt unabhängig und wird durch Zytokine wie IL-10 vermittelt. Die so erzeugten Tr-TCC wurden dann auf die Expression von regulatorischen T Zell Markern, wie CD25 und Foxp3, hin untersucht und mit nTregs verglichen. Interessanterweise wiesen die Phänotypen der Tr-TCC und nTreg Zellen einige Ähnlichkeiten zueinander auf. Mit B:T-Zell-Co-Kultur-Assays konnte dargestellt werden, daß die generierten Tr-TCCs, abhängig vom Zell-Kontakt, B Zellen zur Sekretion von IgG4 induzieren. Weitere Experimente haben verdeutlicht, daß sowohl memory und als auch naive B Zellen erforderlich sind, um eine signifikante Menge an IgG4 zu produzieren. Es konnte gezeigt werden, daß Moleküle wie GITR, GITR-L, TGF-β, IL-10 und Foxp3 eine wichtige Rolle bei der Induktion von diesem Immunglobulin spielen. Weiterhin wurde nachgewiesen, daß die Blockierung von GITR Molekülen selektiv die IgG4 Produktion verhindert, wie es auch bei neutralisierenden Antikörpern gegen GITR-L, IL-10 und TGF-β der Fall ist. Interessanterweise konnte die vom anti-GITR Antikörper induzierte IgG4 Blockade durch einen Überschuß an rekombinanten IL- 10, nicht aber durch rTGF-β rückgängig gemacht werden. Der Bedarf an Foxp3 bei diesem Prozeß war sehr überraschend, da Tr-1 Zellen Foxp3 nicht konstitutiv exprimieren und über eine aktive funktionelle Rolle dieses Transkriptionsfaktors bislang nichts bekannt ist. Um dies zu untersuchen wurde die Menge an Foxp3 während des Tr- TCC-Generierungs Prozesses gemessen. Hierbei wurde festgestellt, daß die Menge an konstitutiven Foxp3 nach jeder Stimulationsrunde zunimmt und mit der Fähigkeit von T Zellen korreliert IgG4 zu induzieren. Der funktionelle Bedarf dieses Transkriptionsfaktors konnte außerdem durch Ausschalten mit spezifische siRNAs nachgewiesen werden. Diese Tr-TCC Zellen induzierten IgG2 anstelle des IgG4. Weiterhin konnte mit Hilfe von isolierten nTreg von unbehandelten gesunden Spendern IgG4 induziert werde, jedoch in geringerer Mengen als durch Tr-TCC. In weiterer Übereinstimmung war die IgG4 Induktion durch nTreg (CD4+CD25+) ebenfalls GITR und IL-10 abhängig. Isolierte CD4+CD25- Effektor T Zellen induzierten die Produktion von IgG2, ein Ergebnis das gut mit dem „silencing“ von Foxp3 in Tr-TCC korreliert. In den letzten Experimenten konnte gezeigt werden, daß die B Zell-Aktivierung durch Toll-like-Rezeptoren die Fähigkeit von Tr-TCC beeinträchtigt IgG4 zu induzieren. Diese Ergebnisse bieten eine Hypothese, warum die Onchozerca Infektionen unterschiedlich verlaufen können: Hypo-Responsiv (hohes IgG4 und niedrige Pathologie) gegenüber dem Hyper-Responsiv (niedriges IgG4, hohes IgE und schwere Erkrankung)

Emotional burnout of specialists in socio-occupational professions: contemporary views on the problem

Представлено сучасні підходи до проблеми вигорання у представників соціономічних професій у контексті емоційної праці, емоційного і когнітивного дисонансу. Проаналізовано й узагальнено наукову літературу з питань вигорання, визначено три основні його компоненти: емоційне і/або фізичне виснаження, зниження продуктивності праці і надмірна деперсоналізація. На основі аналізу визначень вигорання встановлено його зв’язок з емоційною працею (регуляцією і вираженням емоційних станів). Обстоюється думка, що фахівці соціономічних професій особливо вразливі до вигорання, оскільки тривалий час перебувають у стані, коли необхідно постійно контролювати свої емоції, брати на себе відповідальність і відчувати невизначеність, працюючи з іншими людьми. Емоційну працю розглянуто як предиктор вигорання у фахівців соціономічних професій. На основі аналізу літератури, присвяченої проблемам вигорання та емоційного дисонансу, висловлено припущення, що вимоги щодо емоційної праці зумовлюють різноманітність проявів синдрому виго-рання (у тому числі виснаження, цинізму, зниження продуктивності праці та погіршення самопочуття), а знання симптомів вигорання дало б змогу фахівцям соціономічних професій запобігти виникненню та загостренню цього стану.This article deals with the modern approaches to the problem of burnout of helping professionals in the context of emotional labor, emotional and cognitive dissonance. The burnout literature is reviewed, compared, and summarized. The definition of burnout is proposed including three components: emotional and/or physical exhaustion, lowered work productivity, and excessive depersonalization. Based on an analysis of the definitions of burnout, the paper focuses on the connec-tion of burnout and emotional work (regulation of feelings and emotional expression). It is also maintained a fact that helping professionals are especially vulnerable to burnout because of the necessity to control own emotions for a long time, to take responsibilities, and to feel uncertainties they encounter while working with others. The current study discussed emotional labor as a predictor of burnout of helping professionals. On the basis of the literature on burnout and emotional dissonance, the author of this article hypothesized that emotional job demands would explain variances of burnout (i.e., exhaustion and cynicism). Knowledge of abovementioned syndromes would help socionomy professionals to avoid emergence and aggravation of emotional burnout.Представлены современные подходы к проблеме выгорания у представителей социономических профессий в контексте эмоциональной труда, эмоционального и когнитивного диссонанса. Проанализированы и обобщены научную литературу по вопросам выгорания, определены три основные его компоненты: эмоциональное и / или физическое истощение, снижение производительности труда и чрезмерная деперсонализация. На основе анализа определений выгорания установлена ​​его связь с эмоциональной трудом (регуляцией и выражением эмоциональных состояний). Отстаивается мнение, что специалисты социономических профессий особенно уязвимы к выгоранию, поскольку длительное время находятся в состоянии, когда необходимо постоянно контролировать свои эмоции, брать на себя ответственность и чувствовать неопределенность, работая с другими людьми. Эмоциональный труд рассмотрен как предиктор выгорания у специалистов социономических профессий. На основе анализа литературы, посвященной проблемам выгорания и эмоционального диссонанса, высказано предположение, что требования по эмоциональной труда обусловливают разнообразие проявлений синдрома выгорания (в том числе истощения, цинизма, снижение производительности труда и ухудшение самочувствия), а знание симптомов выгорания позволило бы специалистам социономических профессий предотвратить возникновение и обострение этого состояния

Mycorrhizal Symbiosis for Sustainable Optimization of Tropical Agriculture: A Review of Research

Excessive application of chemical fertilizers and other agrochemicals can cause significant imbalances in soils and agricultural ecosystems. To minimize these impacts, biofertilizers and organic fertilizers are needed to maintain a sustainable production system. The use of subterranean microorganisms in agriculture to stimulate plant growth and improve yields has recently received increasing interest. In this context, mycorrhizae represent a viable solution to mitigate these adverse effects. Mycorrhizal fungi are able to form a symbiotic relationship with the roots of plants in the environment. Mycorrhizal fungus helps the plant to absorb nutrients and water. In addition, mycorrhizal fungi play a crucial role in storing carbon (C) in the soil. Most previous studies have just considered the effects of AMF species on a specific crop in one particular area but have not assessed the balance of AMF in production systems in tropical agriculture. This consideration should allow for the optimization of cropping practices through a review of the work on the use of AMF in tropical agriculture production systems. In this paper, we will discuss, through different examples of experiments carried out in the tropics, the performance of different strategies for managing the potential of AMF to maintain a sustainable production system

Hyperreactive onchocerciasis is characterized by a combination of Th17-Th2 immune responses and reduced regulatory T cells

<div><p>Clinical manifestations in onchocerciasis range from generalized onchocerciasis (GEO) to the rare but severe hyperreactive (HO)/sowda form. Since disease pathogenesis is associated with host inflammatory reactions, we investigated whether Th17 responses could be related to aggravated pathology in HO. Using flow cytometry, filarial-specific cytokine responses and PCR arrays, we compared the immune cell profiles, including Th subsets, in individuals presenting the two polar forms of infection and endemic normals (EN). In addition to elevated frequencies of memory CD4⁺ T cells, individuals with HO showed accentuated Th17 and Th2 profiles but decreased CD4⁺CD25^hiFoxp3⁺ regulatory T cells. These profiles included increased IL-17A⁺, IL-4⁺, RORC2⁺ and GATA3⁺CD4⁺ T cell populations. Flow cytometry data was further confirmed using a PCR array since Th17-related genes (IL-17 family members, IL-6, IL-1β and IL-22) and Th2-related (IL-4, IL-13, STAT6) genes were all significantly up-regulated in HO individuals. In addition, stronger <i>Onchocerca volvulus</i>-specific Th2 responses, especially IL-13, were observed <i>in vitro</i> in hyperreactive individuals when compared to GEO or EN groups. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network instigating the development of severe onchocerciasis.</p></div

Helminth antigens differentially modulate the activation of CD4(+) and CD8(+) T lymphocytes of convalescent COVID-19 patients in vitro

BACKGROUND: The coronavirus disease 2019 (COVID-19) is a respiratory disease caused by SARS-CoV-2, a recently discovered strain of coronavirus. The virus has spread rapidly, causing millions of death worldwide. Contrary to the predictions, prevalence and mortality due to COVID-19 have remained moderate on the African continent. Several factors, including age, genetics, vaccines, and co-infections, might impact the course of the pandemic in Africa. Helminths are highly endemic in Sub-Saharan Africa and are renowned for their ability to evade, skew, and suppress human immune responses through various immune-modulatory mechanisms. Such effects will likely impact SARS-CoV-2 transmission and disease progression. METHODS: Here, we analyzed in vitro the impact of antigen extracts from three major helminth parasites, including Onchocerca volvulus, Brugia malayi, and Ascaris lumbricoides, on the immune reactivity to SARS-CoV-2 peptides in COVID-19 patients. Activation of CD4+ and CD8+ T cells was investigated using flow cytometry to monitor the expression of CD137 (4-1BB) and CD69. Cytokine expression, including IL-6, IL-10, IFN-γ, and TNFα, was measured by Luminex in cell culture supernatants. RESULTS: We observed that helminth antigens significantly reduced the frequency of SARS-CoV-2-reactive CD4+ T helper cells. In contrast, the expression of SARS-CoV-2-reactive CD8+ T cells was not affected and even significantly increased when PBMCs from COVID-19 patients living in Benin, an endemic helminth country, were used. In addition, stimulation with helminth antigens was associated with increased IL-10 and a reduction of IFNγ and TNFα. CONCLUSION: Our data offer a plausible explanation for the moderate incidence of COVID-19 in Africa and support the hypothesis that helper T cell-mediated immune responses to SARS-CoV-2 are mitigated in the presence of helminth antigens, while virus-specific cytotoxic T cell responses are maintained

Genetic diversity and virulence factors of Gram-negative bacilli isolated at the CHU-Z in Abomey-Calavi/So-Ava (Benin)

Nosocomial infections are increasingly recurrent in health facilities and represent a serious public health concern. Apart from patients, health workers are also at high risk of infection. The risk factors associated with this type of infection are still not fully characterized. The present study aimed at characterizing Gram-negative bacillus strains isolated from surfaces and medico-technical equipment at the CHU-Z in Abomey-Calavi/Sô-Ava. 128 samples were collected by dry swabbing in five departments of the Abomey-Calavi University Hospital Center. Identification of the strains and antibiograms were done using the API 20 E Gallery and CASFM recommendations. The pathogenic potential of the isolates was evaluated by i) analyzing the biofilm formation ability and ii) examining the presence of the beta-lactam resistance gene (blaSHV). In addition, E. coli strains were analyzed for their enterohemorrhagic potential through the screening for the gene encoding for shigatoxin (stx). The proportion of contaminated samples by enterobacteria strains was 23.43%. Twelve species of Gram-negative bacillus were identified with a high predominance of Klebsiella oxytoca (20%), followed by Acinetobacter baumanni (16.66%), Chryseomonas luteola (13.33%). Most strains were resistant to tetracycline (87%) and ceftriaxone (80%). However, most of them were sensitive to norfloxacin (17%), ciprofloxacin (17%), and imipenem (13%). All strains of Escherichia coli, Enterobacter sakazakii, Klebsiella terrigena, Serratia rubidaea, Citrobacter youngae and 50% of Chryseomonas luteola formed biofilm. The results of PCR amplification showed that 6.66% of the strains carry the blaSHV gene and none of E. coli strains have the gene coding for Shigatoxin. These data suggest a significant risk of severe infections for patients and health workers at the CHU-Z in Abomey-Calavi/Sô-Ava. Additional investigations are required to better characterize the presence of pathogenic strains in hospital environment

Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

<div><p>Helminth parasites are known to be efficient modulators of their host’s immune system. To guarantee their own survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterized. Here we investigate the impact of plasma and affinity-purified IgG/IgG4 fractions from endemic normals (EN) and LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states were investigated by monitoring the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) respectively by flow cytometry and ELISA. We could show that the activation of granulocytes was inhibited in the presence of plasma from EN and Mf+ individuals whereas those of Mf- and CP presented no effect. This inhibitory capacity was impaired upon depletion of IgG in Mf+ individuals but persisted in IgG-depleted plasma from EN, where it strongly correlated with the expression of IgA. In addition, IgA-depleted fractions failed to suppress granulocyte activation. Strikingly, affinity-purified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. Reduction of both the affinity to, and the suppressive properties of anti-inflammatory IgG4 on granulocytes was reached only when FcγRI and II were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP, natively exhibit FcγRI/II-dependent suppressive properties on granulocytes. Our findings suggest that quantitative and qualitative alterations in IgG4 molecules are associated with the different clinical phenotypes in LF endemic regions.</p></div

Depletion of IgG4 abrogates the suppressive capacity of IgG positive fractions from plasma of LF infected individuals.

<p>Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone (dark bars) or in presence of 2.5 μg/ml of IgG4 negative fractions obtained from IgG enriched fractions (n = 8) (light bars) from EN (A), Mf+ (B), Mf- (C), CP (D) or the corresponding IgG4 positive fractions (n = 8) (grey bars) and increasing concentration (1.25 μg/ml, 2.5 μg/ml and 5 μg/ml) of IgG4 positive fractions from different groups (E). Cells were stained with anti-CD63 and HLADR antibodies. Activated granulocytes were characterized as CD63+/HLADR- cells. The release of histamine (F), neutrophil elastase (G) and eosinophil cationic protein (H) in supernatants from cultures with IgG4 positive fractions was assessed after 18 hours. Bars represent means ± SEM. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test;*: p<0.05; **: p<0.01; ***: p<0.001.</p

Preferential expression of IgG4 in Mf+.

<p>Plasma samples from EN (n = 14) and LF infected Mf+ (n = 14), Mf-(n = 14) and CP (n = 14) patients were diluted and analyzed for the expression of IgG1 (A), IgG2 (B), IgG3 (C), IgG4 (D), IgE (E), IgA (F) and IgM (G) using Luminex-based immunoassay. Bars depict the plasmatic antibody expressions as mean ± SEM. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p<0.05; **: p<0.01; ***: p<0.001.</p