Chronic helminth infection burden differentially affects haematopoietic cell development while ageing selectively impairs adaptive responses to infection

Throughout the lifespan of an individual, the immune system undergoes complex changes while facing novel and chronic infections. Helminths, which infect over one billion people and impose heavy livestock productivity losses, typically cause chronic infections by avoiding and suppressing host immunity. Yet, how age affects immune responses to lifelong parasitic infection is poorly understood. To disentangle the processes involved, we employed supervised statistical learning techniques to identify which factors among haematopoietic stem and progenitor cells (HSPC), and both innate and adaptive responses regulate parasite burdens and how they are affected by host age. Older mice harboured greater numbers of the parasites’ offspring than younger mice. Protective immune responses that did not vary with age were dominated by HSPC, while ageing specifically eroded adaptive immunity, with reduced numbers of naïve T cells, poor T cell responsiveness to parasites, and impaired antibody production. We identified immune factors consistent with previously-reported immune responses to helminths, and also revealed novel interactions between helminths and HSPC maturation. Our approach thus allowed disentangling the concurrent effects of ageing and infection across the full maturation cycle of the immune response and highlights the potential of such approaches to improve understanding of the immune system within the whole organism

Experimental vaccination for onchocerciasis and the identification of early markers of protective immunity

Onchocerciasis, caused by Onchocerca volvulus remains a major public health and socio-economic problem across the tropics, despite years of mass drug administration (MDA) with Ivermectin to reduce disease burden. Through modelling, it has been shown that elimination cannot be achieved with MDA alone and additional tools are needed, such as vaccination, which remains the most cost-effective tool for long-term disease control. The feasibility behind vaccination against O. volvulus can be demonstrated in the Litomosoides sigmodontis mouse model, which shows that vaccine induced protection can be achieved with immunisation using irradiated L3, the infective stage of L. sigmodontis and with microfilariae (Mf), the transmission stage of the parasite. There is further evidence of protective immunity in humans, with individuals living in endemic areas that show no signs of infection despite being exposed to the parasite (endemic normal). The protective efficacy of promising vaccine candidates were evaluated using an immunisation time course in the L. sigmodontis model, using either DNA plasmid or peptide vaccines. In immunisation experiments in L. sigmodontis, Mf numbers are used as a measure of protection and marks the end of an immunisation time course. However, when changes in gene expression were measured at the end of an immunisation time course, in attempts to identify gene signatures that could be used as markers of protection (correlates of protection) in the blood, no gene signatures were found to be associated with protection. This suggest that at the end of an immunisation time course, when protection is measured (change in Mf numbers), it is too late in infection to measure changes in immune pathways being triggered. Changes in gene expression were therefore measured in blood samples collected throughout an immunisation time course in the L. sigmodontis model, in order to identify the time point in an immunisation experiment which are the most indicative of protection. Two independent immunisation time courses were used, either using irradiated L3 or Mf as vaccine against L. sigmodontis, as these elicit the greatest protection. This generated a large high dimensional dataset, that was too large and complex for a differential fold-change analysis. Therefore, an analysis pipeline was created using machine learning algorithms, to detect changes in gene expression throughout the time courses to detect markers of protection. The 6 hour time point following immunisation showed the greatest change in gene expression, with the analysis pipeline identifying known pathways associated with vaccine-induced immunity. The pipeline was applied to gene expression data from human samples obtained from individuals living in endemic areas who were either infected with O. volvulus or endemic normal (naturally protected), this was to identify pathways associated with protective immunity in humans. When comparing vaccine induced immunity seen in mice and natural protective immunity in humans there was some overlap in pathways being triggered, suggesting that similar pathways are needed for protection and that if a vaccine can trigger the right pathways in mice, it is likely to be effective in humans. Overall the machine learning analysis of the gene expression data, not only shows that it is feasible to measure change in gene expression in blood during filarial infections, but that during an immunisation time course it is the early time points following immunisation that are the most predictive of vaccine efficacy (protection outcome). One of the vaccine candidates, cysteine protease inhibitor-2 (CPI), is a known immuno-modulator that inhibits MHC-II antigen presentation on antigen presenting cells such as dendritic cells (DC). This candidate has consistently been shown to induce protection if its immuno-modulatory active site was modified. In in vitro studies, it was shown that modification of the active site of CPI rescues antigen presentation in DC. This shows the importance of DC activation before the onset of infection, demonstrating the importance of triggering protective responses early in infection, and provides insight on how one of the vaccine candidates achieves protection

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration.

BACKGROUND/AIM: In rat pancreatic islets, beta-cell gene expression, survival and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. METHODS: Rat pancreatic islets were cultured 1-7 days at G5, G10 or G30 with or without 3 µM of the GK activator Ro 28-0450 (Ro). After culture, beta-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H autofluorescence, intracellular calcium concentration and insulin secretion were tested in the absence or presence of Ro 28-0450. RESULTS: Prolonged culture of rat islets at G5 or G30 instead of G10 triggered beta-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected beta-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to beta-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10, but failed at restoring beta-cell glucose responsiveness after culture at G5 or G30. CONCLUSIONS: Pharmacological GK activation prevents the alteration of beta-cell survival and function by long-term culture at G5, but mimics glucotoxicity when added to G10. The complex effects of glucose on the beta-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se

Habitat restoration weakens negative environmental effects on telomere dynamics

Habitat quality can have far-reaching effects on organismal fitness, an issue of concern given the current scale of habitat degradation. Many temperate upland streams have reduced nutrient levels due to human activity. Nutrient restoration confers benefits in terms of invertebrate food availability and subsequent fish growth rates. Here we test whether these mitigation measures also affect the rate of cellular ageing of the fish, measured in terms of the telomeres that cap the ends of eukaryotic chromosomes. We equally distributed Atlantic salmon eggs from the same 30 focal families into 10 human-impacted oligotrophic streams in northern Scotland. Nutrient levels in five of the streams were restored by simulating the deposition of a small number of adult Atlantic salmon Salmo salar carcasses at the end of the spawning period, while five reference streams were left as controls. Telomere lengths and expression of the telomerase reverse transcriptase (TERT) gene that may act to lengthen telomeres were then measured in the young fish when 15 months old. While TERT expression was unrelated to any of the measured variables, telomere lengths were shorter in salmon living at higher densities and in areas with a lower availability of the preferred substrate (cobbles and boulders). However, the adverse effects of these habitat features were much reduced in the streams receiving nutrients. These results suggest that adverse environmental pressures are weakened when nutrients are restored, presumably because the resulting increase in food supply reduces levels of both competition and stress

Glucose et stress oxydatif dans les cellules beta pancréatiques : rôle du zinc et des métallothionéines

Glucose stimulation of insulin secretion by pancreatic β cells is essential to maintain glucose homeostasis. In addition to this short-term effect, glucose is also important to maintain β cell survival and differentiated phenotype. Indeed, both chronic hypo- and hyper-glycemia alter β-cell gene expression, survival and function. Similarly, in vitro, rat β-cell gene expression, function and survival are optimally preserved by culture in the presence of 10 mM glucose (G10) and markedly impaired by culture in either lower (2 or 5 mmol/l glucose, G2 or G5) or higher (30 mmol/l glucose, G30) glucose concentrations. It therefore seems that glucose stimulation exerts beneficial effects on β cell function and survival between G2 and G10 and a deleterious effect on these parameters between G10 and G30. The precise molecular mechanisms behind such phenotypical plasticity are poorly understood, but they could involve an increase in oxidative stress. Indeed, mRNA levels of oxidative stress-response genes like Metallothionein 1a/2a (Mt1a/2a), Heme oxygenase 1 (Hmox1) or c-Mycfollow an asymmetric V-shaped profile similar to that of β cell dysfunction and apoptosis. We therefore hypothesized that extreme glucose concentrations increases oxidative stress in β cells. In a first study, we developed a method to measure the β cell redox status in our experimental conditions. Our results showed that mt-HyPer, besides its expected sensitivity to H2O2, was also highly pH-sensitive. As glucose stimulation increases mitochondrial pH in β cells, that probe could not be used in our experimental model. In contrast, the fluorescence ratio of mt-roGFP1, which measures the thiol/disulfide equilibrium, was only slightly affected by pH. Using that probe, we demonstrated that mitochondrial thiol oxidation in rat β cells reversibly increases when glucose is lowered from 10 to 2 mmol/l. In contrast, acutely increasing glucose concentration from 10 to 30 mmol/l did not increase mt-roGFP1 oxidation in β cell. In a second study, I first demonstrated that 18 to 24h culture of rat islet cell clusters in the présence of G5 or G30 vs. G10 increases mitochondrial glutathione oxidation, thereby confirming our initial hypothesis. I also tested the effect of ZnCl2, a potent inducer of Mt1a, on β cell alterations induced by prolonged exposure to low and high glucose concentrations. My results show that addition of 50μM ZnCl2partially reduced mt-roGFP oxidation after 18-24h culture in G5, and tended to do so after culture in G30.Addition of 100μM ZnCl2also significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30. Theses protective effects of ZnCl2 did not correct β cell dysfunction induced by culture in G5 and G30. In a third study, using islets from Mt1/2 knock-out mice, I wanted to determine the role of MT1/2 on the beneficial effects of Zn2+. I also wanted to test whether the deficiency in Mt1/2 increases β cell apoptosis and mitochondrial glutathione oxidation induced by culture in a low glucose concentration. In contrast with what I observed in rat islets, ZnCl2, despite increasing Mt1expression, did not protect wild-type mouse islets from apoptosis induced by culture in a low glucose concentration, thereby preventing me from studying the role of MT1/2 expression in this effect. Moreover, deficiency in Mt1/2 in mouse islets did not increase apoptosis induced by culture in a low glucose concentration. These results help us to understand the molecular mechanisms underlying the plasticity of the β cell phenotype and may help in the development of new therapeutic strategies for T2D.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Role of activating transcription factor 3 in low glucose- and thapsigargin-induced apoptosis in cultured mouse islets.

In vitro, survival and function of rat pancreatic β-cells are optimally preserved in the presence of 10 mmol/l glucose (G10) and markedly altered by prolonged culture at either 2 mmol/l glucose (G2) or 30 mmol/l glucose (G30). The increase in islet cell apoptosis in G2 and G30 vs. G10 is preceded by parallel increases in the mRNA levels of the integrated stress response (ISR) gene activating transcription factor 3 (Atf3) and its putative target and proapoptotic gene growth arrest- and DNA damage-inducible gene 153 (Gadd153/Chop). In this study, we used islets from Atf3 knockout (Atf3(-/-)) mice to test the role of ATF3 in the stimulation of islet cell apoptosis under conditions associated with ISR activation. The glucose sensitivity of Atf3(-/-) and WT islets for the stimulation of insulin secretion and Xbp1 mRNA splicing during 18h culture was similar, demonstrating that glucose metabolism was unaffected by Atf3 deletion. However, the stimulation of islet cell apoptosis by the SERCA pump inhibitor thapsigargin was slightly but significantly reduced in Atf3(-/-) vs. WT islets despite similar level of expression of Gadd153 and Gadd34 mRNA. Also, the stimulation of islet cell apoptosis by 7 days of culture in G2 was slightly but significantly reduced in Atf3(-/-) vs. WT islets, and this effect was accompanied by a significant reduction in Gadd153 mRNA expression. In conclusion, the increase in Atf3 gene expression induced by thapsigargin and low glucose concentrations slightly contributes to the stimulation of islet cell apoptosis under these culture conditions

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration

BACKGROUND/AIM: In rat pancreatic islets, beta-cell gene expression, survival and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. METHODS: Rat pancreatic islets were cultured 1-7 days at G5, G10 or G30 with or without 3 µM of the GK activator Ro 28-0450 (Ro). After culture, beta-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H autofluorescence, intracellular calcium concentration and insulin secretion were tested in the absence or presence of Ro 28-0450. RESULTS: Prolonged culture of rat islets at G5 or G30 instead of G10 triggered beta-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected beta-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to beta-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10, but failed at restoring beta-cell glucose responsiveness after culture at G5 or G30. CONCLUSIONS: Pharmacological GK activation prevents the alteration of beta-cell survival and function by long-term culture at G5, but mimics glucotoxicity when added to G10. The complex effects of glucose on the beta-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se

Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects after prolonged culture in a low non-stimulating glucose concentration.

AIMS/HYPOTHESIS: Pancreatic beta cells chronically exposed to low glucose concentrations show signs of oxidative stress, loss of glucose-stimulated insulin secretion (GSIS) and increased apoptosis. Our aim was to confirm the role of mitochondrial oxidative stress in rat islet cell apoptosis under these culture conditions and to evaluate whether its reduction similarly improves survival and GSIS. METHODS: Apoptosis, oxidative stress-response gene mRNA expression and glucose-induced stimulation of mitochondrial metabolism, intracellular Ca(2+) concentration and insulin secretion were measured in male Wistar rat islets cultured for 1 week in RPMI medium containing 5-10 mmol/l glucose with or without manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP) or N-acetyl-L-: cysteine (NAC). Oxidative stress was measured in islet cell clusters cultured under similar conditions using cytosolic and mitochondrial redox-sensitive green fluorescent protein (roGFP1/mt-roGFP1). RESULTS: Prolonged culture in 5 vs 10 mmol/l glucose increased mt-roGFP1 (but not roGFP1) oxidation followed by beta cell apoptosis and loss of GSIS resulting from reduced insulin content, mitochondrial metabolism, Ca(2+) influx and Ca(2+)-induced secretion. Tolbutamide-induced, but not high K(+)-induced, Ca(2+) influx was also suppressed. Under these conditions, MnTBAP, but not NAC, triggered parallel ∼50-70% reductions in mt-roGFP1 oxidation and beta cell apoptosis, but failed to protect against the loss of GSIS despite significant improvement in glucose-induced and tolbutamide-induced Ca(2+) influx. CONCLUSIONS/INTERPRETATION: Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects during culture in a low glucose concentration. Thus, targeting beta cell survival may not be sufficient to restore insulin secretion when beta cells suffer from prolonged mitochondrial oxidative stress, e.g. in the context of reduced glucose metabolism

Protective Antioxidant and Antiapoptotic Effects of ZnCl2 in Rat Pancreatic Islets Cultured in Low and High Glucose Concentrations

Aim/Hypothesis: Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl2, a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations. Methods: Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNELpositive b-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing ‘‘redox sensitive GFP’’ targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA. Results: As observed for Mt1a mRNA levels, b-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50–100 mM ZnCl2 to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late b-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions. Conclusion: ZnCl2 reduces mitochondrial oxidative stress and improves rat b-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS

Glucose regulation of islet stress responses and beta-cell failure in type 2 diabetes.

Pancreatic beta-cells exposed to high glucose concentrations display altered gene expression, function, survival and growth that may contribute to the slow deterioration of the functional beta-cell mass in type 2 diabetes. These glucotoxic alterations may result from various types of stress imposed by the hyperglycaemic environment, including oxidative stress, endoplasmic reticulum stress, cytokine-induced apoptosis and hypoxia. The glucose regulation of oxidative stress-response and integrated stress-response genes in cultured rat islets follows an asymmetric V-shaped profile parallel to that of beta-cell apoptosis, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations. These observations suggest that both types of stress could play a role in the alteration of the functional beta-cell mass under states of prolonged hypoglycaemia and hyperglycaemia. In addition, beta-cell demise under glucotoxic conditions may also result from beta-cell hypoxia and, in vivo, from their exposure to inflammatory cytokines released locally by non-endocrine islet cells. A better understanding of the relative contribution of each type of stress to beta-cell glucotoxicity and of their pathophysiological cause in vivo may lead to new therapeutic strategies to prevent the slow deterioration of the functional beta-cell mass in glucose intolerant and type 2 diabetic patients