Early IL-10 production is essential for syngeneic graft acceptance

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival. J. Leukoc. Biol. 92: 259-264; 2012.FAPESPFAPESPCNPqCNPqFCTFC

dietary supplementation with high doses of regular vitamin d3 safely reduces diabetes incidence in nod mice when given early and long term

High doses of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) prevent diabetes in the non-obese diabetic (NOD) mouse but also elicit unwanted calcemic side-effects. Because immune cells themselves can convert vitamin D3 into 1,25(OH)2D3 locally, we hypothesized that dietary vitamin D3 can also prevent disease. Thus, we evaluated whether dietary administration of high doses of regular vitamin D3 (800 IU per day) during different periods of life (pregnancy and lactation, early-life (3-14 weeks of age), or lifelong (3-35 weeks of age)) safely prevents diabetes in NOD mice. We found that only lifelong treatment raised serum 25-hydroxyvitamin D3 from 173 nmol/L in controls to 290 nmol/L, without inducing signs of calcemic or bone toxicity, and significantly reduced diabetes development in both male and female NOD mice. This diabetes protection by vitamin D3 correlated with preserved pancreatic insulin content and improved insulitis scores. Moreover, vitamin D3 treatment decreased interferon-γ-positive CD8+ T-cells and increased CD4+(CD25+)FoxP3+ T-cells in pancreatic draining lymph nodes. In conclusion, this study shows for the first time that high doses of regular dietary vitamin D3 can safely prevent diabetes in NOD mice when administered lifelong, although caution is warranted with regards to administering equivalently high doses in humans

Characterization of the inflammatory response in the syngeneic and allogeneic graft in an experimental model of skin transplantation.

A inflamação é um evento intrínseco ao transplante que é desencadeado após o dano causado pela cirurgia. No presente trabalho realizou-se a caracterização fenotípica e funcional das células inflamatórias presentes no enxerto, após o transplante alogênico ou singênico de pele em camundongos. Os resultados obtidos mostraram diferenças significativas na produção de citocinas pró e anti-inflamatórias entre o transplante alogênico e singênico, diferenças já detectáveis nas primeiras 24 horas pós-transplante. Mostrou-se que existe produção aumentada de IL-10 no transplante singênico em relação ao transplante alogênico indicando que a produção de IL-10 no enxerto possui um importante papel para o aceite de transplantes de pele. Além disso, na ausência de IL-10, a rejeição de enxertos alogênicos apresentou-se acelerada e surpreendentemente uma grande parcela dos enxertos singênicos não foi aceita e apresentava aspecto de fibrose com grande deposição de colágeno. O conjunto dos dados indica que a IL-10 possui um importante papel regulatório na inflamação local do enxerto.Inflammation is an intrinsic event of transplantation that occurs due to to damage caused by surgery. In this study, phenotypic and functional characterization of inflammatory cells present in the graft was performed, after allogeneic or syngeneic skin transplantation in mice. Our results show significant differences in the production of pro and anti-inflammatory cytokines between the syngeneic and allogeneic grafts, detectable as early as 24 hours after transplantation. Higher production of IL-10 was shown in the syngeneic grafts in comparison to allogeneic grafts, indicating that production of IL-10 in the graft is important for acceptance. This is reinforced by data that shows that in the absence of IL-10, rejection of allografts is accelerated and that, surprisingly, a high percentage of the syngeneic grafts is not accepted as well, or shows fibrosis with high deposition of collagen. Taken together, this data indicates that IL-10 has an important regulatory function in the local inflammation of the graft

NOVEL IMMUNOTHERAPIES IN TYPE 1 DIABETES: EXPLORING ANTIGEN-SPECIFIC AND NON-SPECIFIC APPROACHES

Type 1 Diabetes (T1D) is a chronic autoimmune disease characterized by loss of tolerance towards beta-cell antigens (Ags). The complete pathogenesis of the disease is still not fully understood but what is known is that the clinical onset of the disease is preceded by a silent asymptomatic phase in which beta-cells are progressively destroyed by auto-reactive effector T (Teff) cells, finally culminating in chronic hyperglycemia and diagnosis of the disease. There is currently no cure for T1D and that represents a major predicament for the public health care system as the rates and incidences of this disease increase. Therefore, discovering therapies which target the etiology of the disease and not only treat the clinical symptoms of T1D becomes imperative. That is not simple as T1D is a complex disease triggered by the interaction of multiple factors such as genetic predisposition, environmental triggers and relevant antigenic exposure. As such, due to this multitude of causative factors it is difficult to select therapies which will target such a broad range of variables, without for example, causing generalized immunosuppression. Furthermore, because T1D is the result of loss of tolerance, one should also consider Ag-specific therapies that will re-establish tolerance towards disease relevant auto-Ags. Thus, the main purpose of this work was to establish different therapeutic strategies that would prevent or revert T1D in the NOD mice which spontaneously develop diabetes. Several therapeutic protocols using both Ag-specific as well as Ag-non-specific mono-therapies have been successful in preventing or reverting T1D in animal models. Up-to-date, however, mono-therapies have done quite poorly in the clinical setting. There are several possible explanations as to the reasons for these failures. For one, the complexity of the disease itself does not facilitate the choice of a therapeutic agent and to add difficulty to this conundrum, pre-clinical studies are in their majority employed in a genetically identical group of animals and thus success in these settings does not mean they will translate into humans that possess a much broader pool and variability of genes. Thus, it is reasonable to rationalize that CT which bring together different therapeutic agents and target different arms of the pathogenic attack may be more efficient at combatting disease. Hence, we have taken this hypothesis into consideration and set up a CT using systemic low dose anti-CD3 together with orally administered GM L.lactis secreting PINS and IL10 (LL-PINS+IL10). We have shown in Chapters 4 and 5 that CT was able to stably reverse diabetes in 59% of recently diagnosed NOD mice by reinduction of tolerance to PINS. Initial beta-cell mass at start of therapy seems to be an important factor that determines the success rate of CT, reversal of diabetes in mice with starting glycemia below 350 mg/dL was significantly higher compared to mice with starting glycemia above 350 mg/dL, indicatives of higher and lower remaining beta-cell mass at start of therapy. Furthermore, we demonstrated that the addition of Anakinra treatment on top of CT enhanced diabetes reversal in mice with starting glycemia below 350 mg/dL, all mice that received triple therapy normalized whereas in CT alone 70% of mice normalized. In addition to these observations, our data indicate that the main mechanism by which CT controls disease is by increasing the frequencies of CD4+CD25+Foxp3+ Tregs in the pancreatic draining lymph nodes (PLN) and pancreas of CT-cured mice. Importantly, Tregs responded specifically to PINS and not to other irrelevant Ags such as ovalbumin (OVA). These cells also appear to be crucial for the maintenance of tolerance by CT, as depletion of the Foxp3+ population results in diabetes recurrence.In another set of experiments described in Chapter 6, we wanted to establish if different doses of regular vitamin D supplementation given at different periods in life were able to prevent diabetes. Data from epidemiological studies have largely indicated that vitamin D supplementation during early life correlates to protection from T1D while deficiency is associated with higher risk for disease. Most of these studies however emphasize the importance of vitamin D supplementation in at-risk individuals who are vitamin D deficient or insufficient, but do not clarify the effects of increasing 25(OH)D levels in subjects who are already vitamin D sufficient. The age at which vitamin D supplements should be administered for most effectiveness is also not clear, as clinical data yield contradictory results. For example, vitamin D insufficiency is more prevalent in pregnant/lactating women and while some studies point that vitamin D supplementation during this period decreases the risk of T1D in offspring, other reports observe no effect. An even more fundamental issue that generates intense debate is what should be considered as a vitamin D sufficient status and what should be the guidelines for vitamin D supplementation. Thus taking all of these issues into consideration and to address some of these questions, we first performed a dose titration test in which NOD mice received from 2,000 IU up to 8,000 IU of vitamin D3 daily, to evaluate whether these doses presented any toxic or calcemic effects. We observed that all the doses above 2,000 IU induced severe weight loss and 25(OH)D levels that were clearly toxic. Therefore, we selected lower doses of 1,250 IU and 800 IU/daily given at different time-points; during intra-utero/neonatal life, short-term (from 3 wks of age until 14 wks of age) and long-term (from 3wks of age until 35 wks of age). Our data show that only long-term vitamin D3 supplementation (with both the 800 IU as well as the 1,250 IU doses) can significantly prevent diabetes. Of interest, control mice received a vitamin D3 sufficient diet of 4 IU/daily and presented normal 25(OH) levels of a median of 71 ng/dL which increased to 116 ng/mL and 124ng/mL, when mice received 800 IU and 1,250 IU of vitamin D3 respectively. These increased levels of serum 25(OH) correlated with protection from disease. Moreover, we also verified that long-term vitamin D supplementation did not cause noticeable calcemic effects, no vascular calcifications were observed in kidney nor in heart and bone parameters were normal and even improved in vitamin D supplemented mice. Important changes in the immunological profiles were also observed, a significant decrease in IFN-g+CD8+Tcells while inversely an increase in CD4+Foxp3+ Tregs occurred. In summary, our data set should be of added value in the elaboration of future clinical trials as well as establishment of guidelines for vitamin D supplementation in the prevention of T1D.As a general conclusion, the current data presented in this thesis show promising therapeutic approaches for the prevention and intervention of T1D. It also indicates that different therapeutics approaches should be employed considering the time-point of intervention. We have shown for the first time that oral regular vitamin D3 supplementation can prevent diabetes when given long-term and that this does not seem to induce harmful side-effects. A more drastic approach seems necessary once disease has already been established. Here, (CT may bring new hope to this field. We have in fact shown that our CT approach which combines an Ag-specific together with a systemic mmunomodulator can re-introduce beta-cell specific tolerance and establish long lasting normoglycemia in cured animals. In the near future, we hope to fine-tune these findings in order to move on to clinical studies.nrpages: 154status: publishe

Vitamin D and diabetes.

There is no doubt that vitamin D deficiency is the cause of several metabolic bone diseases, but vitamin D status is also linked to many major human diseases including immune disorders. Mounting data strengthen the link between vitamin D and diabetes, in particular T1D and T2D. Despite some inconsistencies between studies that associate serum 25(OH)D levels with the risk of developing T1D or T2D, there seems to be an overall trend for an inverse correlation between levels of 25(OH)D and both disorders. There is also compelling evidence that 1,25(OH)2D regulates b-cell function by different mechanisms, such as influencing insulin secretion by regulating intracellular levels of Ca2+, increasing β-cell resistance to apoptosis, and perhaps also increasing β-cell replication. The capacity of vitamin D, more specifically 1,25(OH)2D, to modulate immune responses is of particular interest for both the therapy and prevention of diabetes. In the case of T1D, vitamin D supplementation in prediabetic individuals could help prevent or reduce the initiation of autoimmune processes possibly by regulating thymic selection of the T-cell repertoire, decreasing the numbers of autoreactive T cells, and inducing Treg cells. Although immune modulation is generally discussed for the treatment of T1D, it is also relevant for T2D. Indeed, recent studies have shown that T2D patients have increased systemic inflammation and that this state can induce β-cell dysfunction and death. Supplementation trials with regular vitamin D for the protection against the development of T1D and T2D have generated some contradictory data, but many weaknesses can be identified in these trials as most were underpowered or open-labeled. However, the overwhelming strength of preclinical data and of the observational studies make vitamin D or its analogues strong candidates for the prevention or treatment of diabetes or its complications. However, proof of causality needs well-designed clinical trials and if positive, adequate dosing, regimen, and compound studies are needed to define the contribution of vitamin D status and therapy in the global diabetes problem. There are many confounding factors that need to be taken into consideration when translating successful vitamin D therapies in animal models into humans, for example, gender, age, lifestyle, and genetic background. To come to solid conclusions on the potential of vitamin D or its analogues in the prevention of or therapy for all forms of diabetes, it is clear that large prospective trials with carefully selected populations and end points will be needed, but should also receive high priority.info:eu-repo/semantics/publishe

Heart health and microorganisms: the unexpected beat

The human body is colonized by an extremely complex ecosystem composed of many commensal organisms such as viruses, bacteria and fungi, which together are called microbiota. The microbiota, in particular the gut microbiota, has important metabolic functions which influence the integrity of mucosal barrier and homeostasis of the immune system. Microbial imbalance or dysbiosis has been identified as a potential risk factor for susceptibility to several chronic metabolic diseases, including diabetes mellitus, obesity and cardiovascular disease (CVD). Diets based on high intake of whole-plant foods, e.g. the Mediterranean diet, and the current prevailing Western-style diet have different effects in the gut microbiota composition influencing the abundance of different bacterial communities in the gut. The advances in science are showing a growing importance of the gut in the regulation of the immune system and its effects are related to development and also to protection against several diseases. In CVD, as in other disorders, the immune system plays at least a partial role in its pathology. Recently gut microbiota alterations, mainly during obesity induced by high fat diet have been linked to metabolic syndrome due systemic low grade inflammation and consequently, might play a role in CVD development. In this chapter, we discuss the role of nutrition influencing gut microbiota and the consequences on the immune system for cardiovascular health.info:eu-repo/semantics/publishe

Effects of Vitamin D on Antigen-specific and Antigen-non-specific Immune Modulation: Relevance for Type 1 Diabetes

Vitamin D is a fat-soluble precursor of the circulating 25-hydroxyvitamin D₃ (25(OH)D₃)which can be converted by the 1α-hydroxylase (1α(OH)ase) enzyme into the bioactive hormonal metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), generally known to promote bone mineralization through its ability to enhance calcium absorption from the gut. Importantly, in humans, vitamin D is mainly derived from endogenous production of vitamin D₃ from ultraviolet (UV) radiation exposure to the skin while a small part (<10%) is obtained via dietary intake of dairy products and fatty fish (1). Taking these factors into account, geographic distribution and seasonality, skin pigmentation, age, and lifestyle may predispose certain populations to be at a higher risk of developing vitamin D insufficiency or deficiency (2). The first valid reports correlating the importance of an adequate vitamin D status to optimal human health originate from the early part of the 20th century, when vitamin D was described to prevent and treat the bone disease rickets. Since then, the findings that vitamin D receptors (VDR) are present in many body tissues and that vitamin D metabolizing enzymes can be found in various cells outside the kidney, including the intestine, prostate, immune cells, and within the skin itself (reviewed in reference 3), have revolutionized the vitamin D business. In this review, we will mainly focus on vitamin D as a component of immune regulation and on the role of vitamin D in antigen-specific and non-specific therapies with potential relevance for type 1 diabetes (T1D).status: publishe

Intestinal barrier and gut microbiota: Shaping our immune responses throughout life.

The gastrointestinal (GI) tract is considered the largest immunological organ in the body having a central role in regulating immune homeostasis. Contrary to earlier belief, the intestinal epithelial barrier is not a static physical barrier but rather strongly interacts with the gut microbiome and cells of the immune system. This intense communication between epithelial cells, immune cells and microbiome will shape specific immune responses to antigens, balancing tolerance and effector immune functions. Recent studies indicate that composition of the gut microbiome affects immune system development and modulates immune mediators, which in turn affect the intestinal barrier. Moreover, dysbiosis may favor intestinal barrier disruption and could be related to increased susceptibility to certain diseases. This review will be focused on the development of the intestinal barrier and its function in host immune defense and how gut microbiome composition throughout life can affect this role.info:eu-repo/semantics/publishe

Immunometabolism: A target for the comprehension of immune response toward transplantation.

Organ transplantation is a life-saving procedure, however predicting graft survival is still challenging. Understanding immune-cell pathobiology is critical to the development of effective therapies to prevent rejection. Over the recent years it has become progressively evident that the complex nature of immune cell behavioral dynamics is strongly dependent on cellular metabolism, which in turn, relies on competition for nutrients, oxygen and metabolites with other immune cells and microbiota. Furthermore, the influence of the inflammatory state can lead to substantial changes in conditions within the tissue micro-environment. Considering the context of immunity, alterations in metabolic pathways (glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, the fatty acid oxidation and synthesis, and the amino acid metabolic pathways) will influence the production of different sets of cytokines and affect transplantation outcome. It is now known that naïve, resting and effector cells acquire different metabolic profiles and studies have shown that specifically targeting some of these metabolic routes can prevent differentiation of effector T cells in favor of Tregs. Ultimately, to develop effective therapies that will prevent graft loss and understanding how cell metabolism impacts the fate and function of immune cells is now a critical point of discussion. The distinct metabolic features and requirements observed in effector and suppressive cell subsets offer promising opportunities for selective regulation of the immune responses in transplantation and will be discussed in this review.info:eu-repo/semantics/publishe

OR.71. Combination Therapy of Short-term Low-dose Anti-CD3 and Oral Administration of Genetically-modified Lactococcus Lactis Secreting Insulin and IL-10 Enhances Remission in New-onset Diabetic NOD Mice

info:eu-repo/semantics/publishe