In addition to host defense, alternative functions of immune cells are emerging. Immune cells are crucial during healing of injured tissue, in formation of new blood vessels, angiogenesis, and also in maintaining the balance in inflammation having immune regulating functions. Over the last decade a higher degree of heterogeneity and plasticity of immune cells have been reported and immune cells develop different characteristics in different situations in vivo. This thesis investigates roles for immune cells in situations of muscle hypoxia and reduced blood perfusion, wound healing in skin and at sites of transplantation of allogeneic islets of Langerhans and on top of this, ways to steer immune cell function for future therapeutic purposes. A specific neutrophil subset (CD49d+VEGFR1+CXCR4high) was found to be recruited to VEGF-A released at hypoxia and these neutrophils were crucial for functional angiogenesis. In muscle with restricted blood flow macrophages were detected in perivascular positions and started to express aSMA and PDGFR1b and were found to directly assist in blood flow regulation by iNOS-dependent NO production. This essential function in muscle regain of function could be boosted by plasmid overexpression of CXCL12 where the effect of these macrophages chaperoning the vasculature was amplified improving limb blood flow regulation. The effect on macrophages accelerating tissue regeneration being amplified by CXCL12 was tested in a model of cutaneous wound healing where the administration of CXCL12 was optimized for high bioavailability. In the skin, CXCL12-treatment induced accumulation of TGFb-expressing macrophages close to the wound driving the healing process, and subsequently the wounds healed with an efficiency never reported before. In the last study means to circumvent systemic immune suppressive therapy required in allogeneic transplantation was investigated. Allogeneic islets of Langerhans transplanted to muscle were immediately destroyed by the host immune system. Co-transplanting islets and CCL22-encoding plasmids we could curb this fast rejection for 10 days by accumulating CD4+CD25+FoxP3+ regulatory T lymphocytes at the site for transplantation preventing islet grafts from being attacked by the host cytotoxic T lymphocytes. In summary this thesis outlines distinct immune cell subsets being essential for regain of tissue function in hypoxia, ischemia and post injury and ways to amplify specific immune cell functions in these situations that are feasible for clinical use.

A privação dos hormônios sexuais, natural ou induzida, contribui para o aparecimento de diversas desordens metabólicas e endócrinas. Esse estudo investigou se a suplementação em dose única com DHEA, esteróide mais abundante em humanos, melhora a sensibilidade à insulina, bem como sua secreção e ou tolerância à glicose em ratas castradas alimentadas com dieta hiperlipídica (OHL). A castração induziu a perda da proteção fisiológica das fêmeas contra o ganho de peso. O tratamento com DHEA não promoveu alterações sobre esse parâmetro, porém, corrigiu a elevação na concentração de insulina plasmática e o índice HOMA IR, além da constante de decaimento de glicose, kitt. Os animais castrados apresentaram aumento da área da ilhota. DHEA não alterou essa condição. No entanto, as ilhotas das ratas tratadas com DHEA apresentaram aumento do grau de fosforilação da proteína Akt e melhora da capacidade secretória estática de insulina. Esse estudo sugere o uso do DHEA como alternativa protetora sobre a sensibilidade a insulina em fêmeas desprovidas de ovários. / Natural and induced privation of sexual hormones contributes to the development of several metabolic and endocrine disorders. The present study evaluated if DHEA supplementation, the most abundant steroid in humans, would improve the insulin sensitivity and secretion as well as the glucose tolerance, in high fat diet fed ovariectomized rats (OHL). Ovariectomy (OVX) reduced the physiological female protection against the weight gain. Although no effect upon adipose depot-specific action of DHEA has been found, DHEA has corrected the blood insulin levels and HOMA IR. In addition, DHEA has improved peripheral insulin action by the glucose disappearance rate, kitt. The islets area was increased in all ovariectomized groups. Pancreatic islets from DHEA-treated rats showed an increased in the Akt serine phosphorylation status and restored glucose-stimulated insulin secretion. Our results suggest that DHEA can promote protective effects by increasing the insulin sensitivity in females castrated rats exposed to health risk factors.

O transplante (Tx) de ilhotas pancreáticas (IP) é uma atraente alternativa para o tratamento do diabetes melito tipo 1. No entanto, para evitar a rejeição há necessidade de imunossupressão. Uma nova idéia de tolerância surge a partir do paradoxo imunológico, onde a mãe, imunologicamente competente, não rejeita o embrião durante a gravidez. Uma das hipóteses é que células da placenta expressam a molécula IDO, a qual protege o embrião do ataque imunológico materno. O objetivo do estudo foi analisar o efeito da indução da expressão da IDO em IP em transplante experimental de IP. Para tanto, as seguintes etapas de padronização foram necessárias. Etapa 1: Padronização da perfusão e digestão do tecido pancreático de rato e determinação do método para a purificação das IP, comparando-se diferentes gradientes de densidade: descontínuo de Ficoll, contínuo de Ficoll e contínuo de iodixanol. Foi demonstrado que o gradiente contínuo de iodixanol fornece maior pureza e maior número de IP íntegras e funcionais. Etapa 2: Padronização do Tx experimental de IP sob a cápsula renal para avaliação do número mínimo de IP transplantadas para reverter o diabetes induzido por estreptozotocina, definido como glicemia >300mg/Kg. Foram transplantadas entre 200 a 3.000 IP por experimento. A rejeição das IP foi analisada pela sobrevida das IP (permanência da glicemia <300mg/dL), tanto em Tx isogênico (Lewis-Lewis) como em alogênico (Sprague-Dawley-Lewis). Para reverter o diabetes foram necessárias no mínimo 2.500 IP. No transplante entre ratos isogênicos (n=6) não houve rejeição das IP. Já no transplante entre animais alogênicos (n=12), as IP apresentaram uma curta sobrevida pós-Tx (11±1 dias; p<0,01 vs. Tx isogênico). Dez dias pós-Tx, houve um grande infiltrado de macrófagos e linfócitos T no enxerto alogênico e uma diminuição significativa da expressão de insulina (p<0,001 vs. Tx isogênico). Etapa 3: Construção do vetor de expressão para IDO. A partir de RNA extraído de placenta de rata no 10º dia de gestação, foi amplificada a seqüência completa do cDNA para IDO, utilizando-se RT-PCR. Em seguida, o cDNA para IDO foi inserido em vetor de expressão (vetor-IDO). Etapa 4: Transfecção do vetor-IDO nas IP. O vetor-IDO foi introduzido nas IP através de lipofecção (Lipofectamina 2000), testando-se diferentes concentrações do vetor-IDO (0, 0,5, 1 e 10 ng/uL) e diferentes períodos de incubação (1h, 15h e 24h). A expressão de IDO nas IP foi confirmada por RT-PCR e imuno-histoquímica. A incubação com 10 ng/uL de vetor-IDO durante 24h foi eficaz para induzir a expressão de IDO nas IP, confirmada a nível de RNAm (RT-PCR) e de proteína (imuno-histoquímica). A eficiência da transfecção em nível funcional foi confirmada pela degradação de triptofano em cultura (dosagem de triptofano por HPLC). Etapa 5: Onze transplantes alogênicos (Sprague-Dawley-Lewis) com IP transfectadas com vetor-IDO foram realizados para analisar o efeito da IDO. Três animais foram sacrificados para análise de imuno-histoquímica e 8 animais foram acompanhados por 45 dias. A sobrevida das IP transfectadas com vetor-IDO foi significativamente maior comparada com a sobrevida de IP não-transfectadas (p<0,01). O estudo conclui que a expressão da IDO protege as IP aumentando a sobrevida das IP. / Transplantation (Tx) of pancreatic islets (PI) is an attractive alternative of treatment for type 1 diabetes mellitus. However, continuous immunossupression is necessary in order to avoid allograft rejection. A new idea of tolerance is based on the immunological paradox, during pregnancy, in that the mother, immunologically competent, does not reject the semi-allogeneic fetus. The hypothesis is that the placenta produces IDO molecules, which protect the embryos against the maternal immunologic attack. The aim of this study was to analyze the effect of the induction of the IDO expression into PI in an experimental model of PI transplantation. The following steps for standardization were necessary. Step 1: Besides the standardization of the rat pancreas perfusion and digestion, the best method for purification of the PI was determined, comparing several density gradients: Ficoll discontinuous, Ficoll continuous and iodixanol continuous. The iodixanol continuous gradient was able to provide high purity and a high number of intact and functional PI. Step 2: The transplantation of the PI between rats was established determining the minimal number of PI to reverse the diabetes (glycemia > 300mg/dL) induced by streptozotocin. In addition, the rejection was analyzed by PI survival (time with glycemia <300mg/dL) in syngeneic (Lewis-Lewis) and allogeneic (Sprague-Dawley-Lewis) transplantation. To reverse the diabetes at least 2,500 PI were necessary. Transplantation between syngenic rats (n=6) disclosed no rejection of the PI. In the allogeneic transplantation (n=12), the PI had a short survival (11±1 days). Ten days post-Tx, a higher number of macrophages and T lymphocytes were observed in the grafts, accompanied by very low insulin expression. Step 3: The expression vector for IDO was constructed from RNA extracted from rat placenta. RT-PCR was carried out to amplify the IDO cDNA, which was inserted into expression vector (IDO vector). Step 4: The IDO vector was introduced into PI through lipofection (Lipofectamine 2000) analyzing several concentrations of the IDO vector (0, 0.5, 1.0 and 10 ng/uL) and several periods of incubation (1h, 15h e 24h). The IDO expression in PI was confirmed by RT-PCR and immunohistochemistry. The incubation with 10 ng/uL of IDO vector during 24h was efficient to induce IDO expression in PI. The function of the IDO was confirmed by tryptofan degradation in culture (measurement of tryptofan by HPLC). Step 5: Eleven allogenic transplants (Sprague-Dawley to Lewis) of PI expressing IDO were performed to analyze the effect of the IDO in the rejection. Eight animals were accompanied for 45 days, whereas three were sacrificed after 10 days for immunohistochemistry analysis. Finally, the survival of the PI expressing IDO was significantly higher than nontransfected PI. The study concludes that the induction of the IDO into PI protects the PI increasing the PI survival.

Nous avons découvert que les DC pathologiques (LCH DC) qui s’accumulent dans les granulomes de patients atteints d’histiocytose langerhansienne (LCH) produisent l’IL-17A (Coury et al, Nat Med 2008). In vitro, les LCH DC fusionnent en cellules géantes (MGC) sous l’influence de leur production autocrine d’IL-17A. In vivo, les granulomes de LCH DC et MGC détruisent les tissus. Pendant la thèse, nous avons étudié les transcriptomes des monocytes, DC, traitées ou non par l’IL-17A et des LCH DC. L’IL-17A induit BCL2A1, un membre de la famille Bcl-2 qui prolonge la survie des DC. Elle induit aussi les chémokines CCL20 et CCL2, qui regroupent les DC avant leur fusion. L’induction du récepteur nucléaire LXR-α, de protéines impliquées dans le métabolisme, le transport et le stockage des lipides témoignent d’un profond remodelage lipidique. Après la confirmation de ces régulations par PCR et western, les goutelettes lipidiques sont quantifiées à l’huile rouge, puis l’analyse lipidomique révèle l’augmentation de phospholipides, triglycérides, esters de cholestérol et cholestérol par l’IL-17A dans les DC qui sont aussi capables de capturer du palmitate extracellulaire fluorescent. Les transcriptomes des DC traitées à l’IL-17A et des LCH DC sont similaires. La simvastatine qui bloque la synthèse de cholestérol tue les LCH DC. Ce travail établit pour la première fois que l’IL-17A affecte profondément le métabolisme lipidique des DC, une activité qui pourrait avoir d’importantes applications dans les maladies chroniques inflammations associées à une dérégulation lipidique comme l’athérosclérose, l’obésité, la tuberculose et la LCH / We found that the pathological DCs (DC LCH), accumulating in the granulomas of patients affected with Langerhans cell histiocytosis (LCH), produce IL-17A (Coury et al, Nat Med 2008). In vitro, LCH DCs form multinucleated giant cells (MGC) by a fusion process, under the influence of their IL-17A autocrine production. In vivo, granulomas, which are mainly composed of LCH DCs and MGCs, destroy tissues of the patients. During the PhD, we studied the transcriptomes of monocytes, DCs, treated or not with IL-17A, and LCH DCs. IL-17A induceed BCL2A1, a member of the Bcl-2 family that prolonged DC survival. It also induced the CCL2 and CCL20 chemokines that clustered DC, a process required to license DC fusion. The inductions of LXR-α nuclear receptors, proteins involved in metabolism, transport and storage of lipids signed a deep lipid remodeling induced by IL-17A in DCs. We confirmed these regulations by PCR and western studies, then the lipid droplets were quantified after Oil Red-O staining. Further lipidomic analysis revealed an increase of phospholipids, triglycerides, cholesteryl esters and cholesterol by IL-17A in DCs, which are also able to capture extracellular fluorescent palmitate. Transcriptomes of DCs treated with IL-17A and LCH DCs were similar. Simvastatin, which inhibits the synthesis of cholesterol, killed LCH DC. For the first time, this work establishes that IL-17A profoundly affects the lipid metabolism of DCs, an activity that may have important applications in chronic inflammations associated with lipid deregulations such as atherosclerosis, obesity, tuberculosis and LCH

<p>Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. </p><p>An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. </p><p>In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified.</p><p>The thrombin inhibitor melagatran completely blocked the IBMIR in an <i>in vitro</i> tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. </p><p>These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.</p>

<p>Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity.</p><p>In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation<i> in vivo</i>. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF <i>in vitro</i> efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated <i>in vivo</i> in patients transplanted with isolated islets.</p><p>There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets <i>in vitro</i> and<i> in vivo</i>, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials. </p>

Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified. The thrombin inhibitor melagatran completely blocked the IBMIR in an in vitro tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.

Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity. In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation in vivo. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF in vitro efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated in vivo in patients transplanted with isolated islets. There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets in vitro and in vivo, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials.

Clinical islet transplantation is today an established alternative treatment for a selected group of type 1 diabetes patients. The predominant technique for transplantation is infusion of islets in the liver via the portal vein. Obstacles to advancing islet transplantation include limited engraftment resulting from an immediate blood-mediated inflammatory reaction (IBMIR), a life-long need for immunosuppression and the shortage of organs available. In this thesis, innate and adaptive immunity were explored in allogeneic and xenogeneic settings, with the long-term goal of preventing islet graft destruction. Methods for studying immune responses to islets in blood and engrafted islets in liver tissue (intragraft gene expression) were developed and refined. The innate response to human islets and exocrine tissue in ABO-compatible blood was characterized up to 48 h using a novel whole-blood model. Physiological changes in the blood during incubations were explored and adjusted to allow prolonged experiments. Increased production of chemokines targeting CXCR1/2, CCR2 and CXCR3 was observed, accompanied by massive intra-islet neutrophil infiltration. Notably, endocrine and exocrine tissue triggered a similarly strong innate immune response. Two studies of adult porcine islet transplantation to non-human primates (NHPs) were performed. Expression of immune response genes induced in liver tissue of non-immunosuppressed NHPs (≤72 h) was evaluated after porcine islet transplantation. Up-regulation of CXCR3 mRNA, together with IP-10, Mig, MIP-1α, RANTES, MCP-1 and cytotoxic effector molecule transcripts, was associated with T-cell and macrophage infiltration at 48-72 h. Long-term survival (&gt;100 days) of adult porcine islets in a NHP model was later demonstrated using T-cell-based immunosuppression, including co-stimulatory blockade (anti-CD154 mAb). Graft failure was associated with increased levels of circulating, indirectly activated T cells, non-Gal pig-specific IgG and gene transcripts of inflammatory cytokines. Microarray analysis of the response to inflammatory cytokines in cultured porcine islets identified genes involved in cell death, immune responses and oxidative stress; this gene pattern coincided with physiological changes (decrease in insulin and ATP content). In summary, allogeneic whole-blood experiments and xenogeneic in vivo studies underscored the importance of preventing early inflammation and cell-recruitment to avoid islet graft loss in islet transplantation. Long-term survival of porcine islets in NHPs was shown to be feasible using T-cell-directed immunosuppression, including anti-CD154 mAb.

Amyloid is defined as extracellular protein aggregates with a characteristic fibrillar ultra-structure, Congo red affinity and a unique x-ray diffraction pattern. At present, 25 different human amyloid fibril proteins have been identified, and amyloid aggregation is associated with pathological manifestations such as Alzheimer’s disease, spongiform encephalopathy and type 2 diabetes. Amyloid aggregation triggers apoptosis by incorporation of early oligomers in cellular membranes, causing influx of ions. Amyloid is the only visible pathological islet alteration in subjects with type 2 diabetes, and islet amyloid polypeptide (IAPP) is the major islet amyloid fibril component. IAPP is produced by beta-cells and co-localized with insulin in the secretory granules. Both peptides are synthesised as pro-molecules and undergo proteolytic cleavage by the prohormone convertase 1/3 and 2. Although IAPP is the main amyloid constituent, both proIAPP and proIAPP processing intermediates have been identified in islet amyloid. The aim of this thesis was to study the role of impaired processing of human proIAPP in early islet amyloidogenesis. Five cell lines with individual processing properties were transfected with human proIAPP and expression, aggregation and viability were studied. Cells unable to process proIAPP into IAPP or to process proIAPP at the N-terminal processing site accumulated intracellular amyloid-like aggregates and underwent apoptosis. Further, proIAPP immunoreactivity was detected in intracellular amyloid-like aggregates in betacells from transgenic mice expressing human IAPP and in transplanted human beta-cells. ProIAPP was hypothesized to act as a nidus for further islet amyloid deposition, and to investigate this theory, amyloid-like fibrils produced from recombinant IAPP, proIAPP and insulin C-peptide/A-chain were injected in the tail vein of transgenic mice expressing the gene for human IAPP. Pancreata were recovered after 10 months and analysed for the presence of amyloid. Both IAPP and proIAPP fibrils but not des-31,32 proinsulin fibrils, caused an increase in affected islets and also an increase of the amyloid amount. This finding demonstrates a seeding capacity of proIAPP on IAPP fibrillogenesis. IAPP has been known for some time to trigger apoptosis in cultured cells, and a novel method for real time detection of apoptosis in beta-cells was developed. Aggregation of recombinant proIAPP and proIAPP processing intermediates were concluded to be inducers of apoptosis as potent as IAPP fibril formation. From the results of this study, a scenario for initial islet amyloidogenesis is proposed. Initial amyloid formation occurs intracellularly as a result of alterations in beta-cell processing capacity. When the host cell undergoes apoptosis intracellular proIAPP amyloid becomes extracellular and can act as seed for further islet amyloid deposition.