Final Report Summary - MACROPHAGES IN T1D (Functional profiling and therapeutic tolerization of macrophages in type 1 diabetes)
SUMMARY DESCRIPTION OF THE PROJECT OBJECTIVES
Type 1 diabetes (T1D) is a chronic condition that results from the specific destruction of pancreatic beta cells by the immune system. As a consequence, affected individuals can no longer regulate their blood sugar levels and become dependent on glucose monitoring and insulin injections. Despite careful disease management, serious neuronal and vascular long-term complications can often not be avoided. This debilitating disease is an important and growing public health concern in Europe: it is predicted that by 2020 approximately 24.400 individuals will be diagnosed with T1D annually.
It is thought that T cells are the principal effector subset that drives beta cell decay within the islets of Langerhans. The importance of innate immunity and in particular macrophages has historically attracted a minor degree of attention. Nevertheless, convincing data exist that macrophages significantly contribute to T1D pathogenesis.
Recent evidence from clinical trials has exposed the failure of strategies for inducing peripheral T cell tolerance, most recently with anti-CD3 antibody therapy. While genetically defined thymic escape and consequential T cell autoimmunity may be very important in type 1 diabetes, we hypothesize that T cell-mediated recruitment and activation of macrophages represents a secondary yet significant pathological pathway.
The present project’s key objectives therefore were:
1. Macrophage profiling in pancreas and PDLN around diabetes onset
2. Investigating the role of macrophages in antigen drainage and spreading
3. Proof of concept study - tolerance induction by tolerogenic macrophages
DESCRIPTION OF THE WORK PERFORMED SINCE THE BEGINNING OF THE PROJECT
As projected in the original application (see Figure 1 in attachment), the first months would be devoted to the introduction of the NOD mouse model for type 1 diabetes, the technique of islet isolation and initial experiments on macrophage detection and profiling in the pancreatic islets during diabetes progression.
1. NOD mice were never bred or maintained in our facility and therefore diabetes incidence needed to be carefully monitored. Female mice were purchased from two different suppliers (Charles River and Taconic) and their blood glucose values were monitored from the age of 9 weeks until development of overt diabetes (value over 300mg/dl). Standard immunohistochemistry and immunofluorescence was performed on diabetic and non-diabetic animals at the end of the experiment.
2. The technique of islet isolation was introduced into the Elewaut lab and optimized to yield sufficient quantities of pure islets for flow cytometric analysis of islet infiltrates. Initial results were obtained that give a first impression of the phenotype of macrophages that infiltrate the islets around onset.
3. Immunofluorescent staining was developed and optimized for detection of macrophages in pancreatic islets.
DESCRIPTION OF THE MAIN RESULTS ACHIEVED
The progression of diabetes was plotted in two different graphs. The first graph (Figure 2), shows averaged blood glucose values per group from a different supplier. Although a non-significant lagging period appears to exist in the Charles River group, we can conclude that average blood glucose values rise in similar fashion in cohorts from both suppliers as time progresses. These data serve as a first indication that both NOD strains from the two suppliers can be used and that the most economic option should be chosen.
Next, data were plotted in diabetes incidence curves, which allow for easy comparison of progression rates with published data. At week 30, total incidence for Taconic mice was 50%, while 60% of Charles River mice were diabetic at that time (Figure 3). From comparison with the ‘standard’ curves obtained from the Jackson Laboratories (Figure 4), it can be concluded that both progression rate as final diabetes incidence are substantially lower on our colony. It was suggested by the authors of this report that if the female incidence is <60% by 30 weeks, the presence of a pathogen or other immunomodulatory agent in the colony is indicated.
In order to attempt lowering the bacterial load, the next batch of mice was housed in individually ventilated cages, treated under laminar flow hoods and provided with sterilized bedding, water and food. Theoretically, these conditions should accelerate and aggravate disease, yet we noted only a mild increase (Figure 5). The seven non-diabetic mice were used for islet isolation and macrophage detection at week 24 of age.
Initially, two mice were subjected to the protocol of islet isolation as outlined in the original application. The procedure immediately worked well (Figure 6), yet the yield of infiltrating cells after digestion was too low to assess by flow cytometry (data not shown). Therefore, five mice pancreata were pooled and subjected to the islet isolation procedure. This approach proved successful, as a sizeable macrophages population could be detected by flow cytometry (Figure 7). In comparison with mesenteric lymph nodes, a clear relative shift was observed within the F4/80+ macrophage population from a ‘resident’ phenotype toward an inflammatory phenotype. The pancreatic population showed loss of Ly6C- cells (3.4% vs 7.5%), the appearance of a Ly6C+ population, more CD49b expression (1.5% vs 0.5%) which is an integrin on inflammatory macrophages. Furthermore, a relative decrease was noted in CD43, a marker for resident macrophages (3.8% vs 7.4%), while CCR7 expression increased within the macrophage compartment (3.5 vs 1.8%). It can be concluded that flow cytometric analysis of the infiltrating macrophage population is achievable as proposed and that this population has the phenotypic characteristics of inflammatory macrophages.
Finally, the non-diabetic mice from the experiment depicted in Figure 2-3 were used to optimize a pancreatic staining for macrophages on frozen sections and assess whether these mice did in fact show signs of macrophage infiltration. Staining on spleen sections showed that the F4/80 staining was specific (Figure 8). Subsequent analysis of the non-diabetic mice from Figure 2-3 showed that all mice had macrophage infiltration, which would make them suitable subjects for analysis of this population in the islets. Macrophage infiltration could also be seen on whole-mount staining of isolated islets, indicating that a sufficient large infiltrate is present despite the relatively low diabetes incidence as observed.
Type 1 diabetes (T1D) is a chronic condition that results from the specific destruction of pancreatic beta cells by the immune system. As a consequence, affected individuals can no longer regulate their blood sugar levels and become dependent on glucose monitoring and insulin injections. Despite careful disease management, serious neuronal and vascular long-term complications can often not be avoided. This debilitating disease is an important and growing public health concern in Europe: it is predicted that by 2020 approximately 24.400 individuals will be diagnosed with T1D annually.
It is thought that T cells are the principal effector subset that drives beta cell decay within the islets of Langerhans. The importance of innate immunity and in particular macrophages has historically attracted a minor degree of attention. Nevertheless, convincing data exist that macrophages significantly contribute to T1D pathogenesis.
Recent evidence from clinical trials has exposed the failure of strategies for inducing peripheral T cell tolerance, most recently with anti-CD3 antibody therapy. While genetically defined thymic escape and consequential T cell autoimmunity may be very important in type 1 diabetes, we hypothesize that T cell-mediated recruitment and activation of macrophages represents a secondary yet significant pathological pathway.
The present project’s key objectives therefore were:
1. Macrophage profiling in pancreas and PDLN around diabetes onset
2. Investigating the role of macrophages in antigen drainage and spreading
3. Proof of concept study - tolerance induction by tolerogenic macrophages
DESCRIPTION OF THE WORK PERFORMED SINCE THE BEGINNING OF THE PROJECT
As projected in the original application (see Figure 1 in attachment), the first months would be devoted to the introduction of the NOD mouse model for type 1 diabetes, the technique of islet isolation and initial experiments on macrophage detection and profiling in the pancreatic islets during diabetes progression.
1. NOD mice were never bred or maintained in our facility and therefore diabetes incidence needed to be carefully monitored. Female mice were purchased from two different suppliers (Charles River and Taconic) and their blood glucose values were monitored from the age of 9 weeks until development of overt diabetes (value over 300mg/dl). Standard immunohistochemistry and immunofluorescence was performed on diabetic and non-diabetic animals at the end of the experiment.
2. The technique of islet isolation was introduced into the Elewaut lab and optimized to yield sufficient quantities of pure islets for flow cytometric analysis of islet infiltrates. Initial results were obtained that give a first impression of the phenotype of macrophages that infiltrate the islets around onset.
3. Immunofluorescent staining was developed and optimized for detection of macrophages in pancreatic islets.
DESCRIPTION OF THE MAIN RESULTS ACHIEVED
The progression of diabetes was plotted in two different graphs. The first graph (Figure 2), shows averaged blood glucose values per group from a different supplier. Although a non-significant lagging period appears to exist in the Charles River group, we can conclude that average blood glucose values rise in similar fashion in cohorts from both suppliers as time progresses. These data serve as a first indication that both NOD strains from the two suppliers can be used and that the most economic option should be chosen.
Next, data were plotted in diabetes incidence curves, which allow for easy comparison of progression rates with published data. At week 30, total incidence for Taconic mice was 50%, while 60% of Charles River mice were diabetic at that time (Figure 3). From comparison with the ‘standard’ curves obtained from the Jackson Laboratories (Figure 4), it can be concluded that both progression rate as final diabetes incidence are substantially lower on our colony. It was suggested by the authors of this report that if the female incidence is <60% by 30 weeks, the presence of a pathogen or other immunomodulatory agent in the colony is indicated.
In order to attempt lowering the bacterial load, the next batch of mice was housed in individually ventilated cages, treated under laminar flow hoods and provided with sterilized bedding, water and food. Theoretically, these conditions should accelerate and aggravate disease, yet we noted only a mild increase (Figure 5). The seven non-diabetic mice were used for islet isolation and macrophage detection at week 24 of age.
Initially, two mice were subjected to the protocol of islet isolation as outlined in the original application. The procedure immediately worked well (Figure 6), yet the yield of infiltrating cells after digestion was too low to assess by flow cytometry (data not shown). Therefore, five mice pancreata were pooled and subjected to the islet isolation procedure. This approach proved successful, as a sizeable macrophages population could be detected by flow cytometry (Figure 7). In comparison with mesenteric lymph nodes, a clear relative shift was observed within the F4/80+ macrophage population from a ‘resident’ phenotype toward an inflammatory phenotype. The pancreatic population showed loss of Ly6C- cells (3.4% vs 7.5%), the appearance of a Ly6C+ population, more CD49b expression (1.5% vs 0.5%) which is an integrin on inflammatory macrophages. Furthermore, a relative decrease was noted in CD43, a marker for resident macrophages (3.8% vs 7.4%), while CCR7 expression increased within the macrophage compartment (3.5 vs 1.8%). It can be concluded that flow cytometric analysis of the infiltrating macrophage population is achievable as proposed and that this population has the phenotypic characteristics of inflammatory macrophages.
Finally, the non-diabetic mice from the experiment depicted in Figure 2-3 were used to optimize a pancreatic staining for macrophages on frozen sections and assess whether these mice did in fact show signs of macrophage infiltration. Staining on spleen sections showed that the F4/80 staining was specific (Figure 8). Subsequent analysis of the non-diabetic mice from Figure 2-3 showed that all mice had macrophage infiltration, which would make them suitable subjects for analysis of this population in the islets. Macrophage infiltration could also be seen on whole-mount staining of isolated islets, indicating that a sufficient large infiltrate is present despite the relatively low diabetes incidence as observed.