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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster I14 - Unveiling Macrophage Dynamics: A Potential Therapeutical Target for Type 1 Diabetes

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Type 1 Diabetes (T1D) pathogenesis is characterized by the destruction of insulin producing beta-cells within the pancreatic Islets of Langerhans resulting in insulin shortage. This damage of beta-cells is largely connected to autoimmune-mediated attack. Stemming from this our view of T1D pathogenesis has shifted towards identifying the connection between beta cells and the adaptive immune system of the body. Macrophages play a multifaceted role in both innate and adaptive immune response and are suspected to be the crucial players in T1D pathogenesis. Activated macrophages are traditionally classified as pro-inflammatory (M1), and anti-inflammatory (M2) phenotypes; but exist on a spectrum dependent on the stimulating factors present in their microenvironments. In T1D, M1 macrophages can contribute to disease development, and subsequent beta cell death, through the exacerbation of inflammatory responses via cytokines such as IL-1β. Conversely, M2 macrophages can stimulate a protective effect, resolving inflammation and promoting tissue repair through the release of factors such as IL-10 and TGF-β. The balance between these macrophage subsets is critical in determining the outcome of T1D. Therapeutically, targeting macrophage polarization presents a promising avenue for T1D intervention. Thus, the focus of this project is to investigate the role of macrophages in the development of T1D and identifying a viable therapeutic target. Preceding therapies that leverage relative states is a need for greater understanding of macrophage involvement in the pathogenesis of T1D.

Materials and

Methods:  In this two-phase study we establish a timeline of macrophage-disease involvement and leverage this to deliver a polarizing nano-immunotherapy. Using the female NOD-ShiLtJ polygenic mouse model, we conducted a time course study with female mice aged to weeks 4, 8, 12, 15, 18 and 20, with n=4 mice. The blood-glucose levels were checked for each mouse prior to sacrifice, with levels 150-250 mg/dL considered pre-diabetic and >250 mg/dL considered diabetic. Following sacrifice at the respective timepoints, the pancreas was excised, embedded in paraffin and subsequently sectioned to get 5 μm thick slices. These sections were then immunostained with anti-CD68 and anti-insulin antibodies to identify macrophages, and insulin-producing i.e. functional beta cells, respectively, while DAPI was used to stain cell nuclei. Fluorescent images were then acquired using a Nikon fluorescent microscope to identify the populations of macrophages (red), functional beta cells (green), and cell nuclei (blue). The cell populations were counted and analyzed digitally using ImageJ software (NIH). Next, the macrophages will be further stained using the M1 and M2 polarization markers to analyze the sub-populations. In the following phase, standard conjugation protocols will be used to chemically conjugate iron oxide nanoparticles to an immunomodulatory agent. Following delivery of the nano-immunotherapeutic agent, will be monitored by in-vivo magnetic particle imaging. Disease development is monitored via blood-glucose levels. Animals are sacrificed at similar time points to the above, the pancreas excised, and analysis performed. At this stage, analysis involves immunostaining for previously known M1 and M2 markers for the variable polarized macrophages.

Results, Conclusions, and Discussions: Figure 1A shows the graphical timeline for the analysis of macrophage population in the pancreatic tissue and figure 1B is the schematic representation of the methodology undertaken during the course of this study. The immunofluorescent images showed a significant influx of macrophages by 8 weeks (Figure 1C) which clearly indicate their role in the initial stages of T1D pathogenesis. For week 4, the beta cells were seen to be almost intact with unhindered reduction of insulin and minimal macrophage cells. By week 8, the beta cells were observed as being infiltrated by a significant population of macrophages and other immune cells while the insulin signal was still prominent. This is indicative of immune system activation, with macrophages acting as “first responders”. By week 12, although the macrophage migration is still active, the insulin signal has started to decrease. These results suggest that beta cells are significantly damaged. The quantitative analyses of the images (from ImageJ) indicate peak macrophage migration at week 8 of disease development (Figure 1D). Though further work is still necessary, these findings are in line with current literature on T1D pathogenesis - which indicate lowered insulin secretion, initiation of beta-cell destruction, in NOD females by 12 weeks of age. By identifying this therapeutic intervention point, we present an opening for delivery of polarizing nano immunotherapies which can target macrophages at the point of greatest therapeutic efficacy. This work addresses a significant shortcoming in the T1D research, by establishing a timeline of macrophage-dependent T1D involvement, and further elucidates specific timepoints for maximum therapeutic efficacy. This study identifies that the macrophages are pivotal in the initiation of the autoimmune cascade of T1D. Understanding their complex roles opens new possibilities for therapeutic interventions aimed at modulating immune responses and preserving beta cell function in T1D. If successful, this approach should be able to prevent macrophage polarization to M1 phenotype resulting in either delayed onset of T1D or complete prevention.

Acknowledgements (Optional):