Biomaterial immunological niches as sensors for progressive experimental autoimmune encephalomyelitis progression and treatment efficacy

Introduction:: Multiple sclerosis is an autoimmune disease of the central nervous system. Relapsing-remitting forms of the disease are defined by periods of neurological disability followed by periods of partial or full recovery of function, whereas progressive forms are defined by continuous tissue damage and loss of function. While there are effective treatments for relapsing-remitting forms, treatments for progressive forms are few and limited in efficacy. A major barrier to treatment discovery is the lack of tissue-specific immune monitoring as brain and spinal cord biopsies are highly invasive and blood biopsies lack disease-specific biomarkers. Thus, there is a clinical need for new methods of monitoring disease progression. Our approach to address this need is to use subcutaneously-implanted polycaprolactone microporous scaffolds as a distal engineered tissue that serves as an immunological niche that can be longitudinally biopsied for biomarkers of disease progression. The Shea lab has previously shown that in relapsing-remitting multiple sclerosis murine models—experimental autoimmune encephalomyelitis—these scaffolds become vascularized and allow for continuous influx and efflux of innate and adaptive immune cells. Gene expression at the scaffold niche during both early and peak of disease reflected disease state [1]. Thus, we hypothesize these scaffolds would also create immunological niches that capture progressive experimental autoimmune encephalomyelitis disease progression and allow for monitoring of immune dynamics during treatment.


Materials and Methods:: Microporous scaffolds are fabricated by compression molding polycaprolactone and salt porogen leaching. C57BL/6 mice were given progressive experimental autoimmune encephalomyelitis via an adoptive transfer of myelin-reactive T cells and controls were given ovalbumin (OVA)-reactive T cells. Scaffolds were implanted subcutaneously and explanted at various timepoints throughout disease progression. Treatment with an anti-VLA-4 antibody began 7 days after the adoptive transfer and was given intraperitoneally every other day. RNA from explanted scaffolds was isolated for bulk RNA sequencing. T-tests were used to screen out non-significantly different genes between groups of interest (Study 1: OVA or myelin groups, Study 2: OVA or myelin and anti-VLA-4 or isotype control treated). Gene signatures were developed using elastic-net regularization of a multinomial regression model using the glmnet r package. 100-fold cross-validation was used to identify the optimal tuning parameter for the regularized model. Differentially expressed genes were identified using DESeq2 and functional enrichment of those genes was performed with clusterProfiler. Single cell RNA sequencing of the scaffolds 7 and 9 days after adoptive transfer was done using the 10X Chromium Controller platform for library preparation, and libraries were sequenced on the Illumina NovaSeq. Cell type identification and analysis was done using Seurat v3. CellChat was used to identify cell-cell communication networks.


Results, Conclusions, and Discussions:: Results and Conclusions:

Gene expression from bulk RNA sequencing obtained a 28 gene signature that differentiates mice with progressive experimental autoimmune encephalomyelitis and mice that received control OVA-reactive T cells at both 9 and 14 days after disease induction. Differentially expressed genes over time in the diseased group had enrichment for metabolic and proteolytic processes as well as innate and adaptive immune cell regulation. Single cell RNA sequencing at the scaffolds confirmed both innate and adaptive cell types are present (Fig. 1A) and identified cell-cell communication networks between antigen-activated T cells and innate antigen-presenting cells. Thus, we concluded scaffold gene expression captures disease progression by monitoring immune dynamics during subclinical phases to peak symptomatic disease. Because we identified significant T cell-mediated processes at the scaffold, we aimed to modulate these processes using a VLA-4 blocking antibody to prevent pathogenic T cell migration to the central nervous system (Fig. 1B). A 33 gene signature from bulk RNA sequencing at the scaffold differentiated healthy mice, pre-symptomatic pre-treatment mice, and anti-VLA-4 and isotype control treated mice (Fig. 1C). Functional enrichment of differentially expressed genes between anti-VLA-4 treated and isotype control mice for GO terms shows metabolic enrichment and KEGG pathway analysis confirms neurodegenerative processes/diseases. Gene set variation analysis (GSVA)-identified pathways show the scaffold captures immune changes in the subclinical phase of disease and during anti-VLA4 treatment. Thus, we conclude treatments modifying symptomatic disease, such as anti-VLA-4, can also be monitored at the scaffold’s immunological niche.

Discussion:

These studies show the potential to use these microporous scaffolds as sensors for biomarker identification in multiple sclerosis disease progression and treatment efficacy. While MRI reports on disease progression via visualizing tissue damage, scaffold biopsy would allow for a minimally-invasive sampling of immunological changes that would otherwise be unattainable, which would help facilitate a precision medicine approach to treating multiple sclerosis. Future studies using treatments that stratify into responders and non-responders would further support the scaffold as a distal immunological niche that can be sampled to show differences in disease state and treatment efficacy.


Acknowledgements (Optional): :

References (Optional): : [1] Morris, A.H., et al. "Engineered immunological niches to monitor disease activity and treatment efficacy in relapsing multiple sclerosis." Nature communications 11.1 (2020): 3871.