Understanding and tuning the immune response in Volumetric Muscle Loss (VML) injury

Ricardo Whitaker

doi:10.17918/00010683

Volumetric Muscle Loss (VML) is a debilitating condition characterized by the sudden loss of 20% or more of muscle mass, leading to tissue impairment. These types of injuries are commonly seen in soldiers on active combat, however, are also frequent in victims of motor vehicle accidents, and on sarcoma patients following tumor removal. The current standard of care including surgery and physical therapy is ineffective. A novel treatment currently in clinical trials based on the implantation of a naturally-derived scaffold provided marginal improvement. The lack of available treatments stems from the poor understanding of the cellular and molecular processes governing VML pathology. Recent seminal papers have revealed a dysfunctional macrophage phenotype following critical size VML injuries. Nonetheless, several gaps in knowledge remain: 1) The complete timeline of macrophage dysfunction from onset to resolution has not yet been uncovered. 2) Other large injury model showed systemic changes in immune response; however such changes have never been investigated in the context of VML. 3) Although a dysfunctional macrophage phenotype has been investigated, the mechanism driving this process has never been uncovered. 4) There are no current treatments which take into consideration these changes in macrophage phenotype, or other immune cells. Therefore, the goal of this thesis is to fill in these gaps in knowledge to instruct better therapeutics. To recreate a below (2 mm) and above (4 mm) critical size injuries, here called regenerative and fibrotic, respectively, we created wounds in the quadriceps of C57B/6 mice using biopsy punches. We firstly confirmed the validity of the model via Masson's Trichrome staining, sGAG quantification and muscle mass 28 days post injury. Once the model has been confirmed, we tracked macrophage phenotype over 28 days at the site of injury using a comprehensive multidimensional flow cytometry panel. Fibrotic macrophages consistently expressed higher levels of inflammatory markers including PD-L1, CD38, CD9 and CXCR4. On the other hand, Fibrotic macrophages downregulated reparative markers CD163, CD301b and CD206 compared to regenerative group. Nonetheless, inflammatory marker CD86 was upregulated, and reparative marker Arg1 downregulated in the Regenerative group. We employed hierarchical clustering analysis to further characterize macrophage subpopulations. A few significantly different clusters emerged on day 1 and day 3, however several clusters were observed on Day 7. These clusters revealed the presence of several hybrid phenotypes present in both injury groups. To further characterize these macrophages we FACS-sorted fibrotic and regenerative macrophages at days 1 and 3 and analyzed their gene expression via nanoString. Fibrotic macrophages at Day 1 showed a broad gene downregulation, which partially held up to Day 3, particularly due to the downregulation of reparative and phagocytosis related genes. We also evaluated previously unchecked changes in immune response, particularly macrophage phenotype following both injuries. Bone marrow macrophage showed no difference among fibrotic, regenerative and naïve groups. Nonetheless, splenic macrophages in Fibrotic injuries showed upregulation of several inflammatory markers CD38, PD-L1 and CXCR4 as well as reparative marker CD206. To evaluate possible mechanisms for these changes, we investigated the presence of systemic cytokines, chemokines, and growth factors in blood serum. We observed an increase in concentration of inflammatory cytokines IL-1b, IL-12 and IL-15 at day 1 in the regenerative group, as well as upregulation of G-CSF at day 1 and IL-13 at day 3 on fibrotic group. Next, we evaluated immune cell presence in bone marrow and spleen. We observed an increase in macrophages and natural killer cell populations in the bone marrow of fibrotic mice. Although no mechanism for these systemic changes have been uncovered, as well as its impact on the local injury, these results shine a light on other possible deleterious consequence of VML injury never studied before. To establish the mechanism driving this dysfunctional macrophage phenotype in the muscle, we analyzed whole muscle tissue gene expression using the same nanoString panel. Fibrotic injuries consistently presented a decrease in genes associated with phagocytosis and extracellular matrix remodeling. On the other hand, genes responsible for the transcription of important chemokines, primarily for neutrophil trafficking, such as Cxcl1, Cxcl2 and Cxcl3 were upregulated in fibrotic injuries. Taken together these results, in addition to the upregulation of G-CSF and the vast literature on neutrophil modulation of macrophage phenotype, we investigated immune cell accumulation to the site of injury. Although several cell types are present at higher levels in fibrotic injury, the most prevalent and the only change preceding changes in macrophage phenotype, was an increase in neutrophils levels in the fibrotic group on day 1. Next, we evaluated if fibrotic neutrophils were phenotypically different from regenerative neutrophils. nanoString of FACSsorted neutrophils at day 3 showed only 8 genes differently expressed among 259. Analysis of neutrophil mode of death, another potent macrophage modulator, showed a slight increase in apoptosis among fibrotic neutrophils versus regenerative at 6 hours post injury. To confirm that neutrophils assist in macrophage dysfunction, we conducted several neutrophil depletion studies. Neutrophil depletion on day 3 rescued the expression of important reparative markers CD163 and CD301b, and it decreased the expression of inflammatory markers CD38 and PD-L1 at day 7, as seen by flow cytometry. Nonetheless, clustering analysis of nanoString data on FACS-sorted macrophages at day 3 demonstrated little to no improvements in gene expression of macrophages following neutrophil depletion. To evaluate the effects of neutrophil depletion on tissue regeneration, histological analysis was performed on day 28. Neutrophil depletion had little to no effect on muscle regeneration as seen by Masson's Trichrome, H&E and Laminin staining. These results demonstrate that neutrophils negatively impact macrophage phenotype, however neutrophil depletion and the subsequent partial recovery of macrophage phenotype is not sufficient for tissue regeneration. We believe that a therapeutic approach that addresses the issues with macrophage phenotype via the mechanism uncovered thus far, as well as providing a platform for cell growth would be able to bridge the current gap for VML treatment. Based on the previous results, a macrophage-based cell therapy is a promising therapeutic strategy. Exogenous macrophages could facilitate neutrophil uptake via efferocytosis, as well as modulate endogenous macrophage phenotype via cell to cell interaction or mediated via release of different soluble factors. Nonetheless, macrophage cell therapy faces two main challenges: 1) Retention of implanted cells. 2) Maintenance of a desired phenotype. In the final portion of my thesis, I have investigated drug candidates to modulate macrophage phenotype, as well as delivery vehicles to enhance macrophage retention at the site of injury. To establish a best drug candidate to modulate macrophage phenotype I have assessed several drugs, here denoted as Drugs A-H as well as unpolarized macrophage control, M0. The flow cytometry markers and genes analyzed via PCR were chosen based on in vivo differences between regenerative and fibrotic injuries. Macrophage phenotype at days 1 and 3 following one day of drug administration assessed via PCR and flow cytometry indicated Drug G as best candidate due to its ability to upregulate important reparative markers, such as CD301b. Conditioned media experiments confirmed Drug G as the best candidate to modulate nearby macrophages. Although we have observed no changes in efferocytotic capacity following any of the drug treatments, Drug G treated macrophages still maintained a more reparative phenotype when cocultured with neutrophils at a 2:1 ratio (neutrophils : macrophages). Through every metric, Drug G treated macrophages possessed the best phenotype for cell therapy following critical size VML. The next step was to evaluate different biomaterials on their capacity to foster implanted macrophages retention at the site of injury. Here we tested a commercially available ECM scaffold (Surgifoam) as well as a gelatin based photo-crosslinkable hydrogel, using methacrylic acid as crosslinker (GelMA). A million IL-4 polarized macrophages (GFP+) were seeded or encapsulated in each material and implanted in a critical size VML injury, alongside acellular and untreated controls. Macrophages seeded on Surgifoam scaffold showed the highest recovery, at days 1 and 7, with nearly 100,000 macrophages recovered by day 7, compared to around 25,000 in GelMA group. These biomaterials also influenced local macrophage phenotype. GelMA treated groups, with or without cells, decrease expression of inflammatory markers CD9, CD38, PD-L1, CD163 and CD206, compared to other groups. In addition, GelMA treated injuries, with or without cells, increased both monocyte subtypes accumulation at the site of injury by day 7. These results demonstrate the feasibility of either Surgifoam or GelMA to increase cell retention compared to existing methods. It also indicates intrinsic immunomodulatory properties of these materials in the absence of cells. In summary, in this work we have demonstrated the previously unknown phenomena: 1) Macrophage dysfunction initiates 1 day after injury and persists for over 28 days. 2) Local muscle injury lead to several changes in immune response systemic, which although not assessed here, may have repercussion on muscle regeneration. 3) Neutrophils are surprisingly only a minor modulator of macrophage phenotype and seem to have little to no effect on muscle regeneration. 4) Biomaterial-mediated macrophage cell therapy is a possible avenue for VML treatment.

Understanding and tuning the immune response in Volumetric Muscle Loss (VML) injury

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Understanding and tuning the immune response in Volumetric Muscle Loss (VML) injury

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