Eosinophils Respond to Extracellular Matrix Treated Muscle Injuries but are Not Required for Macrophage Polarization.

The immune response to decellularized extracellular matrix (ECM) muscle injury is characterized by Th2 T cells, Tregs, M2-like macrophages, and an abundance of eosinophils. Eosinophils have previously been described as mediators of muscle regeneration but inhibit skin wound healing. In addition to response to wounding, a large number of eosinophils respond to biomaterial-treated muscle injury, specifically in response to decellularized ECM. ECM treatment of muscle wounds has been associated with positive outcomes in tissue regeneration, but the detailed mechanisms of action are still being evaluated. Here, this work investigates the role of these eosinophils in terms of their immunologic phenotype and subsequent effect on the local tissue microenvironment. These cells have a mixed phenotype showing both type-2 and regulatory gene upregulation and but are not required for macrophage polarization. Beyond the local tissue, ECM treatment is seen to induce a transient flux of eosinophils to the lungs but prevented a trauma-associated neutrophilia in the lungs of injured mice. This work believes this local and systemic immunomodulation contributes to the regenerative effects of the material and such distal tissue effects should be considered in therapeutic design and implementation.

Basic immunologic study as a foundation for engineered therapeutic development.

Bioengineering and drug delivery technologies play an important role in bridging the gap between basic scientific discovery and clinical application of therapeutics. To identify the optimal treatment, the most critical stage is to diagnose the problem. Often these two may occur simultaneously or in parallel, but in this review, we focus on bottom-up approaches in understanding basic immunologic phenomena to develop targeted therapeutics. This can be observed in several fields; here, we will focus on one of the original immunotherapy targets-cancer-and one of the more recent targets-regenerative medicine. By understanding how our immune system responds in processes such as malignancies, wound healing, and medical device implantation, we can isolate therapeutic targets for pharmacologic and bioengineered interventions.

Development of a predictive algorithm for patient survival after traumatic injury using a five analyte blood panel.

Severe trauma can induce systemic inflammation but also immunosuppression, which makes understanding the immune response of trauma patients critical for therapeutic development and treatment approaches. By evaluating the levels of 59 proteins in the plasma of 50 healthy volunteers and 1000 trauma patients across five trauma centers in the United States, we identified 6 novel changes in immune proteins after traumatic injury and further new variations by sex, age, trauma type, comorbidities, and developed a new equation for prediction of patient survival. Blood was collected at the time of arrival at Level 1 trauma centers and patients were stratified based on trauma level, tissues injured, and injury types. Trauma patients had significantly upregulated proteins associated with immune activation (IL-23, MIP-5), immunosuppression (IL-10) and pleiotropic cytokines (IL-29, IL-6). A high ratio of IL-29 to IL-10 was identified as a new predictor of survival in less severe patients with ROC area of 0.933. Combining machine learning with statistical modeling we developed an equation ("VIPER") that could predict survival with ROC 0.966 in less severe patients and 0.8873 for all patients from a five analyte panel (IL-6, VEGF-A, IL-21, IL-29, and IL-10). Furthermore, we also identified three increased proteins (MIF, TRAIL, IL-29) and three decreased proteins (IL-7, TPO, IL-8) that were the most important in distinguishing a trauma blood profile. Biologic sex altered phenotype with IL-8 and MIF being lower in healthy women, but higher in female trauma patients when compared to male counterparts. This work identifies new responses to injury that may influence systemic immune dysfunction, serving as targets for therapeutics and immediate clinical benefit in identifying at-risk patients.

Conserved and tissue-specific immune responses to biologic scaffold implantation.

Upon implantation into a patient, any biomaterial induces a cascade of immune responses that influences the outcome of that device. This cascade depends upon several factors, including the composition of the material itself and the location in which the material is implanted. There is still significant uncertainty around the role of different tissue microenvironments in the immune response to biomaterials and how that may alter downstream scaffold remodeling and integration. In this study, we present a study evaluating the immune response to decellularized extracellular matrix materials within the intraperitoneal cavity, the subcutaneous space, and in a traumatic skeletal muscle injury microenvironment. All different locations induced robust cellular recruitment, specifically of macrophages and eosinophils. The latter was most prominent in the subcutaneous space. Intraperitoneal implants uniquely recruited B cells that may alter downstream reactivity as adaptive immunity has been strongly implicated in the outcome of scaffold remodeling. These data suggest that the location of tissue implants should be taken together with the composition of the material itself when designing devices for downline therapeutics. STATEMENT OF SIGNIFICANCE: Different tissue locations have unique immune microenvironments, which can influence the immune response to biomaterial implants. By considering the specific immune profiles of the target tissue, researchers can develop implant materials that promote better integration, reduce complications, and improve the overall outcome of the implantation process.

Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Intersection of Immunity, Metabolism, and Muscle Regeneration in an Autoimmune-Prone MRL Mouse Model.

Due to the limited capacity of mammals to regenerate complex tissues, researchers have worked to understand the mechanisms of tissue regeneration in organisms that maintain that capacity. One example is the MRL/MpJ mouse strain with unique regenerative capacity in ear pinnae that is absent from other strains, such as the common C57BL/6 strain. The MRL/MpJ mouse has also been associated with an autoimmune phenotype even in the absence of the mutant Fas gene described in its parent strain MRL/lpr. Due to these findings, the differences between the responses of MRL/MpJ versus C57BL/6 strain are evaluated in volumetric muscle injury and subsequent material implantation. One salient feature of the MRL/MpJ response to injury is robust adipogenesis within the muscle. This is associated with a decrease in M2-like polarization in response to biologically derived extracellular matrix scaffolds. In pro-fibrotic materials, such as polyethylene, there are fewer foreign body giant cells in the MRL/MpJ mice. As there are reports of both positive and negative influences of adipose tissue and adipogenesis on wound healing, this model can provide an important lens to investigate the interplay between stem cells, adipose tissue, and immune responses in trauma and material implantation.