Implantation of medical devices and biomaterials can help restore form and function of missing or damaged tissue. It is known that the immune system plays a critical role in both positive and negative outcomes of these implanted materials. The foreign body response is characterized by protein deposition and clotting followed by macrophage inflammation, frustrated phagocytosis, giant cell formation, and ultimately fibrosis. This can inhibit the function of implanted devices (e.g. Insulin pumps) as well as cosmesis (e.g. capsular contracture in breast implants) and persistent inflammation has been associated with more severe outcomes in some patients, including emergence of autoimmune-like pathologies. On the other hand, the immune system plays a constructive role in tissue remodeling and regeneration and is needed for the positive effects of some biomaterials, such as extracellular matrix-based scaffolds in muscle repair. Given these factors, we sought to understand potential variations in post-operative complications in individuals with primary and secondary immune disorders - both autoimmune and immunodeficiencies. This preliminary observational study using electronic health record mining showed increased complication odds for individuals with both autoimmune conditions and immunodeficiencies, with variations dependent upon the individual's sex and age as well as the type of material implanted. Future prospective studies could yield improved insight into both mechanisms of immune response to materials in humans and identify potential risk factors for individual patients undergoing plastics and reconstructive surgeries.

ABSTRACT Extracellular vesicle (EV) research has revealed EV involvement in a variety of biological processes, with potential applications in both fundamental research and therapeutic development. EVs of bacterial origin have been implicated in host‐immune interactions in the development of a variety of diseases. Despite differences in the membranes of bacterial and mammalian cells, past studies on EV storage conditions have focused on mammalian EVs. In this work, we evaluated the effects of storage temperature on the properties of EVs derived from gram‐positive (Bifidobacterium longum) and gram‐negative (Escherichia coli) bacteria and stored for a period of 8 weeks. It was hypothesized that storage over time would impact the integrity of the EV samples, particularly at higher temperatures. B. longum EV identity was confirmed by western blotting for lipoteichoic acid, GroEL, and RecA, while E. coli EV identity was confirmed by western blotting for lipopolysaccharide, GroEL, and Flagellin. Using nanoparticle tracking analysis (NTA), we observed no effect of storage on E. coli EV size or concentration but found that storage at 4°C for 4–8 weeks led to increases in B. longum EV size. No significant changes in particle size or concentration after storage were observed with microfluidic resistive pulse sensing (MRPS). An apparent increase in macrophage activation by the EVs was observed after 4 weeks of storage, followed by an apparent decrease in macrophage activation after 8 weeks of storage. Although EV protein concentration was stable with storage, the intensity and definition of some protein bands in gel electrophoresis appeared to decrease over the storage period. Overall, even short‐term storage of bacterial EVs led to changes in EV functionality, and size measurements alone were not necessarily indicative of EV stability.

Self-antigens that are obscure to immune cells under homeostasis, are exposed following tissue damage and can trigger autoimmunity. Our previous work showed that conventional type 1 dendritic cells (cDC1s) mediate immunoregulation after traumatic skeletal muscle injury. Here, we found that, immune responses to injury in cDC1-depleted mice (Batf3 -/-) mirror those of autoimmune mice (Aire -/-). Mechanistically, we determined that cDC1s prime killer regulatory T cells (CD8+/-Ly49+Tregs) which express Ly49 inhibitory receptors and HELIOS. These killer-like Tregs are clonally diverse and carry T cell receptors associated with self-reactivity and response to hydrophobicity. cDC1 or CD8 deletion promoted CD62L+CCR7+ naïve T cell retention and B cell recruitment to injured muscle. These naïve T cells, which are implicated in autoimmunity, strongly correlate with B cell abundance in the muscle and are selectively pruned by CD8+/-Ly49+ Tregs. Furthermore, clinically used materials that promote wound healing enrich CD8+/-Ly49+ Treg function whereas those that are associated with pathology promote naïve T cell and B cell accumulation. We hypothesize that CD8+/-Ly49+ Tregs maintain self-tolerance after tissue damage and avert autoimmunity by eliminating naive T cells and preventing pathogenic B cell activation.

Excess systemic inflammation can often be lethal in septic and trauma patients due to the onset of multiple organ dysfunction syndrome (MODS). As of right now, there are no effective immunomodulatory therapeutics that can promote survival within this patient population. Pro-regenerative extracellular matrix (ECM) biomaterials have shown success for the treatment of local inflammation but have not been fully explored for treating systemic inflammation. Here, we demonstrate the efficacy of an intravenously delivered infusible ECM (iECM) material, which promotes increased survival in a murine model of MODS by decreasing systemic mediators of inflammation. Lung and kidney failure are associated with higher mortality in MODS compared to other organ failures, and we demonstrate that iECM localizes primarily to kidney and lung tissues during systemic inflammation induced by endotoxin. iECM successfully lowered vascular permeability within lung tissue and lowered levels of inflammatory cytokine signaling, such as IL-6, verified via ELISA and gene expression analyses. We also demonstrated that immune cell infiltration into lung tissue was modulated with iECM treatment, with an increase in neutrophil retention in the lung and decreases in pro-inflammatory macrophage presence. In summation, iECM improves survival from severe systemic inflammation by decreasing the local and systemic inflammatory signaling pathways that contribute to MODS. These results provide a strong rationale for translational studies of iECM treatment in systemic inflammatory syndromes, including sepsis and trauma.

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.

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.

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 significanceDifferent 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.

Dynamic pathogen exposure may impact the immunological response to SARS-CoV-2 (SCV2). One potential explanation for the lack of severe SCV2-related morbidity and mortality in Southeast Asia is prior exposure to related betacoronaviruses. Recent discoveries of SCV2-related betacoronaviruses from horseshoe bats (Rhinolophus sinicus) in Thailand, Laos, and Cambodia suggest the potential for bat-to-human spillover exposures in the region. In this work, serum antibodies to protein constructs from SCV2 and a representative bat coronavirus isolated in Cambodia (RshSTT182) are measured in pre-pandemic Cambodian human sera using ELISA assays. Of 293 Cambodian samples tested (N = 131 with acute malaria, n = 162 with acute undifferentiated febrile illness), 32 (10.9%) are seropositive for SCV2 based on established Spike and receptor-binding domain (RBD) cutoffs. Within SCV2 seropositive samples, 16 (50%) have higher antibody levels to antigens from the representative virus RshSTT182 versus SCV2 antigens; competitive binding ELISA assays demonstrate inhibition of reactivity to SCV2 Spike after pre-incubation with RshSTT182 Spike. Surrogate virus neutralization tests demonstrate that 8/30 (26.7%) SCV2 ELISA positive pre-pandemic Cambodian samples have neutralizing activity against SCV2, while 14/30 (46.7%) have activity against other SCV2-related betacoronaviruses. These data suggest that exposure to related betacoronaviruses may elicit cross-reactive immunity to SCV2 prior to the global pandemic.

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.

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.

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.

Abstract COVID-19 vaccine research in immune-deficient/disordered people (IDP) has primarily focused on cancer and organ transplantation populations. We followed 195 IDP with varied immune disorders and 35 healthy volunteers (HV) from April 2021-April 2022. Anti-spike IgG and angiotensin-converting enzyme 2 pseudo-neutralization were measured though six months post-dose 3. Anti-spike IgG was detected in 93% of IDP by six months post-dose 3. IgG dynamics in IDP and HV were similar, though median IgG levels for IDP were < 33% of HV at all timepoints. IgG concentrations were lower against Omicron BA.1 than other variants at all timepoints. Pseudo-neutralization capacity was modestly correlated with anti-spike IgG concentration, but was especially low for Omicron BA.1. Post-vaccination adverse events were minimal. Results were largely unaffected by participants’ immunomodulatory medication and treatments. COVID-19 vaccines are safe, induce anti-spike IgG in most IDP, and should be more strongly recommended for people with immunodeficiencies.

During wound healing and surgical implantation, the body establishes a delicate balance between immune activation to fight off infection and clear debris and immune tolerance to control reactivity against self-tissue. Nonetheless, how such a balance is achieved is not well understood. Here we describe that pro-regenerative biomaterials for muscle injury treatment promote the proliferation of a BATF3-dependent CD103 + XCR1 + CD206 + CD301b + dendritic cell population associated with cross-presentation and self-tolerance. Upregulation of E-cadherin, the ligand for CD103, and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Muscle injury recruited natural killer cells that produced Xcl1 when stimulated with fragmented extracellular matrix. Without cross-presenting cells, T-cell activation increases, pro-regenerative macrophage polarization decreases and there are alterations in myogenesis, adipogenesis, fibrosis and increased muscle calcification. These results, previously observed in cancer progression, suggest a fundamental mechanism of immune regulation in trauma and material implantation with implications for both short- and long-term injury recovery.

Murphy roths large (MRL) mice are known to regenerate skin and skeletal muscle wounds without scar formation but are also susceptible to metabolic dysfunction and obesity. How biomaterials influence immune cell recruitment and immune-adipocyte interaction after muscle injury in MRL mice is yet to be delineated. To investigate this, we performed volumetric muscle loss surgeries (VML) in C57BL/6 (B6) and MRL mice and evaluated immune responses to injury, regenerative (decellularized extracellular matrix, ECM) and fibrotic (polyethylene, PE) material implants using multiparametric flow cytometry, RNA sequencing, and histopathology. In MRL but not B6 mice, we observed white fat deposition at the site of VML. This was correlated with enrichment of adipogenesis gene sets including Adipoq, Cebpa, Pparg and Acsl1. In addition, F4/80-CD68+ macrophages were significantly more abundant in MRL mice at 3 weeks post-injury in PE and saline groups. The same treatments also highly potentiated expression of the lipid associated macrophage gene signature comprising Trem2, Ly6c2, Cd9, Cd63 and Lyz2. Ly6G+ neutrophils were the second most abundant cell type (~ 25% of CD45+ cells) which was corroborated by increased abundance of Vav1, Ccl2, Adam8 and Rac2. In contrast, ECM implantation produced a largely SiglecF+ eosinophilic response (~ 60% of CD45+ cells) and inhibited CD68+ macrophage prevalence and also potentiated expression of the pro-regenerative Arg1, Chil3, Retnla and Gata3 in both B6 and MRL. Taken together, our results suggest a mechanistic role for CD68+ macrophages in promoting adipogenesis after VML. Moving forward, we will analyze adaptive immune responses to injury and material implants using abovementioned methods. Intramural Research Program, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health

Abstract Introduction Previously, we reported a strong increase in the recruitment of eosinophils at injured muscle when treated with a biologic scaffold implant. As such, we sought to characterize the surface marker expression of these eosinophils to understand their role at the site of injury. Method Volumetric muscle loss (VML) model was performed on C57BL/6 mice and was subsequently treated with a biologic scaffold (decellularized extracellular matrix). After 7 and 21 days, post-injury, implanted material along with injured tissue was dissected and converted to single cell suspension for flow cytometry. Results Compared to uninjured mice, at both 7 and 21 days time point, mice that received biological scaffold featured significantly increased presence of eosinophils at the site of injury. These eosinophils had a ring-shaped nucleus which has been previously associated with regulatory eosinophils. Eosinophils at the site of injury showed time dependent upregulation of CD101 (Mean fold change from uninjured muscle after 7 days post implant was 2.73 and the same was 9.90 after 21 days, (p<0.0001)), and CD62L (Mean fold change from uninjured muscle after 7 days post implant was 3.65 and the same was 11.64 after 21 days(p<0.0001)) expression. Interestingly, the same pattern was not observed with blood eosinophils except that the blood eosinophils at 3-week timepoint were featuring a significant increase in CD62L expression compared with 1 week time point, suggesting potential return of homeostasis in circulatory compartment. Conclusion Here, we describe time-dependent transformation in eosinophil phenotype in response to biologic scaffold implantation, including upregulation of adhesion molecule CD62L and a regulatory marker CD101.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Described herein are findings in 16 patients who had their dysfunctioning mechanical prostheses in the mitral valve position replaced. The mechanical prostheses had been in place from 2 to 157 months (mean 80). All but 1 patient had the mechanical prosthesis excised because of prosthetic stenosis or regurgitation or both secondary to prosthetic thrombus (despite warfarin therapy) on cloth-ring "pannus" overlaying the orifice or parabasilar detachment ("leak"). The dysfunction was the result of non-infected causes in 13 patients and to infective causes in 3. Three patients (19%) died in the early post-operative period; the other 13 patients survived >1 year. The prostheses were excised in a variety of methods by the explanting surgeons. The best procedure to excise the mechanical prosthesis appears to be mainly operator dependent.

CD206, also known as mannose receptor C type 1, is used frequently as a marker to identify the M2 polarized macrophages in the investigation of immune responses to biomaterials. M2 macrophages help with tissue repair, inflammation reduction, building immune tolerance, and defense against excessive inflammation. CD206 is found on the surface of M2 macrophages and dendritic cells, and functions as a pattern recognition receptor for a variety of connective tissues in health, including pulmonary tuberculosis, liver fibrosis, and others. Previous research has shown a role of CD206 in internalization of collagen fragments in tumors. In this study, we aim to determine whether CD206 is more than just an M2 marker in response to biomaterials. We have found that adaptive immune cells as well as cross-presenting dendritic cells are critical for CD206 induction in biomaterial-treated muscle injury. In Batf3−/− mice, where CD206 expression is lost, we find hemosiderin deposition suggesting defects in phagocytosis. In future studies, we will use CD206-deficient to directly evaluate its role in scaffold remodeling at the site of injury.

Research on coronavirus disease 2019 vaccination in immune-deficient/disordered people (IDP) has focused on cancer and organ transplantation populations. In a prospective cohort of 195 IDP and 35 healthy volunteers (HV), antispike immunoglobulin G (IgG) was detected in 88% of IDP after dose 2, increasing to 93% by 6 months after dose 3. Despite high seroconversion, median IgG levels for IDP never surpassed one-third that of HV. IgG binding to Omicron BA.1 was lowest among variants. Angiotensin-converting enzyme 2 pseudo-neutralization only modestly correlated with antispike IgG concentration. IgG levels were not significantly altered by receipt of different messenger RNA–based vaccines, immunomodulating treatments, and prior severe acute respiratory syndrome coronavirus 2 infections. While our data show that three doses of coronavirus disease 2019 vaccinations induce antispike IgG in most IDP, additional doses are needed to increase protection. Because of the notably reduced IgG response to Omicron BA.1, the efficacy of additional vaccinations, including bivalent vaccines, should be studied in this population.