The omentum is a visceral adipose tissue in the peritoneal cavity that contains fat associated lymphoid cluster (FALCs), which are important sites for the T and B cell responses to foreign antigens in the peritoneal cavity and a portal for leukocytes trafficking into the peritoneum. Compared with other lymphoid structures, little is known about the organization of these clusters and how microbial antigens access this site. After intraperitoneal immunization with a replication deficient strain of Toxoplasma gondii, type 1 conventional dendritic cells (cDC1) were a critical source of interleukin 12 that induced innate lymphoid cells to produce interferon gamma required to recruit inflammatory monocytes to the FALCs. In addition, the migration of infected peritoneal macrophages into the FALCs and their localization in T and B cell rich areas accompanied the priming of parasite-specific T cells. While cDC1s in the omentum are not required for CD8+ T cell priming, nearest neighbor cluster analysis showed that activated CD8+ T cells preferentially cluster around cDC1 at early time points. Further, computational modeling approaches have been used to better understand the essential role of cDC1 required to support the expansion of a protective, antigen-specific CD8+ T cell response to immunization. To conclude, in the FALCs cDC1 have distinct roles in the co-ordination of the innate and adaptive responses to vaccination against microbial pathogens.
Endowing CAR T cells with ectopic expression of immunomodulatory molecules has been shown to increase therapeutic efficacy. An outstanding question is how to genetically integrate these effector molecules into CAR T cells in a clinically feasible manner. We developed genetically integrated systems that combine autonomous antigen-induced production of an immunomodulatory molecule along with constitutive CAR expression in a single lentiviral vector (Uni-Vect). We demonstrate that designer CAR T cells that respond specifically to target antigen, exclusively upregulate the inducible module. Focusing on clinical translation, these systems comprise human-only genetic elements and retain in vivo anti-tumor activity. Here we present implementations of the Uni-Vect instilling enhanced efficacy or safety of CAR T cells. By introducing antigen-driven secretion of an immunomodulatory cytokine IL-12, enhanced antitumor activity of CAR T cells was achieved. To autonomously limit toxicities associated with CAR T cell therapy, we coupled clinically used CAR with in-situ secretion of a tocilizumab-based antibody. Compared to two-component viral vector systems, the Uni-Vect achieves homogenous modification of cell products and improves CAR T-cell manufacturing work-flows. This Uni-Vect is broadly-applicable and easily amenable to construct and screen combinations of immune receptors and modulators of interest, while our featured implementations demonstrated enhanced CAR T cell functions.
Dramatic losses of lymphocytes by apoptosis results in the propagation of septic complications that ultimately leads to reduced patient survival. Targeting and reprogramming of immune cell populations such as T cells in vivo using nanoparticles has the potential to restore adaptive immune cell deficits that impair sepsis recovery and subsequent survival over the long term. Delivery of nucleic acids to immune cells has widespread potential to correct or functionally reprogram genetic aberrations present in a variety of diseases. In many cases, the introduction of genetic materials into cells relies heavily on physical or viral approaches. But physical methods and viral vector based delivery are very limited. High toxicity, unwanted genetic mutations and immunogenicity posing concerns in clinical translations. In comparison to that, non-viral gene delivery methods have the potential to overcome many of these limitations, particularly regarding toxicity and targetability in vivo. Here, we have prepared immunoplexes (IPs) comprised of acetylated polyethyleneimine (PEI)-plasmid DNA complexes that have been coated with negatively charged poly(ethylene-alt-maleic acid) (PEMA) to minimize non-specific interactions and unwanted toxicities with immune cells. IPs represent a modular gene delivery platform that has the potential to be functionalized with a variety of targeting ligands to achieve high levels of immune cell-specific transfection.
Exosomes are 30-150 nm extracellular vesicles that can transport RNAs, proteins, and lipid mediators to recipient cells via circulation. We hypothesized exosome content would be altered following nerve injury and these alterations could mediate neuropathic pain. To characterize exosome composition following nerve injury, small extracellular vesicles (sEVs) were purified from mouse serum four weeks after spared nerve injury (SNI). Our miRNA profiling showed a distinct miRNA signature in SNI model compared to sham control. Proteomics analysis using tandem mass spectrometry detected 274 gene products. Of these, 24 were unique to SNI model, multiple members of serpin and complement family were detected in exosomes. Neuropathic pain can active the complement cascade and our cytokine profiling showed the upregulation of complement component 5a (C5a) in sEVs from SNI model. Intercellular Adhesion Molecule 1 (ICAM-1), required for the leukocyte recruitment, adhesion and homing of exosomes was also increased in sEVs from SNI model compared to sham control. We observed a differential distribution of C5a and ICAM-1 within serum and sEVs between sham and SNI, indicating changes from local or paracrine to long distance signaling under neuropathic pain. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating neuropathic pain. In vivo studies are ongoing to determine the functional significance of alterations in exosome composition.
Aging leads to increased morbidity and mortality which has become more of a concern as the global aged population increases (>65). Last year, over 12,000 elderly deaths occurred from the flu alone. These deaths may be from the immune system undergoing immunosenescence, a condition of chronic elevated levels of basal inflammation. Consequences of immunosenescence also result in diminished vaccine responses and immune system exhaustion, leading to grave clinical complications. Current research shows a decrease in pro-inflammatory cytokines but the downstream signaling pathways in the context of aging have yet to be evaluated. One critical pathway point in the production of type I interferon (IFN) is the phosphorylation of IRF7 in the TLR 7/8 pathway. We show a decrease in phosphorylated interferon-response factor 7 (pIRF7) in certain innate cells populations following stimulation with LPS/IFN and a TLR7/8 specific agonist. We hypothesize a deficit in pIRF7 in response to agonist will decrease translocation across the nuclear membrane resulting in impaired pro-inflammatory cytokine production, phagocytosis, and antigen presentation in antigen presentation cells from elderly patients compared to young patients (<35). We will utilize flow cytometry and confocal microscopy to measure phagocytosis and immune synapse formation in isolated innate cells. The significance of this research proposal will lead to an impactful strategy for amplification of vaccine response in the elderly.
Macrophages are highly plastic immune cells that precisely control the timing and dosage of angiogenic growth factors. A growing body of evidence suggests that early M1 macrophages initiate vascular sprouting, then switch to the M2 phenotype to stabilize these structures. In pathologies where angiogenesis is inhibited, M1 macrophages may be insufficiently activated and also fail to switch to the M2 phenotype. This M1-to-M2 transition has not been thoroughly studied, but it is necessary to understand how M1 activation affects phenotypic switching. In this study, we first evaluated M1 sensitivity to IL-4, the primary cytokine used to induce M2 polarization, in comparison to unactivated M0 macrophages. Although M1 macrophages displayed greater IL-4Rα surface expression than M0 macrophages, they did not switch to the M2 phenotype more quickly or at lower doses of IL-4. Instead, M1 and M0 macrophages treated with IL-4 (“M1-M2” and “M0-M2”) exhibited differential gene expression and protein secretion. While M0-M2 macrophages upregulated M2 markers CD206 and CCL22, M1-M2 macrophages more highly expressed CCL17, as well as genes associated with angiogenesis like CXCR4 and ANG. Additionally, M1-M2 macrophages secreted more PDGF-BB, a late-acting angiogenic protein, and more CCL17. Furthermore, M1-M2 secretions induced greater endothelial cell migration than M0-M2-conditioned media. Together, these findings indicate a substantial role for M1-M2 macrophages in late-stage angiogenesis.
Macrophages are among the first cells to interact with bone-dwelling biomaterials. While macrophage Wnt ligands are known to modulate inflammatory and stem cell activity in various solid organs, little is known about their role in osseous healing. The objectives of this project were to determine the importance of macrophageal Wnts to osseous healing and characterize the Wnt dependence of pro-osteogenic macrophage activity. To characterize macrophage Wnt expression during osseous healing, macrophages were isolated from femoral implants or fracture callus. Macrophage Wnt expression increased significantly during this time, with modified Ti increasing Wnt. Next, to identify the effects of loss of Wnt signaling, Csf1r-iCre+; Wls-/- (mac-Wls) mice were instrumented with implants or underwent fracture. Flow cytometry revealed decreased proportions of mesenchymal stem cells (MSCs) in both. To characterize the outcome of fracture healing in mac-Wls mice, fractures were analyzed using microCT at 14d, revealing attenuated callus formation. Finally, to identify the mechanism of macrophage activity, exosomes from wild type or mac-Wls macrophages were isolated. MSCs educated with wild type, but not mac-Wls macrophage exosomes, exhibited enhanced genetic markers of osteoblast differentiation. These results reveal that osteogenesis-associated macrophages exhibit enhanced Wnt production, which may hold value to enhancing bone-dwelling implant integration and tissue engineering.
Peptide based immunotherapies present exciting solutions to address the complexities of a variety of autoimmune diseases. Randomized polypeptides have previously demonstrated an ability to act as universal antigens to increase anti-inflammatory T cell responses. In order to formulate a more tunable material, and to assess for the first time how physicochemical properties such as charge, molecular weight, and aggregation influence the immune responses, we designed randomized peptide sequences with the capacity to self-assemble into supramolecular nanofibers. This system provides opportunities for tuning the strength of the T cell response and for directing the response against co-assembled epitopes. We found that a randomized self-assembling peptide, named (KEYA)20Q11, improved the ability of nanofibers to be acquired and presented by antigen presenting cells, and the randomized domain provided both B and T cell epitopes to raise strong antibody titers and produce an anti-inflammatory Th2 helper T cell population. When investigated in vivo, (KEYA)20Q11 raised strong antibody titers and polarizes the T helper population toward a Th2 response. Moreover (KEYA)20Q11 increased titers against co-delivered epitopes, improved the stimulating capacity of other T cell epitopes and polarized a disease specific B cell epitope response. (KEYA)20Q11 is a unique fusion of two materials, a highly ordered system with a highly disordered system, and further exploration will provide valuable ins
Tumor antigen-specific antibody responses are positively associated with favorable clinical outcomes in certain human cancers. Preclinical studies suggest that antibodies raised against tumor associated antigens (TAAs) can mediate tumor cell killing, promote inflammatory repolarization of the tumor microenvironment. However, humoral responses raised through vaccination are often ineffective, largely due to the spontaneous loss of extracellular receptor targets and the lack of cytotoxic T cell responses. Recent studies demonstrated that combining anti-TAA monoclonal antibodies and CD8+ T cell vaccines can induce strong endogenous anti-tumor immunity. We designed multi-epitope nanofibers based on a α-helical peptide nanofiber platform (Coil29) to simultaneously deliver both melanoma-specific Trp2 CD8+ T cell epitopes and B cell epitopes (PEPvIII) against a tumor-specific receptor, the epidermal growth factor receptor variant III (EGFRvIII). These multi-epitope nanofibers can stimulate both strong Trp2-specific CD8+ T cell responses and high titers of epitope-specific IgG antibody responses. The combination of humoral and cellular responses significantly retarded the tumor growth and improved the over survival in a prophylactic murine EGFRvIII-B16 tumor model compared to CFA adjuvanted group, suggesting the therapeutic effect of tumor-targeting antibody responses and potential anti-tumor synergy between humoral and cellular responses.
Eradication of HIV-1 infection remains an unresolved challenge due to the persistence of latently-infected cellular reservoirs as well as a lack of proper antigen presentation and subsequent cytotoxic T cell (CTL) targeting of infected cells. Novel therapies that can overcome several of these defects simultaneously are needed. To this end, we propose the use of non-thermal plasma (NTP) as an agent of immunomodulation. NTP, which is the basis for the expanding field of plasma medicine, is a tool used for manipulating cellular redox and achieving specific clinical outcomes, such as the treatment of cancer. NTP, which includes various reactive oxygen and nitrogen species (RONS) among its effectors, has also been extensively studied as a strategy for enhancing immunogenicity via immunogenic cell death (ICD), which is characterized by increased display or release of DAMPs capable of enhancing antigen presenting cell function. Although NTP was recently shown to inhibit HIV-1 replication, the immunomodulatory potential of NTP in HIV-1-infected cells has not been examined.
Craniomaxillofacial (CMF) bone defects can arise from congenital, post-oncologic, and traumatic injuries. There is an extraordinary unmet need for regenerative strategies for CMF bone defects. Current methods of repair involve autografts or allograft sources for bone. Our laboratory has developed mineralized collagen scaffolds able to promote mesenchymal stromal cell (MSC) osteogenic differentiation and CMF bone regeneration in the absence of exogenous growth factor supplements. While such regenerative medicine strategies offer the potential for improving the scope and speed of CMF bone regeneration, the host inflammatory environment may limit the implant integration and bone regeneration. Therefore, an opportunity exists to develop degradable biomaterials that enhance bone regeneration by modulating the host inflammatory response. My central hypothesis is that scaffold pore size, anisotropy, and incorporation of an allogenic matrix source can modulate macrophage phenotype and resultant MSC osteogenesis. While pore size and anisotropy introduce biophysical cues, incorporation of placenta derived amniotic/chorionic matrix into the collagen scaffold may provide additional biomolecular stimuli to alter macrophage response. Ongoing work is evaluating MSC osteogenesis and macrophage polarization in response to these scaffold variations. This work provides insight on the impact of structural and compositional biomaterial cues on the inflammatory response underlying CMF bone repair.
Mesenchymal stromal cell (MSC) therapy aids in the repair of injured skeletal muscle, and while it is known that T-cells play a pivotal role in regulating muscle repair in dystrophy and self-healing acute injuries, how MSCs influence T-cells in otherwise non-healing severe muscle injuries remains unknown. Here, we tested the hypothesis that MSCs aid in regenerating severely injured skeletal muscle by altering the local T-cell milieu via immunomodulatory paracrine signaling. Using a rodent muscle injury model that leads to persistent fibrosis and long-term loss of muscle function, we observed gradual accumulation of CD45+ leukocytes in muscle that peaked at seven days after injury. At this time point, MSC-treated muscles had significantly lower CD3+ T-cells compared to uninjured controls, and closer inspection revealed that this was due to significantly lower CD8+ cytotoxic T-cells. In vitro, using conditioned media from co-culture of CD3/28 activated PBMCs and MSCs, we found that activated PBMC secretome inhibited myogenic differentiation whereas co-culture with MSCs rescued this effect and led to improved myotube formation. Based on these observations, we tested and proved the hypothesis that antibody based systemic depletion and/or neutralization of CD8+, but not CD4+, T-cells enhances muscle function and reduces fibrotic scar tissue, similarly to MSC treatment, thus leading to effective regeneration.
This study investigates the impact of a mechanically-dynamic scaffolding material on macrophage function, a mechanosensitive host-cell population integral to the success of artificial tissue. We utilize a magnetically-responsive alginate scaffold in combination with a time-varied uniform magnetic field (30 Gauss, 0.5 Hz) to impart cyclic strain onto scaffold associated cells. Primary bone-marrow derived macrophages from BALB/c mice were seeded onto scaffolds in three biochemical environments including control (M0), lipopolysaccharide and interferon-gamma treated (M1), or interleukin-4 and -13 treated (M2). Macrophage phenotype and cytokine secretion were assessed by flow cytometry and multiplex ELISA, respectively. Additionally, cell-free scaffolds were subcutaneously implanted in BALB/c mice (n=5) with or without subsequent magnetic stimulation to investigate the impact of magnetic-actuation on infiltrating host-macrophages. In the M1 environment, magnetic stimulation enhanced the M1 phenotype as shown by increased expression of the T-cell co-stimulatory signal, CD86 and increased secretion of multiple M1 cytokines. When scaffolds were implanted into mice, magnetic stimulation during days 5-8 p.i. enhanced the infiltration of host M1 macrophages as determined by macrophage expression of CD86. Our results indicate that magnetically-actuated scaffolds affect macrophage by enhancing the M1 phenotype."
Clostridium difficile is the number one cause of healthcare-associated infection in the United States and the most lethal acute enteric pathogen. Causing additional concern is the emergence of hypervirulent strains. The elderly (≥65) have the greatest risk of CDI and severe morbidity and mortality due to C. difficile-associated disease (CDAD). Additionally, most CDI recurrent infections occur within the elderly. To better understand why the elderly are at greater risk of severe and recurrent CDI, our lab has developed an aging mouse model of CDI. Preliminary work shows that this aging mouse model has greater morbidity and mortality when challenged with C. difficile compared to young mice. Immune response defects in the aged mouse model have also been observed, including: a sharp initial increase in activated Th17 cells within the small intestine, decreased production of toxin-specific antibodies, and a decreased toxin-specific antibody response to vaccination. Due to these defects, we plan to investigate the inflammatory profiles of Th17s and ILC3s in the intestine during CDI, elucidate Tfh-B cell interactions in the germinal centers of mesenteric lymph nodes during CDI, and identify molecular adjuvants that can improve the immunogenicity of a toxin-based DNA vaccine in the aging mouse model. The findings from this study will lead to improved toxin-based DNA vaccines targeted for individuals at high risk, including the elderly.
How the surrounding microenvironment of a host organism responds to an implant dictates the therapeutic potential of the intervention. If properly controlled, immune reaction to a biomaterial implant can assist the integration of the scaffold into the host tissue. The physical features of an implant, as well as the chemical composition, may influence the host immune response. Without proper integration, inflammation associated with the implantation may lead to tissue necrosis and pathological fibrosis, limiting potential for clinical translation. Implantation of a biomaterial scaffold in vivo stimulates infiltration of leukocytes and perturbs the physiologic homeostasis of the surrounding tissue. In certain scenarios, the host organism attempts to wall off the perceived foreign tissue by creating a vascularized capsule made of collagen-rich neo-matrix around the implant. The integration of the implant with the surrounding tissue then could be hampered by low cellular infiltration and lack of vascularization inside the implant. We demonstrate the ability of a self-assembled peptide hydrogel, based on a beta-sheet motif, to tune the immune microenvironment of a polymeric implant, resulting in a pro-healing transformation post-implantation, with aduquate vascularization.
Pathogens have evolved over time to evade the host immune system in various ways. Specifically, the fungal species Cryptococcus neoformans (CN), following engulfment by phagocytes, has been observed to stay alive within the acidic phagolysosome and escape through a process called vomocytosis. Using this phenomenon, CN disseminates throughout the host, eventually leading to a brain infection called cryptococcal meningitis. This condition annually affects ~220k HIV/AIDS patients and results in ~181k fatalities per year worldwide2. Understanding vomocytosis could lead to the development of new cryptococcal meningitis treatments for at-risk patients, and novel biomaterial particulate vaccines. However, the underlying mechanisms of this phenomenon are poorly understood. To study vomocytosis, a robust method for quantifying phagocytosis and expulsion rates is needed. In this work, we synthesized and characterized a novel multi-fluorophore reporter system for precise monitoring and measurement of phagocytic entry and vomocytic expulsion. A disulfide-cleavable linker-modified coumarin is used to confirm phagocytic entry and an anti-FITC antibody is used to verify extracellular localization of CN. A proof-of-concept microparticle system was used as a proxy to fungal cells for their facile chemical manipulation. This work demonstrated robust fluorescent response of reporters to distinct activation prompts (disulfide reduction and fluorescent antibody binding).
Vaccines delivered across mucosal surfaces have the potential to raise protective local responses at the very mucosal surfaces through which most disease-causing pathogens enter the human body. Yet, the defenses that have evolved to protect these surfaces from pathogens present formidable challenges for non-invasive delivery. This challenge is heighted in the case of immune-active biomaterials, which often have sizes and surface properties not readily amenable to such delivery routes. We have developed molecular strategies to enable the delivery of nanofiber vaccines across the sublingual mucosae that lies under the tongue. Conjugation of low molecular weight polyethylene glycol (PEG) (optimally 2000 – 3000 MW) to self-assembling β-sheet nanofibers enabled sublingual immunization of mice using droplets with multiple adjuvants. We investigated the mechanism behind this finding, and found that PEG decreases interactions of the nanofibers with mucus in vitro, thereby prolonging their residence at the mucosae in vivo to initiate immune responses. We also found that peptides with similar surface properties to PEG could be used as a substitute in designing nanofiber vaccines for sublingual immunization. Additionally, we adapted our materials to design a dissolvable freeze-dried wafer for sublingual immunization, with controllable mechanical properties. These strategies represent valuable progressions towards the future viability of nanomaterials as effective mucosal vaccines.
Ionizable lipid nanoparticles (LNPs) show great promise as vehicles for the intracellular delivery of therapeutic macromolecules, including nucleic acids. LNP formulations are numerous but use common excipients: cholesterol for stability, phospholipid to aid in endosomal escape, and PEG to reduce immunogenicity. Varying excipient combinations can significantly change the physical properties of LNPs, thereby influencing their delivery. In this study, two libraries of LNPs were designed for delivery to T cell. Each formulation contained varying molar ratios of ionizable lipid, cholesterol, helper lipids, and lipid-conjugated PEG and was characterized to determine z-average diameter, pKa, and cargo concentration. Then, Jurkat cells, immortalized human T cells, were treated with each formulation and assessed for in vitro intracellular delivery and cytotoxicity. The optimal excipient conditions in Library A led to the development of a next-generation Library B. Overall, the formulations in Library B demonstrated much greater cargo concentration and z-average diameters than those in Library A, and numerous formulations in Library B outperformed the top-performing formulation from Library A, supporting the predicted trends. Further, all formulations in Library B were observed to exhibit over 80% viability. In conclusion, we report the development of multiple potent LNP formulations for intracellular delivery to T cells with potential future application in immunoengineering.
Traumatic spinal cord injury (SCI) causes deleterious functional loss below the injury site. Persistent M1 and transient M2 macrophage/microglia response after SCI leads to enhanced neurotoxicity and impaired wound heading. After SCI, the injury site is filled with lipid-rich cellular and myelin debris. Macrophages are the predominant phagocyte that are responsible for debris-clearance and become lipid-laden foam cells. Recent studies show that the foam cells have reduced efflux capacity and may become pro-inflammatory and neurotoxic in vivo. It is postulated that persistence of foam cells is associated with persistent inflammation and impaired wound healing after SCI. We found that myelin debris without any other inflammatory stimuli only slightly increased inflammatory response of macrophages. However, in the presence of proinflammatory stimuli, myelin debris was anti-inflammatory when macrophages were co-treated with inflammatory stimuli and myelin debris at the same time. When the macrophages were pre-treated with inflammatory stimuli followed by co-treatment of myelin debris and inflammatory stimuli, then myelin debris will exacerbate inflammation. Meanwhile, dosage of inflammatory stimuli and myelin debris also showed great effect on the inflammatory response of macrophages.
In autoimmune diseases, deleterious immune responses are induced against healthy tissues. Regulatory T-cells (Tregs), mediators of immune tolerance, can prevent these aberrant immune responses to the host. Treatments using patients’ own Tregs have shown some clinical benefit. Yet, low availability of Tregs in combination with their laborious and costly ex vivo manipulation have hindered their widespread application. Therefore, there is a pressing need to develop therapies that can generate Tregs directly in the body while minimizing ex vivo manipulation. Engineering tolerizing biomaterials that can closely emulate a native Treg-generating milieu (e.g., hypoxic tumor microenvironement) could provide a promising strategy to downregulate effector T-cell responses while inducing Tregs. To that end, we engineered syringe-injectable immunosuppressive hypoxia-inducing cryogel (HIC) scaffolds. Our preliminary data suggest that HICs can quickly deplete oxygen (<1hr) and controllably maintain an immunosuppressive hypoxic environement (~1-3% O2) for several days. Furthermore, once conjugated with anti-CD3 antibodies, HICs can effectively stimulate murine primary T-cells and downregulate pro-inflammatory cytokine production. Strikingly, HICs also increased the release of anti-inflammatory cytokines and skewed T-cell differentiation toward Tregs. In conlusion, hypoxia-inducing biomaterials hold great potential for the rational design of effective immunotherapies for autoimmune disorders.
Implanted biomaterials currently elicit an inflammatory response that can transition from normal healing to encapsulation of the material with the foreign body response. Combining novel drug delivery with implanted biomaterials could result in total biomaterial integration. Leveraging biotin-avidin conjugation systems to alter biomaterials and promote macrophage from an inflammatory M1 response to a pro-healing M2 phenotype could stimulate biomaterial integration. We studied the release of biotinylated interleukin-4 from gelatin scaffolds modified by biotinylating, then conjugating with avidin variants. The release of biotinylated interlukein-4 was not significantly different between experimental scaffold groups in 1x PBS, but significantly lower in the presence of excess biotin for scaffolds with ten-fold-molar-excess biotin and CaptAvidin. Both M2a protein levels of CCL22 and CCL18 increased before returning to baseline totals by day fourteen. All experimental groups with biotinylated interleukin-4 had higher total protein secretion of CCL22. Varying amounts of biotin and avidin variants on gelatin sponges resulted in no change to the release of biotinylated interleukin-4 over time. M0 macrophages also began to secrete M2a proteins, suggesting temporal modulation of macrophage behavior and the potential use of the biotin-avidin system for the successful integration of implanted biomaterials. Further studies should be conducted in vivo for bioactivity and functionality.
Regulatory T cell (Treg)-targeted therapeutics are receiving increased attention for their potential to treat autoimmune diseases. Tregs are powerful mediators of tolerance toward self-tissues but are often impaired in disease settings, and therapies restoring Treg numbers and function are needed. We investigated the potential of a polymeric tolerogenic artificial antigen presenting cell (TolAPC) to promote immune tolerance by inducing the formation of antigen-specific Tregs. Inspired by tolerized natural antigen presenting cells, particle-based TolAPCs provide critical protein signals 1 and 2 and a soluble released signal 3 to convert naïve T cells into Tregs. TolAPCs consisting of a microparticle core loaded with tolerogenic factors and surface-conjugated signal proteins were fabricated. Altering the material properties of the core improved protein conjugation and resulted in enhanced ability to bind to naïve T cells and induce Tregs with potent suppressive function in vitro. A single dose of TolAPCs administered intravenously to B6 mice resulted in an increased percentage of polyclonal Tregs within the lymph nodes. Finally, TolAPCs loaded with multiple tolerogenic factors and conjugated with MHC Class II tetramers were shown to induce Tregs in an antigen-specific manner. This TolAPC platform can be adapted to generate antigen-specific Tregs against any antigen of choice, giving it potential as an “off-the-shelf” therapy for a variety of autoimmune diseases.
Injectable hydrogels are useful tools for wound healing application as regenerative scaffolds and delivery depots. Comparing to the traditional nanoporous systems, these hydrogels can retain the structural integrity while allowing for faster cellular infiltration, which then leads to a higher integration with the native tissue, less immune response and better regeneration outcomes. In the previous research, we’ve shown that MAP scaffolds can promote faster wound closure and regeneration as well as eliciting a significantly lower immune response than non-porous hydrogels in vivo. Here, the anti-inflammatory potential of MAP hydrogels was assessed in vitro by measuring the cellular response of RAW264.7 cells and mouse bone marrow-derived macrophages encapsulated in the hydrogel system, comparing to those encapsulated in nanoporous hydrogels and cultured on tissue culture plastics.
Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes the rate limiting step of tryptophan catabolism and tryptophan metabolites directly interact with immune cells to induce anergy of effector T cells. This property, and others, make IDO an attractive protein therapy candidate; however, it faces the challenge of quick clearance from the body. We have addressed this by fusing IDO to galectin-3 (gal3) to act as an anchor. Gal3 is a member of the carbohydrate binding lectin family, with affinity for sugar present in proteins of the extracellular matrix and cell surface receptors. We have demonstrated previously that this fusion anchors to subcutaneous tissue, and we believe that this makes it an excellent candidate as therapeutic for psoriasis. In the current study, we demonstrate the ability of IDO-Gal3 to reduce inflammation in a pre-clinical model of imiquimod-induced psoriasis. The cumulative clinical scores of mice treated with IDO-Gal3 decreased significantly when compared to those of mice treated with a non-therapeutic control. Currently, we hypothesize that IDO-Gal3 is preventing proliferation of T helper 17 cells and promoting induction of regulatory T cells. From this study, we can conclude that IDO-Gal3 is a promising therapeutic for the treatment of imiquimod-induced psoriasis. Further experimentation is required to determine the minimum effective dose in this model and to characterize the cellular mechanism of the decrease in clinical scores.
Rapid advances in engineered biological circuits are motivating the design of new biomedical platforms for translational applications. To date, the design principles behind existing biocircuits are cell-based platforms, which require significant genome or protein engineering. Further, these circuits largely interface with static information, such as the expression levels of genes or the abundance of proteins. By contrast, biology activity consists of dynamic processes that are largely driven by enzymes, including proteases, which drive complex processes in health and disease. Ideally, the next generation of medicine would comprise programmable, activity-based circuits, enabling autonomous therapies and diagnostics that operate on biological information in real-time. We construct a platform for operating on dynamic biological information by: (1) developing a framework for interpreting biological activity (e.g., proteolysis, bacterial replication, etc.) as operable bits of information, (2) constructing a circuit, which uses compressed sensing to process the activity of endogenous proteases as a string of bits, to act as a pan-diagnostic that can differentiate multiple disease states, and (3) implementing "sense-and-respond" circuits to dynamically and autonomously treat resistant bacteria in the context of urinary tract infections. We envision that this work will advance the paradigm of activity-based medicine by providing a powerful tool for harnessing biological activity.
Owing to the extensive diversity of HIV-1 in the human population, a successful HIV vaccine must induce antibodies capable of neutralizing most HIV variants to prevent infection, which thus far has not been achieved by any vaccine formulation. However, engineered HIV vaccines capable of presenting multivalent HIV envelope proteins have shown improvements in the breadth of antibody responses raised. We have developed a vaccine platform using self-assembling peptide nanofibers, by which antigens are presented in a multivalent manner. The self-assembling peptide nanofibers, as shown in previous studies, can elicit potent antibody responses in mice without supplemental adjuvants. They may therefore represent a promising vaccine platform for HIV not only because they allow multivalent antigen presentation like other vaccine nanomaterials, but also because they allow for the fine compositional tuning of the antigens comprising the vaccine. This modular nature of supramolecular peptide materials is especially useful for vaccine development against infectious diseases such as HIV whose immune correlates for protection are not yet clear. In the current study, we conjugated an HIV Env gp120 antigen isolated from an acutely infected individual termed 1086.C to the self-assembled peptide nanofibers, and then we characterized the strength and breadth of antibody responses induced by a self-assembled peptide-based HIV envelope vaccine in the presence and absence CD4+ T cell epitopes.
Monoclonal antibody (mAb) therapies are widely used to treat autoimmune disease, such as rheumatoid arthritis and plaque psoriasis. Despite this clinical success, administration of mAbs is a life-long treatment and the development of anti-drug antibodies limits the efficacy of mAb therapies. Anti-cytokine immunization sidesteps this problem by inducing endogenous antibodies that can provide long-term suppression of targeted inflammatory molecules. We use the immunogenic Q11 (Am-QQKFQFQFEQQ-Ac) self-assembling nanofiber platform in conjunction with a known IL-17 B-cell epitope and a synthetic T-cell epitope (PADRE) to raise antibody responses against endogenous IL-17. These antibody responses were then tested in C57BL/6 mice via the imiquimod-induced psoriasis model, which has been shown to mimic the pathology of plaque psoriasis in humans. We further demonstrate that an IL-17 peptide-based vaccine can reduce disease severity while providing insights into how the phenotype of the antibody response can influence disease outcomes.
Chronically open wounds are a common complication for diabetic patients. These ulcers (DFUs) are notoriously difficult to treat for a variety of reasons, but the major challenge stems from the mystery as to why some wounds respond to treatment and others do not. And although the mechanisms behind improper DFU healing are not fully understood, dysregulated and dysfunctional macrophage activation have been implicated. Changes in macrophage activation state or phenotype are essential for wound healing because they orchestrate the behavior of other important cell types. Over time, macrophages transition from a pro-inflammatory (M1) phenotype to other phenotypes associated with the resolution of healing (M2a) and tissue remodeling (M2c)2. Our goal is to utilize routinely collected debrided wound tissue to assess differences between healing and non-healing DFUs, in order to ultimately develop a predictive assay for a personalized medicine approach. We used Nanostring™ for gene expression analysis of 227 wound healing and macrophage phenotype related genes in debrided tissue from 25 patients collected over time. Healing was measure 12 weeks following samples collection. 129 genes were significantly differentially expressed at p < 0.05 following Welch’s t-tests and Benjamini-Hochberg correction. Gene-set enrichment analyses revealed significant upregulation of groups of genes related bacterial communication, inflammasome activation, M2a, and M2c phenotypes in healing wounds...