The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

IL-4 expressing cells are recruited to nerve after injury and promote regeneration.

Interleukin-4 (IL-4) has garnered interest as a cytokine that mediates regeneration across multiple tissues including peripheral nerve. Within nerve, we previously showed endogenous IL-4 was critical to regeneration across nerve gaps. Here, we determined a generalizable role of IL-4 in nerve injury and regeneration. In wild-type (WT) mice receiving a sciatic nerve crush, IL-4 expressing cells preferentially accumulated within the injured nerve compared to affected sites proximal, such as dorsal root ganglia (DRGs), or distal muscle. Immunohistochemistry and flow cytometry confirmed that eosinophils (CD45+, CD11b+, CD64-, Siglec-F+) were sources of IL-4 expression. Examination of targets for IL-4 within nerve revealed macrophages, as well as subsets of neurons expressed IL-4R, while Schwann cells expressed limited IL-4R. Dorsal root ganglia cultures were exposed to IL-4 and demonstrated an increased proportion of neurons that extended axons compared to cultures without IL-4 (control), as well as longer myelinated axons compared to cultures without IL-4. The role of endogenous IL-4 during nerve injury and regeneration in vivo was assessed following a sciatic nerve crush using IL-4 knockout (KO) mice. Loss of IL-4 affected macrophage accumulation within injured nerve compared to WT mice, as well as shifted macrophage phenotype towards a CD206- phenotype with altered gene expression. Furthermore, this loss of IL-4 delayed initial axon regeneration from the injury crush site and subsequently delayed functional recovery and re-innervation of neuromuscular junctions compared to wild-type mice. Given the role of endogenous IL-4 in nerve regeneration, exogenous IL-4 was administered daily to WT mice following a nerve crush to examine regeneration. Daily IL-4 administration increased early axonal extension and CD206+ macrophage accumulation but did not alter functional recovery compared to untreated mice. Our data demonstrate IL-4 promotes nerve regeneration and recovery after injury.

Wnt signaling enhances macrophage responses to IL-4 and promotes resolution of atherosclerosis.

Atherosclerosis is a disease of chronic inflammation. We investigated the roles of the cytokines IL-4 and IL-13, the classical activators of STAT6, in the resolution of atherosclerosis inflammation. Using Il4-/-Il13-/- mice, resolution was impaired, and in control mice, in both progressing and resolving plaques, levels of IL-4 were stably low and IL-13 was undetectable. This suggested that IL-4 is required for atherosclerosis resolution, but collaborates with other factors. We had observed increased Wnt signaling in macrophages in resolving plaques, and human genetic data from others showed that a loss-of-function Wnt mutation was associated with premature atherosclerosis. We now find an inverse association between activation of Wnt signaling and disease severity in mice and humans. Wnt enhanced the expression of inflammation resolving factors after treatment with plaque-relevant low concentrations of IL-4. Mechanistically, activation of the Wnt pathway following lipid lowering potentiates IL-4 responsiveness in macrophages via a PGE2/STAT3 axis.

Interleukin-4 repairs wear particle induced osteolysis by modulating macrophage polarization and bone turnover.

Periprosthetic osteolysis remains as a major complication of total joint replacement surgery. Modulation of macrophage polarization with interleukin-4 (IL-4) has emerged as an effective means to limit wear particle-induced osteolysis. The aim of this study was to evaluate the efficacy of local IL-4 delivery in treating preexisting particle-induced osteolysis. To this end, recently established 8 week modification of murine continuous femoral intramedullary particle infusion model was utilized. Subcutaneous infusion pumps were used to deliver polyethylene (PE) particles into mouse distal femur for 4 weeks to induce osteolysis. IL-4 was then added to the particle infusion for another 4 weeks. This delayed IL-4 treatment (IL-4 Del) was compared to IL-4 delivered continuously (IL-4 Cont) with PE particles from the beginning and to the infusion of particles alone for 8 weeks. Both IL-4 treatments were highly effective in preventing and repairing preexisting particle-induced bone loss as assessed by µCT. Immunofluorescence indicated a significant reduction in the number of F4/80 + iNOS + M1 macrophages and increase in the number of F4/80 + CD206 + M2 macrophages with both IL-4 treatments. Reduction in the number of tartrate resistant acid phosphatase + osteoclasts and increase in the amount of alkaline phosphatase (ALP) + osteoblasts was also observed with both IL-4 treatments likely explaining the regeneration of bone in these samples. Interesting, slightly more bone formation and ALP + osteoblasts were seen in the IL-4 Del group than in the IL-4 Cont group although these differences were not statistically significant. The study is a proof of principle that osteolytic lesions can be repaired via modulation of macrophage polarization.

IL-4/STAT6 signaling facilitates innate hematoma resolution and neurological recovery after hemorrhagic stroke in mice.

Intracerebral hemorrhage (ICH) is a devastating form of stroke affecting millions of people worldwide. Parenchymal hematoma triggers a series of reactions leading to primary and secondary brain injuries and permanent neurological deficits. Microglia and macrophages carry out hematoma clearance, thereby facilitating functional recovery after ICH. Here, we elucidate a pivotal role for the interleukin (IL)-4)/signal transducer and activator of transcription 6 (STAT6) axis in promoting long-term recovery in both blood- and collagenase-injection mouse models of ICH, through modulation of microglia/macrophage functions. In both ICH models, STAT6 was activated in microglia/macrophages (i.e., enhanced expression of phospho-STAT6 in Iba1+ cells). Intranasal delivery of IL-4 nanoparticles after ICH hastened STAT6 activation and facilitated hematoma resolution. IL-4 treatment improved long-term functional recovery in young and aged male and young female mice. In contrast, STAT6 knockout (KO) mice exhibited worse outcomes than WT mice in both ICH models and were less responsive to IL-4 treatment. The construction of bone marrow chimera mice demonstrated that STAT6 KO in either the CNS or periphery exacerbated ICH outcomes. STAT6 KO impaired the capacity of phagocytes to engulf red blood cells in the ICH brain and in primary cultures. Transcriptional analyses identified lower level of IL-1 receptor-like 1 (ST2) expression in microglia/macrophages of STAT6 KO mice after ICH. ST2 KO diminished the beneficial effects of IL-4 after ICH. Collectively, these data confirm the importance of IL-4/STAT6/ST2 signaling in hematoma resolution and functional recovery after ICH. Intranasal IL-4 treatment warrants further investigation as a clinically feasible therapy for ICH.

Microenvironment-responsive immunoregulatory electrospun fibers for promoting nerve function recovery.

The strategies concerning modification of the complex immune pathological inflammatory environment during acute spinal cord injury remain oversimplified and superficial. Inspired by the acidic microenvironment at acute injury sites, a functional pH-responsive immunoregulation-assisted neural regeneration strategy was constructed. With the capability of directly responding to the acidic microenvironment at focal areas followed by triggered release of the IL-4 plasmid-loaded liposomes within a few hours to suppress the release of inflammatory cytokines and promote neural differentiation of mesenchymal stem cells in vitro, the microenvironment-responsive immunoregulatory electrospun fibers were implanted into acute spinal cord injury rats. Together with sustained release of nerve growth factor (NGF) achieved by microsol core-shell structure, the immunological fiber scaffolds were revealed to bring significantly shifted immune cells subtype to down-regulate the acute inflammation response, reduce scar tissue formation, promote angiogenesis as well as neural differentiation at the injury site, and enhance functional recovery in vivo. Overall, this strategy provided a delivery system through microenvironment-responsive immunological regulation effect so as to break through the current dilemma from the contradiction between immune response and nerve regeneration, providing an alternative for the treatment of acute spinal cord injury.

Distinct release strategies are required to modulate macrophage phenotype in young versus aged animals.

The role of innate immunity and macrophages in the host response to biomaterials has received renewed attention. A context-dependent spectrum of macrophage phenotypes are shown to affect tissue integration and performance of implanted biomaterials and medical devices. Recent studies by our group demonstrated that the host response in aged animals was characterized by delayed macrophage recruitment, differences in marker expression and a shifted pro-inflammatory (M1) response, associated with an unresolved host response in the long-term. The present work sought to study the effects of single and sequential cytokine delivery regimens in aged mice to restore delayed recruitment of macrophages and shift the inflammatory host response towards an M2-like phenotype, using MCP-1 (macrophage chemotactic protein-1) and IL-4 (interleukin-4), respectively. Implantation of cytokine-eluting implants showed a preserved response to MCP-1 in both young and aged animals, restoring delayed macrophage recruitment in aged mice. However, the response elicited by IL-4, sequential delivery of MCP-1/IL-4 and coating components was distinct in young versus aged mice. While single delivery of IL-4 did not counteract the high inflammatory response observed in aged mice, the sequential delivery of MCP-1/IL-4 was capable of restoring both recruitment and shifting the macrophage response towards an M2-like phenotype, associated with decreased implant scarring in the long-term. In young mice, sequential delivery was not as effective as IL-4 alone at promoting an M2-like response, but did result in a reduction of M1 macrophages and capsule deposition downstream. These results demonstrate that a proper understanding of patient/context-dependent biological responses are needed to design biomaterial-based therapies with improved outcomes in the setting of aging.

Development of an Accurate and Proactive Immunomodulatory Strategy to Improve Bone Substitute Material-Mediated Osteogenesis and Angiogenesis.

Background: Treatment of large bone defects represents a major clinical problem worldwide. Suitable bone substitute materials are commonly required to achieve successful bone regeneration, and much effort has been spent to optimize their chemical compositions, 3D architecture and mechanical properties. However, material-immune system interactions are increasingly being recognized as a crucial factor influencing regeneration. Here, we envisioned an accurate and proactive immunomodulation strategy via delivery of IL-4 (key regulator of macrophage polarization) to promote bone substitute material-mediated regeneration. Methods: Four different IL-4 doses (0 ng, 10 ng, 50 ng and 100 ng) were delivered into rat large cranial bone defects at day 3 post-operation of decellularized bone matrix (DBM) material implantation, and the osteogenesis, angiogenesis and macrophage polarization were meticulously evaluated. Results: Micro-CT analysis showed that immunomodulation with 10 ng IL-4 significantly outperformed the other groups in terms of new bone formation (1.23-5.05 fold) and vascularization (1.29-6.08 fold), achieving successful defect bridging and good vascularization at 12 weeks. Histological analysis at 7 and 14 days showed that the 10 ng group generated the most preferable M1/M2 macrophage polarization profile, resulting in a pro-healing microenvironment with more IL-10 and less TNF-α secretion, a reduced apoptosis level in tissues around the materials, and enhanced mesenchymal stem cell migration and osteogenic differentiation. Moreover, in vitro studies revealed that M1 macrophages facilitated mesenchymal stem cell migration, while M2 macrophages significantly increased cell survival, proliferation and osteogenic differentiation, explaining the in vivo findings. Conclusions: Accurate immunomodulation via IL4 delivery significantly enhanced DBM-mediated osteogenesis and angiogenesis via the coordinated involvement of M1 and M2 macrophages, revealing the promise of this accurate and proactive immunomodulatory strategy for developing new bone substitute materials.

Immunomodulatory injectable silk hydrogels maintaining functional islets and promoting anti-inflammatory M2 macrophage polarization.

Islet transplantation is considered the most promising treatment for type 1 diabetes. However, the clinical success is limited by islet dysfunction in long-term culture. In this study, we have utilized the rapid self-gelation and injectability offered by blending of mulberry silk (Bombyx mori) with non-mulberry (Antheraea assama) silk, resulting in a biomimetic hydrogel. Unlike the previously reported silk gelation techniques, the differences in amino acid sequences of the two silk varieties result in accelerated gelation without requiring any external stimulus. Gelation study and rheological assessment depicts tuneable gelation as a function of protein concentration and blending ratio with minimum gelation time. In vitro biological results reveal that the blended hydrogels provide an ideal 3D matrix for primary rat islets. Also, A. assama fibroin with inherent Arg-Gly-Asp (RGD) shows significant influence on islet viability, insulin secretion and endothelial cell maintenance. Furthermore, utility of these hydrogels demonstrate sustained release of Interleukin-4 (IL-4) and Dexamethasone with effective M2 macrophage polarization while preserving islet physiology. The immuno-informed hydrogel demonstrates local modulation of inflammatory responses in vivo. Altogether, the results exhibit promising attributes of injectable silk hydrogel and the utility of non-mulberry silk fibroin as an alternative biomaterial for islet encapsulation.

Functional muscle recovery with nanoparticle-directed M2 macrophage polarization in mice.

Persistence of inflammation, and associated limits in tissue regeneration, are believed to be due in part to the imbalance of M1 over M2 macrophages. Here, we hypothesized that providing a sustained source of an antiinflammatory polarizing cytokine would shift the balance of macrophages at a site of tissue damage to improve functional regeneration. Specifically, IL-4-conjugated gold nanoparticles (PA4) were injected into injured murine skeletal muscle, resulting in improved histology and an ∼40% increase in muscle force compared with mice treated with vehicle only. Macrophages were the predominant infiltrating immune cell, and treatment with PA4 resulted in an approximately twofold increase in the percentage of macrophages expressing the M2a phenotype and an approximately twofold decrease in M1 macrophages, compared with mice treated with vehicle only. Intramuscular injection of soluble IL-4 did not shift macrophage polarization or result in functional muscle improvements. Depletion of monocytes/macrophages eliminated the therapeutic effects of PA4, suggesting that improvement in muscle function was the result of M2-shifted macrophage polarization. The ability of PA4 to direct macrophage polarization in vivo may be beneficial in the treatment of many injuries and inflammatory diseases.

B lymphocytes can be activated to act as antigen presenting cells to promote anti-tumor responses.

Immune evasion by tumors includes several different mechanisms, including the inefficiency of antigen presenting cells (APCs) to trigger anti-tumor T cell responses. B lymphocytes may display a pro-tumoral role but can also be modulated to function as antigen presenting cells to T lymphocytes, capable of triggering anti-cancer immune responses. While dendritic cells, DCs, are the best APC population to activate naive T cells, DCs or their precursors, monocytes, are frequently modulated by tumors, displaying a tolerogenic phenotype in cancer patients. In patients with cervical cancer, we observed that monocyte derived DCs are tolerogenic, inhibiting allogeneic T cell activation compared to the same population obtained from patients with precursor lesions or cervicitis. In this work, we show that B lymphocytes from cervical cancer patients respond to treatment with sCD40L and IL-4 by increasing the CD80+CD86+ population, therefore potentially increasing their ability to activate T cells. To test if B lymphocytes could actually trigger anti-tumor T cell responses, we designed an experimental model where we harvested T and B lymphocytes, or dendritic cells, from tumor bearing donors, and after APC stimulation, transplanted them, together with T cells into RAG1-/- recipients, previously injected with tumor cells. We were able to show that anti-CD40 activated B lymphocytes could trigger secondary T cell responses, dependent on MHC-II expression. Moreover, we showed that dendritic cells were resistant to the anti-CD40 treatment and unable to stimulate anti-tumor responses. In summary, our results suggest that B lymphocytes may be used as a tool for immunotherapy against cancer.

One-pot preparation of polymer microspheres with different porous structures to sequentially release bio-molecules for cutaneous regeneration.

Herein, we reveal a double emulsion method combining the sol-gel method to prepare poly(lactic-co-glycolic acid) microspheres with different porous structures for sequential release of two types of biomolecules. By controlling the ripening time of the emulsion, multiple interconnected chambers could be easily chosen to be either embedded in microspheres or opened to the surface. These two types of microspheres exhibited different kinetics for the release of both small molecules and proteins, where the release from microspheres with open pores (5 day over 90%) was much faster than the release from microspheres with embedded pores (25 day over 90%). After loading with interleukin-4 (IL-4) and melatonin, these microspheres were further encapsulated in a sodium alginate hydrogel to form a patch for cutaneous regeneration. The prepared patch was able to recruit alternatively activated (M2) macrophages in the early stage (fast release of IL-4) and promote the growth of blood vessels in the long term (slow release of melatonin), resulting in significantly enhanced cutaneous regeneration. These results also demonstrate the potential of this novel delivery system to deliver multiple therapeutics and achieve synergistic effects.

Interleukin 4 modulates microglia homeostasis and attenuates the early slowly progressive phase of amyotrophic lateral sclerosis.

Microglia activation is a commonly pathological hallmark of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), a devastating disorder characterized by a selective motor neurons degeneration. Whether such activation might represent a causal event rather than a secondary epiphenomenon remains elusive. Here, we show that CNS-delivery of IL-4-via a lentiviral-mediated gene therapy strategy-skews microglia to proliferate, inducing these cells to adopt the phenotype of slowly proliferating cells. Transcriptome analysis revealed that IL-4-treated microglia express a broad number of genes normally encoded by embryonic microglia. Since embryonic microglia sustain CNS development, we then hypothesized that turning adult microglia to acquire such phenotype via IL-4 might be an efficient in vivo strategy to sustain motor neuron survival in ALS. IL-4 gene therapy in SOD1G93A mice resulted in a general amelioration of clinical outcomes during the early slowly progressive phase of the disease. However, such approach did not revert neurodegenerative processes occurring in the late and fast progressing phase of the disease.

Enhancement of immune response of piglets to PCV-2 vaccine by porcine IL-2 and fusion IL-4/6 gene entrapped in chitosan nanoparticles.

In order to develop a novel effective immunomodulator to enhance pig resistance against post-weaning multi systemic wasting syndrome (PMWS), a recombinant plasmid co-expressing pig interleukin-2 (IL-2) and fusion interleukin-4/6 (IL-4/6) genes, designated VRIL4/6-2, was constructed and encapsulated in chitosan (CS) nanoparticles prepared by the ionotropic gelation method. Then 21-day old piglets were divided into two groups and intramuscularly injected respectively with VPIL4/6-2-CS and saline along with the porcine circovirus-2 (PCV-2) vaccine. The blood was collected from each piglet on days 0, 7, 14, 28, 56 and 84 after vaccination to assay the immunological changes. Content of IgG2a, CD4+, CD8+ T cells increased significantly in the sera or blood of piglets treated with VPIL4/6-2-CS (P<0.05). Furthermore, the expression level of IL-2, IL-4, IL-6, IL-15, TLR-2, TLR-7, Bcl-2, TNF-α, CD45 and STATs (STAT1, STAT2, STAT3, STAT4) genes were significantly elevated in the treated piglets respectively in different days after inoculation (P<0.05). The growth weight gain of the treated piglets was markedly improved in comparison with the controls (P<0.05). These indicate that VPIL-4/6-2 entrapped with chitosan nanoparticles is a safe and promising effective adjuvant to promote the immune response of pig to PCV-2 vaccination.

The vascular endothelial specific IL-4 receptor alpha-ABL1 kinase signaling axis regulates the severity of IgE-mediated anaphylactic reactions.

BACKGROUND: Severe IgE-mediated, food-induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life-threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. OBJECTIVE: We sought to define the interaction and requirement of IL-4 and vascular endothelial (VE) IL-4 receptor α chain (IL-4Rα) signaling in histamine-abelson murine leukemia viral oncogene homology 1 (ABL1)-mediated VE dysfunction and fluid extravasation in the severity of IgE-mediated anaphylactic reactions in mice. METHODS: Mice deficient in VE IL-4Rα and models of passive and active oral antigen- and IgE-induced anaphylaxis were used to define the requirements of the VE IL-4Rα and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926 cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1) approaches were used to define the requirement of this pathway in VE barrier dysfunction. RESULTS: IL-4 exacerbation of histamine-induced hypovolemic shock in mice was dependent on VE expression of IL-4Rα. IL-4- and histamine-induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1-dependent. Development of severe IgE-mediated hypovolemia and shock required VE-restricted ABL1 expression. Treatment of mice with a history of food-induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE-mediated anaphylaxis. CONCLUSION: IL-4 amplifies IgE- and histamine-induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL-4Rα/ABL1-dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE-mediated reactions.

Extra-Large Pore Mesoporous Silica Nanoparticles for Directing in Vivo M2 Macrophage Polarization by Delivering IL-4.

Over the past decade, mesoporous silica nanoparticles (MSNs) smaller than 200 nm with a high colloidal stability have been extensively studied for systemic drug delivery. Although small molecule delivery via MSNs has been successful, the encapsulation of large therapeutic biomolecules, such as proteins or DNA, is limited due to small pore size of the conventional MSNs obtained by soft-templating. Here, we report the synthesis of mesoporous silica nanoparticles with extra-large pores (XL-MSNs) and their application to in vivo cytokine delivery for macrophage polarization. Uniform, size-controllable XL-MSNs with 30 nm extra-large pores were synthesized using organic additives and inorganic seed nanoparticles. XL-MSNs showed significantly higher loadings for the model proteins with different molecular weights compared to conventional small pore MSNs. XL-MSNs were used to deliver IL-4, which is an M2-polarizing cytokine and very quickly degraded in vivo, to macrophages and polarize them to anti-inflammatory M2 macrophages in vivo. XL-MSNs induced a low level of reactive oxygen species (ROS) production and no pro-inflammatory cytokines in bone marrow-derived macrophages (BMDMs) and in mice injected intravenously with XL-MSNs. We found that the injected XL-MSNs were targeted to phagocytic myeloid cells, such as neutrophils, monocytes, macrophages, and dendritic cells. Finally, we demonstrated that the injection of IL-4-loaded XL-MSNs successfully triggered M2 macrophage polarization in vivo, suggesting the clinical potential of XL-MSNs for modulating immune systems via targeted delivery of various cytokines.

Neonatal Immunization with a Single IL-4/Antigen Dose Induces Increased Antibody Responses after Challenge Infection with Equine Herpesvirus Type 1 (EHV-1) at Weanling Age.

Neonatal foals respond poorly to conventional vaccines. These vaccines typically target T-helper (Th) cell dependent B-cell activation. However, Th2-cell immunity is impaired in foals during the first three months of life. In contrast, neonatal basophils are potent interleukin-4 (IL-4) producers. The purpose of this study was to develop a novel vaccine triggering the natural capacity of neonatal basophils to secrete IL-4 and to evaluate if vaccination resulted in B-cell activation and antibody production against EHV-1 glycoprotein C (gC). Neonatal vaccination was performed by oral biotinylated IgE (IgE-bio) treatment at birth followed by intramuscular injection of a single dose of streptavidin-conjugated gC/IL-4 fusion protein (Sav-gC/IL-4) for crosslinking of receptor-bound IgE-bio (group 1). Neonates in group 2 received the intramuscular Sav-gC/IL-4 vaccine only. Group 3 remained non-vaccinated at birth. After vaccination, gC antibody production was not detectable. The ability of the vaccine to induce protection was evaluated by an EHV-1 challenge infection after weaning at 7 months of age. Groups 1 and 2 responded to EHV-1 infection with an earlier onset and overall significantly increased anti-gC serum antibody responses compared to control group 3. In addition, group 1 weanlings had a decreased initial fever peak after infection indicating partial protection from EHV-1 infection. This suggested that the neonatal vaccination induced a memory B-cell response at birth that was recalled at weanling age after EHV-1 challenge. In conclusion, early stimulation of neonatal immunity via the innate arm of the immune system can induce partial protection and increased antibody responses against EHV-1.

Shifts in macrophage phenotype at the biomaterial interface via IL-4 eluting coatings are associated with improved implant integration.

The present study tests the hypothesis that transient, early-stage shifts in macrophage polarization at the tissue-implant interface from a pro-inflammatory (M1) to an anti-inflammatory/regulatory (M2) phenotype mitigates the host inflammatory reaction against a non-degradable polypropylene mesh material and improves implant integration downstream. To address this hypothesis, a nanometer-thickness coating capable of releasing IL-4 (an M2 polarizing cytokine) from an implant surface at early stages of the host response has been developed. Results of XPS, ATR-FTIR and Alcian blue staining confirmed the presence of a uniform, conformal coating consisting of chitosan and dermatan sulfate. Immunolabeling showed uniform loading of IL-4 throughout the surface of the implant. ELISA assays revealed that the amount and release time of IL-4 from coated implants were tunable based upon the number of coating bilayers and that release followed a power law dependence profile. In-vitro macrophage culture assays showed that implants coated with IL-4 promoted polarization to an M2 phenotype, demonstrating maintenance of IL-4 bioactivity following processing and sterilization. Finally, in-vivo studies showed that mice with IL-4 coated implants had increased percentages of M2 macrophages and decreased percentages of M1 macrophages at the tissue-implant interface during early stages of the host response. These changes were correlated with diminished formation of fibrotic capsule surrounding the implant and improved tissue integration downstream. The results of this study demonstrate a versatile cytokine delivery system for shifting early-stage macrophage polarization at the tissue-implant interface of a non-degradable material and suggest that modulation of the innate immune reaction at early stages of the host response may represent a preferred strategy for promoting biomaterial integration and success.

Improving selective targeting to macrophage subpopulations through modifying liposomes with arginine based materials.

The effects of surface modifications on liposomes using a library of arginine derivatives for improved drug delivery were examined. Both unmodified and modified liposomes were tested for their drug delivery properties and propensity for internalization by macrophages. All materials were characterized by dynamic light scattering (DLS) and zeta potential. The resulting liposomes were able to encapsulate doxorubicin with a loading efficiency greater than 90% and cumulative releases of less than 15% after 144 h. The internalization of these particles was examined by loading the liposomes with fluorescein or doxorubicin to test internalization through fluorescence level and half maximal inhibitory concentration (IC50), respectively. RAW 264.7 macrophages were activated with lipopolysaccharide (LPS) or interleukin-4 (IL-4) to induce M1- or M2-like phenotypes. Naïve macrophages were also studied. Most modified liposomes enhanced the cytotoxicity of doxorubicin compared to unmodified liposomes. Macrophage phenotype was also observed to influence the cytotoxicity of doxorubicin entrapped in modified liposomes, with some samples enhancing the cytotoxicity in LPS stimulated macrophages and some enhancing toxicity in IL-4 stimulated cells.

StarPEG-Heparin Hydrogels to Protect and Sustainably Deliver IL-4.

A major limitation for the therapeutic applications of cytokines is their short half-life time. Glycosaminoglycans (GAGs), known to complex and stabilize cytokines in vivo, are therefore used to form 3D-biohybrid polymer networks capable of aiding the effective administration of Interleukin-4, a key regulator of the inflammatory response. Mimicking the in vivo situation of a protease-rich inflammatory milieu, star-shaped poly(ethylene glycol) (starPEG)-heparin hydrogels and starPEG reference hydrogels without heparin are loaded with Interleukin-4 and subsequently exposed to trypsin as a model protease. Heparin-containing hydrogels retain significantly higher amounts of the Interleukin-4 protein thus exhibiting a significantly higher specific activity than the heparin-free controls. StarPEG-heparin hydrogels are furthermore shown to enable a sustained delivery of the cytokine for time periods of more than two weeks. Primary murine macrophages adopt a wound healing supporting (M2) phenotype when conditioned with Interleukin-4 releasing starPEG-heparin hydrogels. The reported results suggest that GAG-based hydrogels offer valuable options for the effective administration of cytokines in protease-rich proinflammatory milieus such as chronic wounds of diabetic patients.