Stephanie Duffort

Infiltration of Proinflammatory M1 Macrophages into the Outer Retina Precedes Damage in a Mouse Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD.

T cells and macrophages responding to oxidative damage cooperate in pathogenesis of a mouse model of age-related macular degeneration

Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis.

In vivo downregulation of innate and adaptive immune responses in corneal allograft rejection by HC-HA/PTX3 complex purified from amniotic membrane.

PURPOSE Heavy chain-hyaluronic acid (HC-HA)/PTX3 purified from human amniotic membrane (AM) was previously observed to suppress inflammatory responses in vitro. We now examine whether HC-HA/PTX3 is able to exert a similar effect in vivo, using murine models for keratitis and corneal allograft rejection. METHODS The in vitro effect of HC-HA/PTX3 was tested using OTII ovalbumin (OVA) transgenic, purified CD4(+) T cells, or IFN-γ/lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cytokine production was measured by ELISA, while cell surface markers and cell proliferation were determined by flow cytometry. In vivo effects of HC-HA/PTX3 were analyzed by quantifying the recruitment of enhanced green fluorescence-labeled macrophages and by measuring the expression of arginase 1 (Arg-1), IL-10, and IL-12 in LPS-induced keratitis in the macrophage Fas-induced apoptosis (Mafia) mouse. The effect of corneal allograft survival in a complete major histocompatibility complex (MHC) mismatched mouse model was assessed by grading corneal opacification. RESULTS In vitro studies demonstrated that HC-HA/PTX3 significantly enhanced the expansion of FOXP3 T cells and suppressed cell proliferation and protein expression of IFN-γ, IL-2, CD25, and CD69 in activated CD4(+) T cells. Furthermore, immobilized HC-HA/PTX3 significantly upregulated IL-10 gene expression but downregulated that of IL-12 and IL-23 in activated RAW264.7 cells. Finally, in vivo subconjunctival injection of HC-HA/PTX3 significantly prolonged corneal allograft survival, suppressed macrophage infiltration, and promoted M2 polarization by upregulating Arg-1 and IL-10 but downregulating IL-12. CONCLUSIONS HC-HA/PTX3 can suppress inflammatory responses in vivo by modulating both innate and adaptive immunity of macrophages and CD4(+) T cells.

A Targeted and Adjuvanted Nanocarrier Lowers the Effective Dose of Liposomal Amphotericin B and Enhances Adaptive Immunity in Murine Cutaneous Leishmaniasis

BACKGROUND Amphotericin B (AmB), the most effective drug against leishmaniasis, has serious toxicity. As Leishmania species are obligate intracellular parasites of antigen presenting cells (APC), an immunopotentiating APC-specific AmB nanocarrier would be ideally suited to reduce the drug dosage and regimen requirements in leishmaniasis treatment. Here, we report a nanocarrier that results in effective treatment shortening of cutaneous leishmaniasis in a mouse model, while also enhancing L. major specific T-cell immune responses in the infected host. METHODS We used a Pan-DR-binding epitope (PADRE)-derivatized-dendrimer (PDD), complexed with liposomal amphotericin B (LAmB) in an L. major mouse model and analyzed the therapeutic efficacy of low-dose PDD/LAmB vs full dose LAmB. RESULTS PDD was shown to escort LAmB to APCs in vivo, enhanced the drug efficacy by 83% and drug APC targeting by 10-fold and significantly reduced parasite burden and toxicity. Fortuitously, the PDD immunopotentiating effect significantly enhanced parasite-specific T-cell responses in immunocompetent infected mice. CONCLUSIONS PDD reduced the effective dose and toxicity of LAmB and resulted in elicitation of strong parasite specific T-cell responses. A reduced effective therapeutic dose was achieved by selective LAmB delivery to APC, bypassing bystander cells, reducing toxicity and inducing antiparasite immunity.

The oxidative stress product carboxyethylpyrrole potentiates TLR2/TLR1 inflammatory signaling in macrophages.

Oxidative stress is key in the pathogenesis of several diseases including age-related macular degeneration (AMD), atherosclerosis, diabetes, and Alzheimers disease. It has previously been established that a lipid peroxidation product, carboxyethylpyrrole (CEP), accumulates in the retinas of AMD patients. Retinal infiltrating macrophages also accumulate in the retinas of both AMD patients and in a murine model of AMD. We therefore investigated the ability of CEP-adducts to activate innate immune signaling in murine bone-marrow derived macrophages (BMDMs). We found that CEP specifically synergizes with low-dose TLR2-agonists (but not agonists for other TLRs) to induce production of inflammatory cytokines. Moreover, CEP selectively augments TLR2/TLR1-signaling instead of TLR2/TLR6-signaling. These studies uncover a novel synergistic inflammatory relationship between an endogenously produced oxidation molecule and a pathogen-derived product, which may have implications in the AMD disease process and other oxidative stress-driven pathologies.

Role of T cell recruitment and chemokine-regulated intra-graft T cell motility patterns in corneal allograft rejection.

Keratoplasty is the primary treatment to cure blindness due to corneal opacification. However, immune‐mediated rejection remains the leading cause of keratoplasty failure. Here, we utilize an in vivo imaging approach to monitor, track, and characterize in real‐time the recruitment of GFP‐labeled allo‐specific activated (Bonzo) T cells during corneal allograft rejection. We show that the recruitment of effector T cells to the site of transplantation determined the fate of corneal allografts, and that local intra‐graft production of CCL5 and CXCL9/10 regulated motility patterns of effector T cells in situ, and correlated with allograft rejection. We also show that different motility patterns associate with distinct in vivo phenotypes (round, elongated, and ruffled) of graft‐infiltrating effector T cells with varying proportions during progression of rejection. The ruffled phenotype was characteristic of activated effectors T cells and predominated during ongoing rejection, which associated with significantly increased T cell dynamics within the allografts. Importantly, CCR5/CXCR3 blockade decreased the motility, size, and number of infiltrating T cells and significantly prolonged allograft survival. Our findings indicate that chemokines produced locally within corneal allografts play an important role in the in situ activation and dynamic behavior of infiltrating effector T cells, and may guide targeted interventions to promote graft survival.

Recruitment of Donor T Cells to the Eyes During Ocular GVHD in Recipients of MHC-Matched Allogeneic Hematopoietic Stem Cell Transplants

PURPOSE The primary objective of the present study was to identify the kinetics and origin of ocular infiltrating T cells in a preclinical model of graft-versus-host disease (GVHD) that induces eye tissue damage. METHODS Graft-versus-host disease was induced using an major histocompatibility complex (MHC)-matched, minor histocompatibility-mismatched hematopoietic stem cell transplant (HSCT) model. This approach, which utilized congenic and EGFP-labeled donor populations, mimics a matched, clinically unrelated donor (MUD) cell transplant. Systemic and ocular GVHD were assessed at varying time points using clinical examination, intravital microscopy, immune phenotype via flow cytometric analyses, and immunohistochemical staining. RESULTS Following transplant, we observed characteristic changes in GVHD-associated immune phenotype as well as clinical signs present in recipients post transplant. Notably, the kinetics of the systemic changes and the ocular damage paralleled what is observed clinically, including damage to the cornea as well as the conjunctiva and lacrimal gland. Importantly, the infiltrate contained predominantly donor CD4 as well as CD8 T cells with an activated phenotype and macrophages together with effector cytokines consistent with the presence of a TH1 alloreactive population. CONCLUSIONS Overall, the findings here unequivocally demonstrated that donor T cells compose part of the corneal and ocular adnexa infiltrate in animals undergoing ocular GVHD. In total, the results describe a novel and promising preclinical model characterized by both systemic and ocular changes as detected in significant numbers of patients undergoing GVHD following allo-HSCT, which can help facilitate dissecting the underlying immune mechanisms leading to damage associated with ocular GVHD.

Loss of MPZL3 function causes seborrhoeic dermatitis‐like phenotype in mice

Seborrhoeic Dermatitis (SD) is a common inflammatory skin disorder. It is chronic and relapsing, affecting 1%–3% of the general adult population.1 SD presents as pink to red greasylooking skin with yellowish scales in seborrhoeic areas such as the scalp, face (nasolabial folds, upper lip, eyelids and eyebrows), retroauricular area and the upper chest. It can be pruritic and socially embarrassing. Current treatment includes antifungals, antiinflammatory and immune modulators, but treatment in some patients may cause adverse effects such as skin atrophy and telangiectasia if used long term. Sebaceous secretion, yeast Malassezia infection and individual susceptibilities such as host immunity and epidermal barrier integrity have all been identified as predisposing factors and may work together to worsen disease. In 2006, Birnbaum et al.2 demonstrated that a frameshift mutation in ZNF750, a zinc finger transcription factor and master regulator of epidermal differentiation, causes early onset (<10 years of age) autosomal dominant seborrhoealike psoriasiform dermatitis with 100% penetrance (OMIM #610227). Yet, how ZNF750 dysfunction causes the SD phenotype remains unknown. We have previously generated knockout mice to determine MPZL3 (myelin protein zerolike 3) function in the skin. Mpzl3 encodes an immunoglobulin protein and is expressed in the suprabasal layers of mouse epidermis, the sebaceous gland and anagen hair follicles.3,4 Not surprisingly, Mpzl3 knockout mice showed various skin abnormalities, including epidermal and sebaceous hyperplasia and hair loss.3–5 Interestingly, these mice also developed early onset skin inflammation with dandrufflike flakes.4 Importantly, ZNF750 was recently shown to directly bind to MPZL3 promoter and activate its transcription in cultured human keratinocytes.6 Therefore, Mpzl3 knockout mice can serve as a useful model to understand SD pathogenesis caused by ZNF750 dysfunction.

An enhanced bioluminescence-based Annexin V probe for apoptosis detection in vitro and in vivo

The process of controlled cellular death known as apoptosis has an important central role not only in normal homeostatic maintenance of tissues, but also in numerous diseases such as cancer, neurodegenerative, autoimmune, and cardiovascular diseases. As a result, new technologies with the capability to selectively detect apoptotic cells represent a central focus of research for the study of these conditions. We have developed a new biosensor for the detection of apoptotic cells, incorporating the targeted selectivity for apoptotic cells from Annexin V with the sensitivity of bioluminescence signal generation from a serum-stable mutant of Renilla luciferase (RLuc8). Our data presents a complete characterization of the structural and biochemical properties of this new Annexin-Renilla fusion protein (ArFP) construct, as well as a validation of its ability to detect apoptosis in vitro. Moreover, this work represents the first report of a bioluminescent Annexin V apoptosis sensor utilized in vivo. With this new construct, we examine apoptosis within disease-relevant animal models of surgery-induced ischemia/reperfusion, corneal injury, and retinal cell death as a model of age-related macular degeneration. In each of these experiments, we demonstrate successful application of the ArFP construct for detection and bioluminescence imaging of apoptosis within each disease or treatment model. ArFP represents an important new tool in the continuously growing kit of technologies for apoptosis detection, and our results from both in vitro and in vivo experiments suggest a diverse range of potential clinically relevant applications including cancer therapeutic screening and efficacy analysis, atherosclerosis and cardiovascular disease detection, and the monitoring of any number of other conditions in which apoptosis has a central role.