Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Optic Nerve Crush and Experimental Glaucoma.

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor ß pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Experimental Glaucoma and Optic Nerve Crush.

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and in human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and on separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor ß pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Ion-Complex Microcrystal Formulation Provides Sustained Delivery of a Multimodal Kinase Inhibitor from the Subconjunctival Space for Protection of Retinal Ganglion Cells.

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma onset and progression, and available pharmaceutical interventions are exclusively targeted at IOP lowering. However, degeneration of retinal ganglion cells (RGCs) may continue to progress despite extensive lowering of IOP. A complementary strategy to IOP reduction is the use of neuroprotective agents that interrupt the process of cell death by mechanisms independent of IOP. Here, we describe an ion complexation approach for formulating microcrystals containing ~50% loading of a protein kinase inhibitor, sunitinib, to enhance survival of RGCs with subconjunctival injection. A single subconjunctival injection of sunitinib-pamoate complex (SPC) microcrystals provided 20 weeks of sustained retina drug levels, leading to neuroprotection in a rat model of optic nerve injury. Furthermore, subconjunctival injection of SPC microcrystals also led to therapeutic effects in a rat model of corneal neovascularization. Importantly, therapeutically relevant retina drug concentrations were achieved with subconjunctival injection of SPC microcrystals in pigs. For a chronic disease such as glaucoma, a formulation that provides sustained therapeutic effects to complement IOP lowering therapies could provide improved disease management and promote patient quality of life.

The role of aquaporin-4 in optic nerve head astrocytes in experimental glaucoma.

PURPOSE: To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). METHODS: C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed. RESULTS: B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). CONCLUSION: Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

Astrocyte responses to experimental glaucoma in mouse optic nerve head.

PURPOSE: To delineate responses of optic nerve head astrocytes to sustained intraocular pressure (IOP) elevation in mice. METHODS: We elevated IOP for 1 day to 6 weeks by intracameral microbead injection in 4 strains of mice. Astrocyte alterations were studied by transmission electron microscopy (TEM) including immunogold molecular localization, and by laser scanning microscopy (LSM) with immunofluorescence for integrin ß1, α-dystroglycan, and glial fibrillary acidic protein (GFAP). Astrocyte proliferation and apoptosis were quantified by Ki67 and TUNEL labeling, respectively. RESULTS: Astrocytes in normal optic nerve head expressed integrin ß1 and α-dystroglycan by LSM and TEM immunogold labeling at electron dense junctional complexes that were found only on cell membrane zones bordering their basement membranes (BM) at the peripapillary sclera (PPS) and optic nerve head capillaries. At 1-3 days after IOP elevation, abnormal extracellular spaces appeared between astrocytes near PPS, and axonal vesical and mitochondrial accumulation indicated axonal transport blockade. By 1 week, abnormal spaces increased, new collagen formation occurred, and astrocytes separated from their BM, leaving cell membrane fragments. Electron dense junctional complexes separated or were absent at the BM. Astrocyte proliferation was modest during the first week, while only occasional apoptotic astrocytes were observed by TEM and TUNEL. CONCLUSIONS: Astrocytes normally exhibit junctions with their BM which are disrupted by extended IOP elevation. Responses include reorientation of cell processes, new collagen formation, and cell proliferation.

A method to quantify regional axonal transport blockade at the optic nerve head after short term intraocular pressure elevation in mice.

Axonal transport blockade is an initial step in retinal ganglion cell (RGC) degeneration in glaucoma and targeting maintenance of normal axonal transport could confer neuroprotection. We present an objective, quantitative method for assessing axonal transport blockade in mouse glaucoma models. Intraocular pressure (IOP) was elevated unilaterally in CD1 mice for 3 days using intracameral microbead injection. Longitudinal sections of optic nerve head (ONH) were immunofluorescently labeled for myelin basic protein (MBP) and amyloid precursor protein (APP), which is transported predominantly orthograde by neurons. The beginning of the myelin transition zone, visualized with the MBP label, was more posterior with elevated IOP, 288.8 ± 40.9 µm, compared to normotensive control eyes, 228.7 ± 32.7 µm (p = 0.030, N = 6 pairs). Glaucomatous regional APP accumulations in retina, prelaminar ONH, unmyelinated ONH, and myelinated optic nerve were identified by objective qualification of pixels with fluorescent intensity greater than the 97.5th percentile value of control eyes (suprathreshold pixels). This method segregated images with APP blockade from those with normal transport of APP. The fraction of suprathreshold pixels was significantly higher following IOP elevation than in normotensive controls in the unmyelinated ONH and myelinated nerve regions (paired analyses, p = 0.02 and 0.003, respectively, N = 12), but not in retina or prelaminar ONH (p = 0.91 and 0.08, respectively). The mean intensity of suprathreshold pixels was also significantly greater in glaucoma than in normotensive controls in prelaminar ONH, unmyelinated ONH and myelinated optic nerve (p = 0.01, 0.01, 0.002, respectively). Using this method, subconjunctival glyceraldehyde, which is known to worsen long-term RGC loss with IOP elevation, also produced greater APP blockade, but not statistically significant compared to glaucoma alone. Systemic losartan, which aids RGC axonal survival in glaucoma, reduced APP blockade, but not statistically significant compared to glaucoma alone. The method provides a short-term assessment of axonal injury for use in initial tests of neuroprotective therapies that may beneficially affect RGC transport in animal models of glaucoma.

Dasatinib inhibits peripapillary scleral myofibroblast differentiation.

Scleral fibroblast activation occurs in glaucomatous and myopic eyes. Here we perform an unbiased screen to identify kinase inhibitors that reduce fibroblast activation to diverse stimuli in vitro and to in vivo intraocular pressure (IOP) elevation. Primary cultures of peripapillary scleral (PPS) fibroblasts from two human donors were screened using a library of 80 kinase inhibitors to identify compounds that inhibit TGFß-induced extracellular matrix (ECM) synthesis. Inhibition of myofibroblast differentiation was verified by alpha smooth muscle actin (αSMA) immunoblot and collagen contraction assay. Inhibition of IOP-induced scleral fibroblast proliferation was assessed by ELISA assay for proliferating cell nuclear antigen (PCNA). The initial screen identified 7 inhibitors as showing>80% reduction in ECM binding. Three kinase inhibitors were verified to reduce TGFß-induced αSMA expression and cellular contractility (rottlerin, PP2, tyrphostin 9). The effect of three Src inhibitors, bosutinib, dasatinib, and SU-6656, on myofibroblast differentiation was evaluated, with only dasatinib significantly inhibiting TGFß-induced ECM synthesis, αSMA expression, and cellular contractility at nanomolar dosages. Subconjunctival injection of dasatinib reduced IOP-induced scleral fibroblast proliferation compared to control (4.9 ± 11.1 ng/sclera with 0.1 µM versus 88.7 ± 38.6 ng/sclera in control, P < 0.0001). Dasatinib inhibits scleral myofibroblast differentiation and there is pharmacologic evidence that this inhibition is not solely due to Src-kinase inhibition.

Rho-Kinase Inhibition Reduces Myofibroblast Differentiation and Proliferation of Scleral Fibroblasts Induced by Transforming Growth Factor ß and Experimental Glaucoma.

PURPOSE: We evaluated prevention of transforming growth factor ß (TGFß)-induced transdifferentiation of cultured scleral fibroblasts to myofibroblasts by rho-associated protein kinase (ROCK) inhibitors. Additionally, we tested whether local delivery of ROCK inhibitors reduced scleral fibroblast proliferation in response to chronic intraocular pressure (IOP) elevation. METHODS: Primary human peripapillary sclera (PPS) fibroblasts were cultured and treated with TGFß to induce myofibroblast transdifferentiation, as determined by immunoblot assessment of α smooth muscle actin (SMA) levels and collagen gel contraction. Cells were treated with the ROCK inhibitors Y27632, fasudil, and H1152 before TGFß treatment. ROCK activity in TGFß-treated fibroblasts and sclera from ocular hypertensive mice was assessed by measuring phosphorylation of the ROCK substrate MYPT1 at Thr696. Fibroblast proliferation following IOP elevation and ROCK inhibitor treatment was assessed by an enzyme-linked immunosorbent (ELISA) assay. RESULTS: ROCK inhibitors H1152 (10µM), Y27632 (10 µM), and fasudil (5µM) reduced SMA expression 72%, 85%, and 68%, respectively. Collagen gel contraction was reduced by 36% (P < 0.001), 27% (P = 0.0003), and 33% (P = 0.0019) following treatment with fasudil (25 µM), Y27632 (10 µM), and H1152 (10µM). ROCK activity induced by TGFß rose 4.74 ± 1.9 times over control at 4 hours (P = 0.0004) and 2.4 ± 0.47-fold (P = 0.0016) in sclera after IOP elevation. Proliferation of scleral fibroblasts after chronic IOP elevation was reduced 77% by Y27632 (P = 0.001) and 84% by fasudil (P = 0.0049). CONCLUSIONS: ROCK inhibitors reduce TGFß-induced myofibroblast transdifferentiation and glaucoma-induced scleral cell proliferation. TRANSLATIONAL RELEVANCE: These findings suggest altered fibroblast activity promoted by ROCK inhibitors could modify scleral biomechanics and be relevant to glaucoma treatment.

Age-Related Changes in Quantitative Strain of Mouse Astrocytic Lamina Cribrosa and Peripapillary Sclera Using Confocal Microscopy in an Explant Model.

Purpose: The purpose of this study was to measure the full-field deformation response to IOP change in the peripapillary sclera (PPS) and astrocytic lamina cribrosa (ALC) of young and old mouse eyes ex vivo. Methods: Thirty-eight transgenic reporter mice with green fluorescent protein-expressing astrocytes were studied at 2 to 4 months and 13 to 15 months old. The ALC and PPS of the explant eyes were imaged using laser scanning microscopy under controlled inflation from 10 to 30 mm Hg. Strains were estimated for the ALC and PPS from imaged volumes using digital volume correlation. Results: ALC strains were significantly greater than zero nasal-temporally for both age groups (mean = 4.3% and 4.0%; each P ≤ 0.004) and significantly greater than zero in the inferior-superior direction for younger mice (P = 0.0004). Younger mice had larger ALC inferior-superior strains than older mice (P = 0.002). The ALC area and perimeter enlarged with inflation in both age groups, with a greater increase in younger than in older mice (all P ≤ 0.004). The ALC nasal-temporal diameter change was greater than inferior-superiorly, and younger mice had greater enlargement nasal-temporally than older. PPS maximum shear strain was greater in the older mice (P = 0.002). The axial lengths of older mice were 14% longer and the PPS was 16% thinner than younger mice (both P = 0.0003). Conclusions: The behavior of the ALC in younger mice with inflation exhibited greater strains and enlargement of ALC area than older mice. Some strain measures in the PPS were greater in older mice, likely related to their longer axial length and thinner PPS.

The effects of age on mitochondria, axonal transport, and axonal degeneration after chronic IOP elevation using a murine ocular explant model.

The purpose of this study was to compare younger and older mice after chronic intraocular pressure (IOP) elevation lasting up to 4 days with respect to mitochondrial density, structure, and movement, as well as axonal integrity, in an ex vivo explant model. We studied 2 transgenic mouse strains, both on a C57BL/6J background, one expressing yellow fluorescent protein (YFP) in selected axons and one expressing cyan fluorescent protein (CFP) in all mitochondria. Mice of 4 months or 14 months of age were exposed to chronic IOP by anterior chamber microbead injection for 14 h, 1, 3, or 4 days. The optic nerve head of globe--optic nerve explants were examined by laser scanning microscopy. Mitochondrial density, structure, and movement were quantified in the CFP explants, and axonal integrity was quantified in YFP explants. In control mice, there was a trend towards decreased mitochondrial density (# per mm2) with age when comparing younger to older, control mice, but this was not significant (1947 ±â€¯653 vs 1412 ±â€¯356; p = 0.19). Mitochondrial density decreased after IOP elevation, significantly, by 31%, in younger mice (p = 0.04) but trending towards a decrease, by 22%, in older mice (p = 0.82) compared to age matched controls. Mitochondrial mean size was not altered after chronic IOP elevation for 14 h or more (p ≥ 0.16). When assessing mitochondrial movement, in younger mice, 5% were mobile at any given time; 4% in the anterograde direction and 1% retrograde. In younger untreated tissue, only 75% of explants had moving mitochondria (mean = 15.8 moving/explant), while after glaucoma induction only 24% of explants had moving mitochondria (mean = 4.2 moving/explant; difference from control, p = 0.03). The distance mitochondria traveled in younger mice was unchanged after glaucoma exposure, but in older glaucoma explants the distance traveled was less than half of older controls (p < 0.0003). In younger mice, mitochondrial speed increased after 14 h of elevated IOP (p = 0.006); however, in older glaucoma explants, movement was actually slower than controls (p = 0.02). In RGC-YFP explants, axonal integrity declined significantly after 4 days of IOP elevation to a similar degree in both younger and older mice. Older mice underwent greater loss of mitochondrial movement with chronic IOP elevation than younger mice, but suffered similar short-term axonal fragmentation in C57BL/6J mice. These transgenic strains, studied in explants, permit observations of alterations in intracellular structure and organelle activity in experimental glaucoma.

Sustained Dorzolamide Release Prevents Axonal and Retinal Ganglion Cell Loss in a Rat Model of IOP-Glaucoma.

PURPOSE: To determine if one injection of a sustained release formulation of dorzolamide in biodegradable microparticles (DPP) reduces retinal ganglion cell (RGC) loss in a rat model of glaucoma. METHODS: We injected either DPP or control microparticles intravitreally in rats. Two days later, unilateral ocular hypertension was induced by translimbal, diode laser treatment by a surgeon masked to treatment group. IOP and clinical exams were performed until sacrifice 6 weeks after laser treatment. RGC loss was measured by masked observers in both optic nerve cross-sections and RGC layer counts from retinal whole mounts. RESULTS: Cumulative IOP exposure was significantly reduced by DPP injection (49 ± 48 mm Hg × days in treated versus 227 ± 191 mm Hg × days in control microparticle eyes; P = 0.012, t-test). While control-injected eyes increased in axial length by 2.4 ± 1.7%, DPP eyes did not significantly enlarge (0.3 ± 2.2%, difference from control, P = 0.03, t-test). RGC loss was significantly less in DPP eyes compared with control microparticle injection alone (RGC axon count reduction: 21% vs. 52%; RGC body reduction: 25% vs. 50% [beta tubulin labeling]; P = 0.02, t-test). CONCLUSIONS: A single injection of sustained release DPP protected against RGC loss and axial elongation in a rat model of IOP glaucoma. TRANSLATIONAL RELEVANCE: Sustained release IOP-lowering medications have the potential to stop glaucoma progression.

Regulation of autoimmune myocarditis by host responses to the microbiome.

The extensive, diverse communities that constitute the microbiome are increasingly appreciated as important regulators of human health and disease through inflammatory, immune, and metabolic pathways. We sought to elucidate pathways by which microbiota contribute to inflammatory, autoimmune cardiac disease. We employed an animal model of experimental autoimmune myocarditis (EAM), which results in inflammatory and autoimmune pathophysiology and subsequent maladaptive cardiac remodeling and heart failure. Antibiotic dysbiosis protected mice from EAM and fibrotic cardiac dysfunction. Additionally, mice derived from different sources with different microbiome colonization profiles demonstrated variable susceptibility to disease. Unexpectedly, it did not track with segmented filamentous bacteria (SFB)-driven Th17 programming of CD4+ T cells in the steady-state gut. Instead, we found disease susceptibility to track with presence of type 3 innate lymphoid cells (ILC3s). Ablating ILCs by antibody depletion or genetic tools in adoptive transfer variants of the EAM model demonstrated that ILCs and microbiome profiles contributed to the induction of CCL20/CCR6-mediated inflammatory chemotaxis to the diseased heart. From these data, we conclude that sensing of the microbiome by ILCs is an important checkpoint in the development of inflammatory cardiac disease processes through their ability to elicit cardiotropic chemotaxis.

Regional Retinal Ganglion Cell Axon Loss in a Murine Glaucoma Model.

Purpose: To determine if retinal ganglion cell (RGC) axon loss in experimental mouse glaucoma is uniform in the optic nerve. Methods: Experimental glaucoma was induced for 6 weeks with a microbead injection model in CD1 (n = 78) and C57BL/6 (B6, n = 68) mice. From epoxy-embedded sections of optic nerve 1 to 2 mm posterior to the globe, total nerve area and regional axon density (axons/1600 µm2) were measured in superior, inferior, nasal, and temporal zones. Results: Control eyes of CD1 mice have higher axon density and more total RGCs than control B6 mice eyes. There were no significant differences in control regional axon density in all mice or by strain (all P > 0.2, mixed model). Exposure to elevated IOP caused loss of RGC in both strains. In CD1 mice, axon density declined without significant loss of nerve area, while B6 mice had less density loss, but greater decrease in nerve area. Axon density loss in glaucoma eyes was not significantly greater in any region in either mouse strain (both P > 0.2, mixed model). In moderately damaged CD1 glaucoma eyes, and CD1 eyes with the greatest IOP elevation exposure, density loss differed by region (P = 0.05, P = 0.03, mixed model) with the greatest loss in the temporal and superior regions, while in severely injured B6 nerves superior loss was greater than inferior loss (P = 0.01, mixed model, Bonferroni corrected). Conclusions: There was selectively greater loss of superior and temporal optic nerve axons of RGCs in mouse glaucoma at certain stages of damage. Differences in nerve area change suggest non-RGC responses differ between mouse strains.

Complete Freund's adjuvant induces experimental autoimmune myocarditis by enhancing IL-6 production during initiation of the immune response.

INTRODUCTION: Complete Freund's Adjuvant (CFA) emulsified with an antigen is a widely used method to induce autoimmune disease in animal models, yet the contribution of CFA to the immune response is not well understood. We compared the effectiveness of CFA with Incomplete Freund's Adjuvant (IFA) or TiterMax Gold Adjuvant (TMax) in experimental autoimmune myocarditis (EAM) in male mice. METHODS: EAM was induced in A/J, BALB/c, and IL6KO BALB/c male mice by injection of the myocarditogenic peptide in CFA, IFA, or TMax on days 0 and 7. EAM severity was analyzed by histology on day 21. In addition, specific flow cytometry outcomes were evaluated on day 21. RESULTS: Only mice immunized with CFA and myocarditogenic peptide on both days 0 and 7 developed substantial myocarditis as measured by histology. We observed a significantly increased level of IL6 in the spleen 3 days after CFA immunization. In the spleen and heart on day 21, there was an expansion of myeloid cells in CFA-immunized mice, as compared to IFA or TMax-immunized animals. Recombinant IL-6 at the time of IFA immunization partially restored susceptibility of the mice to EAM. We also treated EAM-resistant IL-6 knockout mice with recombinant IL-6 around the time of the first immunization, on days -1 to 2, completely restoring disease susceptibility, showing that the requirement for IL-6 coincides with primary immunization. Examining APC populations in the lymph node draining the immunization site evidenced the contribution of IL-6 to the CFA-dependence of EAM was through controlling local dendritic cell (DC) trafficking. CONCLUSIONS: CFA used with myocarditogenic peptide twice is required to induce EAM in both A/J and Balb/c mice. Although IFA and TiterMax induce antibody responses, only CFA preferentially induced autoantigen-specific responses. CFA expands monocytes in the heart and in the spleen. IL-6 signaling is required during short window around primary immunization to induce EAM. In addition, IL-6 deficient mice resistance to EAM could be reversed by injecting IL-6 around first immunization. IL-6 expands dendritic cell and monocytic populations and ultimately leads to a robust T-cell driven immune response in CFA immunized mice.

Macrophages and cardiac fibroblasts are the main producers of eotaxins and regulate eosinophil trafficking to the heart.

Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil-associated diseases. Eosinophils are thought to play a pathogenic role in myocarditis. We investigated the pathways that recruit eosinophils to the heart using a model of eosinophilic myocarditis, in which experimental autoimmune myocarditis (EAM) is induced in IFNγ-/- IL-17A-/- mice. Two conditions are necessary for efficient eosinophil trafficking to the heart: high eotaxin (CCL11, CCL24) expression in the heart and expression of the eotaxin receptor CCR3 by eosinophils. We identified cardiac fibroblasts as the source of CCL11 in the heart interstitium. CCL24 is produced by F4/80+ macrophages localized at inflammatory foci in the heart. Expression of CCL11 and CCL24 is controlled by Th2 cytokines, IL-4 and IL-13. To determine the relevance of this pathway in humans, we analyzed endomyocardial biopsy samples from myocarditis patients. Expression of CCL11 and CCL26 was significantly increased in eosinophilic myocarditis compared to chronic lymphocytic myocarditis and positively correlated with the number of eosinophils. Thus, eosinophil trafficking to the heart is dependent on the eotaxin-CCR3 pathway in a mouse model of EAM and associated with cardiac eotaxin expression in patients with eosinophilic myocarditis. Blocking this pathway may prevent eosinophil-mediated cardiac damage.

Collaborative Interferon-γ and Interleukin-17 Signaling Protects the Oral Mucosa from Staphylococcus aureus.

Infections with Staphylococcus aureus are a continuing and growing problem in community and hospital settings. Preclinical animal modeling of S. aureus relies on experimental infection, which carries some limitations. We describe here a novel, spontaneous model of oral staphylococcal infection in double knockout mice, deficient in the receptors for IL-17 (IL-17RA) and interferon (IFN)-γ (IFNγRI), beginning at 6 to 8 weeks of age. IFNγRI(-/-)IL17RA(-/-) (GRAKO) mice developed progressive oral abscesses. Cytometric methods revealed extensive neutrophilic infiltration of oral tissues in GRAKO mice; further investigation evidenced that IL-17 predominated neutrophil defects in these mice. To investigate the contribution of IFN-γ signaling to this native host defense to S. aureus, we observed perturbations of monocyte recruitment and macrophage differentiation in the oral tissues of GRAKO mice, and CXCL9/chemokine ligand receptor (CXCR)3-driven recruitment of T-cell oral tissues and draining lymph nodes. To address the former finding, we depleted macrophages and monocytes in vivo from IL17RA(-/-) mice using liposomes loaded with clodronate. This treatment elicited oral abscesses, recapitulating the phenotype of GRAKO mice. From these findings, we propose novel collaborative functions of IL-17 and IFN-γ, acting through neutrophils and macrophages, respectively, in native mucocutaneous host defenses to S. aureus.