NAIP-NLRC4 Inflammasomes Coordinate Intestinal Epithelial Cell Expulsion with Eicosanoid and IL-18 Release via Activation of Caspase-1 and -8.

Intestinal epithelial cells (IECs) form a critical barrier against pathogen invasion. By generation of mice in which inflammasome expression is restricted to IECs, we describe a coordinated epithelium-intrinsic inflammasome response in vivo. This response was sufficient to protect against Salmonella tissue invasion and involved a previously reported IEC expulsion that was coordinated with lipid mediator and cytokine production and lytic IEC death. Excessive inflammasome activation in IECs was sufficient to result in diarrhea and pathology. Experiments with IEC organoids demonstrated that IEC expulsion did not require other cell types. IEC expulsion was accompanied by a major actin rearrangement in neighboring cells that maintained epithelium integrity but did not absolutely require Caspase-1 or Gasdermin D. Analysis of Casp1-/-Casp8-/- mice revealed a functional Caspase-8 inflammasome in vivo. Thus, a coordinated IEC-intrinsic, Caspase-1 and -8 inflammasome response plays a key role in intestinal immune defense and pathology.

Lipoxins A4 and B4 inhibit glial cell activation via CXCR3 signaling in acute retinal neuroinflammation.

Lipoxins are small lipids that are potent endogenous mediators of systemic inflammation resolution in a variety of diseases. We previously reported that Lipoxins A4 and B4 (LXA4 and LXB4) have protective activities against neurodegenerative injury. Yet, lipoxin activities and downstream signaling in neuroinflammatory processes are not well understood. Here, we utilized a model of posterior uveitis induced by lipopolysaccharide endotoxin (LPS), which results in rapid retinal neuroinflammation primarily characterized by activation of resident macroglia (astrocytes and Müller glia), and microglia. Using this model, we observed that each lipoxin reduces acute inner retinal inflammation by affecting endogenous glial responses in a cascading sequence beginning with astrocytes and then microglia, depending on the timing of exposure; prophylactic or therapeutic. Subsequent analyses of retinal cytokines and chemokines revealed inhibition of both CXCL9 (MIG) and CXCL10 (IP10) by each lipoxin, compared to controls, following LPS injection. CXCL9 and CXCL10 are common ligands for the CXCR3 chemokine receptor, which is prominently expressed in inner retinal astrocytes and ganglion cells. We found that CXCR3 inhibition reduces LPS-induced neuroinflammation, while CXCR3 agonism alone induces astrocyte reactivity. Together, these data uncover a novel lipoxin-CXCR3 pathway to promote distinct anti-inflammatory and proresolution cascades in endogenous retinal glia.

Eicosanoid and Specialized Proresolving Mediator Regulation of Lymphoid Cells.

Eicosanoids and specialized proresolving mediators (SPMs) regulate leukocyte function and inflammation. They are ideally positioned at the interface of the innate and adaptive immune responses when lymphocytes interact with leukocytes. Receptors for leukotriene B4 (LTB4), prostaglandin E2 (PGE2), and SPMs are expressed on lymphocytes. Evidence points toward an essential role of these lipid mediators (LMs) in direct regulation of lymphocyte functions. SPMs, which include lipoxins, demonstrate comprehensive protective actions with lymphocytes. LTB4 and PGE2 regulation of lymphocytes is diverse and depends on the interaction of lymphocytes with other cells. Importantly, both LTB4 and PGE2 are essential regulators of T cell antitumor activity. These LMs are attractive therapeutic targets to control dysregulated innate and adaptive immune responses, promote lymphocyte antitumor activity, and prevent tumor immune evasion.

Tissue-specific contributions of Tmem79 to atopic dermatitis and mast cell-mediated histaminergic itch.

Atopic dermatitis (AD) is the most common skin disease in children. It is characterized by relapsing inflammation, skin-barrier defects, and intractable itch. However, the pathophysiology of itch in AD remains enigmatic. Here, we examine the contribution of Tmem79, an orphan transmembrane protein linked to AD in both mice and humans. We show that Tmem79 is expressed by both keratinocytes and sensory neurons, but that loss of keratinocytic Tmem79 is sufficient to elicit robust scratching. Tmem79-/- mice demonstrate an accumulation of dermal mast cells, which are diminished following chronic treatment with cyclooxygenase inhibitors and an EP3 receptor antagonist. In Tmem79-/- mice, mast cell degranulation produces histaminergic itch in a histamine receptor 1/histamine receptor 4 (H4R/H1R)-dependent manner that may involve activation of TRPV1- afferents. TMEM79 has limited sequence homology to a family of microsomal glutathione transferases and confers protection from cellular accumulation of damaging reactive species, and may thus play a role in regulating oxidative stress. In any case, mechanistic insights from this model suggest that therapeutics targeting PGE2 and/or H1R/H4R histaminergic signaling pathways may represent useful avenues to treat Tmem79-associated AD itch. Our findings suggest that individuals with mutations in Tmem79 develop AD due to the loss of protection from oxidative stress.

Lipid droplet formation in Mycobacterium tuberculosis infected macrophages requires IFN-γ/HIF-1α signaling and supports host defense.

Lipid droplet (LD) formation occurs during infection of macrophages with numerous intracellular pathogens, including Mycobacterium tuberculosis. It is believed that M. tuberculosis and other bacteria specifically provoke LD formation as a pathogenic strategy in order to create a depot of host lipids for use as a carbon source to fuel intracellular growth. Here we show that LD formation is not a bacterially driven process during M. tuberculosis infection, but rather occurs as a result of immune activation of macrophages as part of a host defense mechanism. We show that an IFN-γ driven, HIF-1α dependent signaling pathway, previously implicated in host defense, redistributes macrophage lipids into LDs. Furthermore, we show that M. tuberculosis is able to acquire host lipids in the absence of LDs, but not in the presence of IFN-γ induced LDs. This result uncouples macrophage LD formation from bacterial acquisition of host lipids. In addition, we show that IFN-γ driven LD formation supports the production of host protective eicosanoids including PGE2 and LXB4. Finally, we demonstrate that HIF-1α and its target gene Hig2 are required for the majority of LD formation in the lungs of mice infected with M. tuberculosis, thus demonstrating that immune activation provides the primary stimulus for LD formation in vivo. Taken together our data demonstrate that macrophage LD formation is a host-driven component of the adaptive immune response to M. tuberculosis, and suggest that macrophage LDs are not an important source of nutrients for M. tuberculosis.

Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.

Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

Although autacoids primarily derived from the cyclooxygenase-2 and 5-lipoxygenase (LOX) pathways are essential mediators of inflammation, endogenous specialized proresolving mediators (SPMs) act as robust agonists of resolution. SPM biosynthesis is initiated by the conversion of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid primarily via the 12/15-LOX pathway. Although 12/15-LOX activity is prominent in the cornea, the role of SPM pathway activation during infection remains largely unknown and is the focus of the current study. Pseudomonas keratitis was induced in resistant BALB/c and susceptible C57BL/6 (B6) mice. Biosynthetic pathways for proinflammatory autacoids and SPMs were assessed. Divergent lipid mediator profiles demonstrate the importance of 15-LOX pathways in the pathogenesis of ocular infectious disease. Results indicate that an imbalance of LOX enzymatic pathways contributes to susceptibility observed in B6 mice where deficient activation of SPM circuits, as indicated by reduced 15-hydroxy-eicosatetraenoic acid and 17-hydroxydocosahexaenoic acid levels, prevented transition toward resolution and led to chronic inflammation. In sharp contrast, BALB/c mice demonstrated a well-balanced axis of 5-LOX/12-LOX/15-LOX pathways, resulting in sufficient proresolving bioactive metabolite formation and immune homeostasis. Furthermore, a novel immunoregulatory role for 15-LOX was revealed in inflammatory cells (polymorphonuclear leukocytes and macrophages), which influenced phagocytic activity. These data provide evidence that SPM circuits are essential for host defense during bacterial keratitis.-Carion, T. W., Greenwood, M., Ebrahim, A. S., Jerome, A., Suvas, S., Gronert, K., Berger, E. A. Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

Local TNFR1 Signaling Licenses Murine Neutrophils for Increased TLR-Dependent Cytokine and Eicosanoid Production.

Neutrophils are generally the first immune cells recruited during the development of sterile or microbial inflammation. As these cells express many innate immune receptors with the potential to directly recognize microbial or endogenous signals, we set out to assess whether their functions are locally influenced by the signals present at the onset of inflammation. Using a mouse model of peritonitis, we demonstrate that neutrophils elicited in the presence of C-type lectin receptor ligands have an increased ability to produce cytokines, chemokines, and lipid mediators in response to subsequent TLR stimulation. Importantly, we found that licensing of cytokine production was mediated by paracrine TNF-α-TNFR1 signaling rather than direct ligand sensing, suggesting a form of quorum sensing among neutrophils. Mechanistically, licensing was largely imparted by changes in the posttranscriptional regulation of inflammatory cytokines, whereas production of IL-10 was regulated at the transcriptional level. Altogether, our data suggest that neutrophils rapidly adapt their functions to the local inflammatory milieu. These phenotypic changes may promote rapid neutrophil recruitment in the presence of pathogens but limit inflammation in their absence.

Maresin 1 Promotes Inflammatory Resolution, Neuroprotection, and Functional Neurological Recovery After Spinal Cord Injury.

Resolution of inflammation is defective after spinal cord injury (SCI), which impairs tissue integrity and remodeling and leads to functional deficits. Effective pharmacological treatments for SCI are not currently available. Maresin 1 (MaR1) is a highly conserved specialized proresolving mediator (SPM) hosting potent anti-inflammatory and proresolving properties with potent tissue regenerative actions. Here, we provide evidence that the inappropriate biosynthesis of SPM in the lesioned spinal cord hampers the resolution of inflammation and leads to deleterious consequences on neurological outcome in adult female mice. We report that, after spinal cord contusion injury in adult female mice, the biosynthesis of SPM is not induced in the lesion site up to 2 weeks after injury. Exogenous administration of MaR1, a highly conserved SPM, propagated inflammatory resolution after SCI, as revealed by accelerated clearance of neutrophils and a reduction in macrophage accumulation at the lesion site. In the search of mechanisms underlying the proresolving actions of MaR1 in SCI, we found that this SPM facilitated several hallmarks of resolution of inflammation, including reduction of proinflammatory cytokines (CXCL1, CXCL2, CCL3, CCL4, IL6, and CSF3), silencing of major inflammatory intracellular signaling cascades (STAT1, STAT3, STAT5, p38, and ERK1/2), redirection of macrophage activation toward a prorepair phenotype, and increase of the phagocytic engulfment of neutrophils by macrophages. Interestingly, MaR1 administration improved locomotor recovery significantly and mitigated secondary injury progression in a clinical relevant model of SCI. These findings suggest that proresolution, immunoresolvent therapies constitute a novel approach to improving neurological recovery after acute SCI.SIGNIFICANCE STATEMENT Inflammation is a protective response to injury or infection. To result in tissue homeostasis, inflammation has to resolve over time. Incomplete or delayed resolution leads to detrimental effects, including propagated tissue damage and impaired wound healing, as occurs after spinal cord injury (SCI). We report that inflammation after SCI is dysregulated in part due to inappropriate synthesis of proresolving lipid mediators. We demonstrate that the administration of the resolution agonist referred to as maresin 1 (MaR1) after SCI actively propagates resolution processes at the lesion site and improves neurological outcome. MaR1 is identified as an interventional candidate to attenuate dysregulated lesional inflammation and to restore functional recovery after SCI.

Rapid induction of inflammatory lipid mediators by the inflammasome in vivo.

Detection of microbial products by host inflammasomes is an important mechanism of innate immune surveillance. Inflammasomes activate the caspase-1 (CASP1) protease, which processes the cytokines interleukin (IL)-1ß and IL-18, and initiates a lytic host cell death called pyroptosis. To identify novel CASP1 functions in vivo, we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD-domain-containing 4) inflammasome. Here we show that systemic inflammasome activation by flagellin leads to a loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (less than 30 min) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4 and CASP1, but is independent of the production of IL-1ß or IL-18. Instead, inflammasome activation results, within minutes, in an 'eicosanoid storm'--a pathological release of signalling lipids, including prostaglandins and leukotrienes, that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by the activation of cytosolic phospholipase A(2) in resident peritoneal macrophages, which are specifically primed for the production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Our results therefore identify eicosanoids as a previously unrecognized cell-type-specific signalling output of the inflammasome with marked physiological consequences in vivo.

Female-Specific Downregulation of Tissue Polymorphonuclear Neutrophils Drives Impaired Regulatory T Cell and Amplified Effector T Cell Responses in Autoimmune Dry Eye Disease.

Immune-driven dry eye disease primarily affects women; the cause for this sex-specific prevalence is unknown. Polymorphonuclear neutrophils (PMN) have distinct phenotypes that drive inflammation but also regulate lymphocytes and are the rate-limiting cell for generating anti-inflammatory lipoxin A4 (LXA4). Estrogen regulates the LXA4 circuit to induce delayed female-specific wound healing in the cornea. However, the role of PMNs in dry eye disease remains unexplored. We discovered an LXA4-producing tissue PMN population in the corneal limbus, lacrimal glands, and cervical lymph nodes of healthy male and female mice. These tissue PMNs, unlike inflammatory PMNs, expressed a highly amplified LXA4 circuit and were sex-specifically regulated during immune-driven dry eye disease. Desiccating stress in females, unlike in males, triggered a remarkable decrease in lymph node PMN and LXA4 formation that remained depressed during dry eye disease. Depressed lymph node PMN and LXA4 in females correlated with an increase in effector T cells (Th1 and Th17), a decrease in regulatory T cells (Treg), and increased dry eye pathogenesis. Ab depletion of tissue PMN abrogated LXA4 formation in lymph nodes, as well as caused a marked increase in Th1 and Th17 cells and a decrease in Tregs. To establish an immune-regulatory role for PMN-derived LXA4 in dry eye, females were treated with LXA4. LXA4 treatment markedly inhibited Th1 and Th17 and amplified Treg in draining lymph nodes, while reducing dry eye pathogenesis. These results identify female-specific regulation of LXA4-producing tissue PMN as a potential key factor in aberrant effector T cell activation and initiation of immune-driven dry eye disease.

Distinct cellular sources of hepoxilin A3 and leukotriene B4 are used to coordinate bacterial-induced neutrophil transepithelial migration.

Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.

Systemic disease during Streptococcus pneumoniae acute lung infection requires 12-lipoxygenase-dependent inflammation.

Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs), and a risk of systemic spread of the bacterium. We investigated host mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-LOX in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12-LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-LOX exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high-level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX-dependent HXA3 production and subsequent PMN transepithelial migration.

Hepoxilin A(3) facilitates neutrophilic breach of lipoxygenase-expressing airway epithelial barriers.

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

Dysregulation of neuroprotective lipoxin pathway in astrocytes in response to cytokines and ocular hypertension​.

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte reactivity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension. By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4's neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

Protective Effects of Lipoxin A4 and B4 Signaling on the Inner Retina in a Mouse Model of Experimental Glaucoma.

Glaucoma is a common neurodegenerative disease characterized by progressive degeneration of retinal ganglion cells (RGCs) and the retinal nerve fiber layer (RNFL), resulting in a gradual decline of vision. A recent study by our groups indicated that the levels of lipoxins A4 (LXA4) and B4 (LXB4) in the retina and optic nerve decrease following acute injury, and that restoring their function is neuroprotective. Lipoxins are members of the specialized pro-resolving mediator (SPM) family and play key roles to mitigate and resolve chronic inflammation and tissue damage. Yet, knowledge about lipoxin neuroprotective activity remains limited. Here we investigate the in vivo efficacy of exogenous LXA4 and LXB4 administration on the inner retina in a mouse model of chronic experimental glaucoma. To investigate the contribution of LXA4 signaling we used transgenic knockout (KO) mice lacking the two mouse LXA4 receptors (Fpr2/Fpr3-/-). Functional and structural changes of inner retinal neurons were assessed longitudinally using electroretinogram (ERG) and optical coherence tomography (OCT). At the end of the experiment, retinal samples were harvested for immunohistological assessment. While both lipoxins generated protective trends, only LXB4 treatment was significant, and consistently more efficacious than LXA4 in all endpoints. Both lipoxins also appeared to dramatically reduce Müller glial reactivity following injury. In comparison, Fpr2/Fpr3 deletion significantly worsened inner retinal injury and function, consistent with an essential protective role for endogenous LXA4. Together, these results support further exploration of lipoxin signaling as a treatment for glaucomatous neurodegeneration.

Regulation of Diseases-Associated Microglia in the Optic Nerve by Lipoxin B4 and Ocular Hypertension.

Background: The resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap. Methods: Cellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of the mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups was assessed by Student's t-test and one-way ANOVA. Results: Single-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment. Conclusion: We identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

Intravenous fish oil lipid emulsion promotes a shift toward anti-inflammatory proresolving lipid mediators.

Parenteral nutrition (PN)-associated liver disease (PNALD) is a life-threatening complication of the administration of PN. The development of PNALD may be partly due to the composition of the lipid emulsion administered with PN: soybean oil-based lipid emulsions (SOLE) are associated with liver disease, while fish oil-based lipid emulsions (FOLE) are associated with prevention and improvement of liver disease. The objective of this study was to determine how the choice of lipid emulsion modified the production of bioactive lipid mediators (LMs). We utilized a mouse model of steatosis to study the differential effect of FOLE and SOLE. We subsequently validated these results in serum samples from a small cohort of human infants transitioning from SOLE to FOLE. In mice, FOLE was associated with production of anti-inflammatory, proresolving LMs; SOLE was associated with increased production of inflammatory LMs. In human infants, the transition from SOLE to FOLE was associated with a shift toward a proresolving lipidome. Together, these results demonstrate that the composition of the lipid emulsion directly modifies inflammatory homeostasis.

Estrogen negatively regulates epithelial wound healing and protective lipid mediator circuits in the cornea.

Estrogen receptors (ERs) are expressed in leukocytes and in every ocular tissue. However, sex-specific differences and the role of estradiol in ocular inflammatory-reparative responses are not well understood. We found that female mice exhibited delayed corneal epithelial wound closure and attenuated polymorphonuclear (PMN) leukocyte responses, a phenotype recapitulated by estradiol treatment both in vivo (topically in male mice) and in vitro (corneal epithelial cell wound healing). The cornea expresses 15-lipoxygenase (15-LOX) and receptors for lipoxin A(4) (LXA(4)), which have been implicated in an intrinsic lipid circuit that regulates corneal inflammation and wound healing. Delayed epithelial wound healing correlated with lower expression of 15-LOX in the regenerated epithelium of female mice. Estradiol in vitro and in vivo down-regulated epithelial 15-LOX expression and LXA(4) formation, while estradiol abrogation of epithelial wound healing was completely reversed by treatment with LXA(4). More important, ERß and ERα selectively regulated epithelial wound healing, PMN cell recruitment, and activity of the intrinsic 15-LOX/LXA(4) circuit. Our results demonstrate for the first time a sex-specific difference in the corneal reparative response, which is mediated by ERß and ERα selective regulation of the epithelial and PMN 15-LOX/LXA(4) circuit. These findings may provide novel insights into the etiology of sex-specific ocular inflammatory diseases.

Dysregulation of Neuroprotective Lipoxin Pathway in Astrocytes in Response to Cytokines and Ocular Hypertension.

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte activity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension (n=40). By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4's neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.