Determination of the FABP5 expression profile in skin lesions of an IMQ-induced psoriasis mouse model using flow cytometry.

The fatty acid-binding protein 5 (FABP5) is a key player in psoriasis development. Therefore, characterizing the expression profile of FABP5 in various cell types within both layers of psoriatic skin is important. Here, we present a protocol that describes steps for an imiquimod-induced psoriasis mouse model and preparation of epidermal and dermal single-cell suspensions. We then detail procedures to detect the FABP5 expression profile in skin keratinocytes and immune cells using intracellular flow cytometry staining. For complete details on the use and execution of this protocol, please refer to Hao et al.1.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis.

One of the hallmarks of intractable psoriasis is neutrophil infiltration in skin lesions. However, detailed molecular mechanisms of neutrophil chemotaxis and activation remain unclear. Here, we demonstrate a significant upregulation of epidermal fatty acid binding protein (E-FABP, FABP5) in the skin of human psoriasis and psoriatic mouse models. Genetic deletion of FABP5 in mice by global knockout and keratinocyte conditional (Krt6a-Cre) knockout, but not myeloid cell conditional (LysM-Cre) knockout, attenuates psoriatic symptoms. Immunophenotypic analysis shows that FABP5 deficiency specifically reduces skin recruitment of Ly6G+ neutrophils. Mechanistically, activated keratinocytes produce chemokines and cytokines that trigger neutrophil chemotaxis and activation in an FABP5-dependent manner. Proteomic analysis further identifies that FABP5 interacts with valosin-containing protein (VCP), a key player in NF-κB signaling activation. Silencing of FABP5, VCP, or both inhibits NF-κB/neutrophil chemotaxis signaling. Collectively, these data demonstrate dysregulated FABP5 as a molecular mechanism promoting NF-κB signaling and neutrophil infiltration in psoriasis pathogenesis.

Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages.

Fats are essential in healthy diets, but how dietary fats affect immune cell function and overall health is not well understood. Mimicking human high-fat diets (HFDs), which are rich in different fatty acid (FA) components, we fed mice various HFDs from different fat sources, including fish oil and cocoa butter. Mice consuming the fish oil HFD exhibit a hair-loss phenotype. Further studies show that omega-3 (n-3) FAs in fish oil promote atypical infiltration of CD207- (langerin-) myeloid macrophages in skin dermis, which induce hair loss through elevated TNF-α signaling. Mechanistically, epidermal fatty acid binding protein (E-FABP) is demonstrated to play an essential role in inducing TNF-α-mediated hair loss by activating the n-3 FA/ROS/IL-36 signaling pathway in dermal resident macrophages. Absence of E-FABP abrogates fish oil HFD-induced murine hair loss. Altogether, these findings support a role for E-FABP as a lipid sensor mediating n-3 FA-regulated macrophage function and skin health.

CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element.

The epigenetic patterns that are established during early thymic development might determine mature T cell physiology and function, but the molecular basis and topography of the genetic elements involved are not fully known. Here we show, using the Cd4 locus as a paradigm for early developmental programming, that DNA demethylation during thymic development licenses a novel stimulus-responsive element that is critical for the maintenance of Cd4 gene expression in effector T cells. We document the importance of maintaining high CD4 expression during parasitic infection and show that by driving transcription, this stimulus-responsive element allows for the maintenance of histone H3K4me3 levels during T cell replication, which is critical for preventing de novo DNA methylation at the Cd4 promoter. A failure to undergo epigenetic programming during development leads to gene silencing during effector T cell replication. Our study thus provides evidence of early developmental events shaping the functional fitness of mature effector T cells.

Acute IL-4 Governs Pathogenic T Cell Responses during Leishmania major Infection.

Leishmania spp. infection is a global health problem affecting more than 2 million people every year with 300 million at risk worldwide. It is well established that a dominant Th1 response (IFN-γ, a hallmark Th1 cytokine) provides resistance, whereas a dominant Th2 response (IL-4, a hallmark Th2 cytokine) confers susceptibility during infection. Given the important role of IL-4 during L. major infection, we used IL-4-neutralizing Abs to investigate the cellular and molecular events regulated by IL-4 signaling. As previously published, neutralization of IL-4 in L. major-infected BALB/c mice (a Leishmania susceptible strain) provided protection when compared with control L. major-infected BALB/c mice. Despite this protection, IFN-γ production by T cells was dramatically reduced. Temporal neutralization of IL-4 revealed that acute IL-4 produced within the first days of infection is critical for not only programming IL-4-producing Th2 CD4+ T cells, but for promoting IFN-γ produced by CD8+ T cells. Mechanistically, IL-4 signaling enhances anti-CD3-induced Tbet and IFN-γ expression in both CD4+ and CD8+ T cells. Given the pathogenic role of IFN-γ-producing CD8+ T cells, our data suggest that IL-4 promotes cutaneous leishmaniasis pathology by not only promoting Th2 immune responses but also pathogenic CD8+ T cell responses. Our studies open new research grounds to investigate the unsuspected role of IL-4 in regulating both Th1 and Th2 responses.

Leishmania major degrades murine CXCL1 - An immune evasion strategy.

Leishmaniasis is a global health problem with an estimated report of 2 million new cases every year and more than 1 billion people at risk of contracting this disease in endemic areas. The innate immune system plays a central role in controlling L. major infection by initiating a signaling cascade that results in production of pro-inflammatory cytokines and recruitment of both innate and adaptive immune cells. Upon infection with L. major, CXCL1 is produced locally and plays an important role in the recruitment of neutrophils to the site of infection. Herein, we report that L. major specifically targets murine CXCL1 for degradation. The degradation of CXCL1 is not dependent on host factors as L. major can directly degrade recombinant CXCL1 in a cell-free system. Using mass spectrometry, we discovered that the L. major protease cleaves at the C-terminal end of murine CXCL1. Finally, our data suggest that L. major metalloproteases are involved in the direct cleavage and degradation of CXCL1, and a synthetic peptide spanning the CXCL1 cleavage site can be used to inhibit L. major metalloprotease activity. In conclusion, our study has identified an immune evasion strategy employed by L. major to evade innate immune responses in mice, likely reservoirs in the endemic areas, and further highlights that targeting these L. major metalloproteases may be important in controlling infection within the reservoir population and transmittance of the disease.

Early vs. late intervention of high fat/low dose streptozotocin treated C57Bl/6J mice with enalapril, α-lipoic acid, menhaden oil or their combination: Effect on diabetic neuropathy related endpoints.

We have previously demonstrated that enalapril, α-lipoic acid and menhaden (fish) oil has potential as a treatment for diabetic peripheral neuropathy. In this study we sought to determine the efficacy of these treatments individually or in combination on multiple neuropathic endpoints in a high fat fed low dose streptozotocin treated mouse, a model of type 2 diabetes, following early or late intervention. Four or twelve weeks after the onset of hyperglycemia, diabetic mice were treated with enalapril, α-lipoic acid, menhaden oil or their combination for 12 weeks. Afterwards, endpoints including glucose tolerance, motor and sensory nerve conduction velocity, thermal nociception, and intraepidermal and cornea nerve fiber density was determined. Glucose clearance was impaired in diabetic mice and significantly improved only with combination treatment and early intervention. Diabetes caused steatosis, slowing of motor and sensory nerve conduction velocity, thermal hypoalgesia and reduction in intraepidermal and cornea nerve fiber density. Treating diabetic mice with enalapril, α-lipoic acid or menhaden oil partially protected diabetic mice from these deficits, whereas the combination of these three treatments was more efficacious following early or late intervention. These studies suggest that a combination therapy may be more effective for treating neural complications of type 2 diabetes.

Effect of Treatment with Salsalate, Menhaden Oil, Combination of Salsalate and Menhaden Oil, or Resolvin D1 of C57Bl/6J Type 1 Diabetic Mouse on Neuropathic Endpoints.

Aims. In this study a streptozotocin induced type 1 diabetes mouse model was used to assess the effectiveness of salsalate, menhaden oil, the combination of salsalate and menhaden oil, or resolvin D1 on neuropathic endpoints. Materials and Methods. Changes in body weight, blood glucose, serum markers for triglycerides, free fatty acids, cholesterol, and resolvin D1, motor and sensory nerve conduction velocities and thermal sensitivity were assessed, as well as performing in vivo confocal microscopy of subepithelial corneal nerves and immunohistochemistry of nerves in the cornea and foot pad. Results. Diabetic animals failed to gain weight and had elevated blood glucose levels. Diabetic mice had slowed nerve conduction velocity, reduced innervation of the foot pad and cornea subepithelial and epithelial layers, and reduced thermal sensitivity. Monotherapy treatment with salsalate, menhaden oil, and resolvin D1 reduced the pathological signs of diabetic neuropathy. The combination of salsalate and menhaden oil also reduced signs of pathology and generated elevated plasma levels of resolvin D1 compared to other groups. Conclusions. Additional studies are needed to determine whether the combination of salsalate and menhaden oil may be more efficacious than monotherapy alone for the treatment of diabetic peripheral neuropathy.

Effect of Inhibition or Deletion of Neutral Endopeptidase on Neuropathic Endpoints in High Fat Fed/Low Dose Streptozotocin-Treated Mice.

Previously we demonstrated that a vasopeptidase inhibitor of angiotensin converting enzyme and neutral endopeptidase (NEP), a protease that degrades vaso- and neuro-active peptides, improves neural function in diabetic rodent models. The purpose of this study was to determine whether inhibition or deletion of NEP provides protection from neuropathy caused by diabetes with an emphasis on morphology of corneal nerves as a primary endpoint. Diabetes, modeling type 2, was induced in C57Bl/6J and NEP deficient mice through a combination of a high fat diet and streptozotocin. To inhibit NEP activity, diabetic C57Bl/6J mice were treated with candoxatril using a prevention or intervention protocol. Twelve weeks after the induction of diabetes in C57Bl/6J mice, the existence of diabetic neuropathy was determined through multiple endpoints including decrease in corneal nerves in the epithelium and sub-epithelium layer. Treatment of diabetic C57Bl/6J mice with candoxatril improved diabetic peripheral neuropathy and protected corneal nerve morphology with the prevention protocol being more efficacious than intervention. Unlike C57Bl/6J, mice deficient in NEP were protected from the development of neuropathologic alterations and loss of corneal nerves upon induction of diabetes. These studies suggest that NEP contributes to the development of diabetic neuropathy and may be a treatable target.

Nicotinamide Riboside Opposes Type 2 Diabetes and Neuropathy in Mice.

Male C57BL/6J mice raised on high fat diet (HFD) become prediabetic and develop insulin resistance and sensory neuropathy. The same mice given low doses of streptozotocin are a model of type 2 diabetes (T2D), developing hyperglycemia, severe insulin resistance and diabetic peripheral neuropathy involving sensory and motor neurons. Because of suggestions that increased NAD(+) metabolism might address glycemic control and be neuroprotective, we treated prediabetic and T2D mice with nicotinamide riboside (NR) added to HFD. NR improved glucose tolerance, reduced weight gain, liver damage and the development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy. In T2D mice, NR greatly reduced non-fasting and fasting blood glucose, weight gain and hepatic steatosis while protecting against diabetic neuropathy. The neuroprotective effect of NR could not be explained by glycemic control alone. Corneal confocal microscopy was the most sensitive measure of neurodegeneration. This assay allowed detection of the protective effect of NR on small nerve structures in living mice. Quantitative metabolomics established that hepatic NADP(+) and NADPH levels were significantly degraded in prediabetes and T2D but were largely protected when mice were supplemented with NR. The data justify testing of NR in human models of obesity, T2D and associated neuropathies.

Corneal Sensitivity to Hyperosmolar Eye Drops: A Novel Behavioral Assay to Assess Diabetic Peripheral Neuropathy.

PURPOSE: Diagnosis of peripheral neuropathy (PN), which affects approximately 50% of the diabetic population, is subjective, with many patients seeking a diagnosis only after presenting with symptoms. Recently, in vivo confocal microscopy of subepithelial corneal nerve density has been promoted as a surrogate marker for early detection of PN, but imaging of corneal nerves requires sophisticated instrumentation, expertise in confocal imaging, cooperative patients, and automated analysis tools to derive corneal nerve density. As an alternative, we developed a simple screening method that is based on the sensitivity of corneal nerves to cause reflex eyelid squinting in response to hyperosmolar eye drops. METHODS: Eyes of control and type 2 diabetic rats were given an eye drop of a 290- to 900-mOsm solution, and the ocular response was video recorded. Other neuropathic end points including nerve conduction velocity and subepithelial cornea nerve density were determined. RESULTS: Motor and sensory nerve conduction velocity and total nerve fiber length of corneal nerves in the subepithelial layer were significantly decreased in diabetic rats. Applying the hyperosmotic solutions to the ocular surface caused an osmolarity-dependent increase in squinting of the treated eye in control rats. Squinting was almost totally blocked by preapplication of proparacaine or N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide, a transient receptor potential melastatin-8 channel blocker. Squinting in response to the 900-mOsm solution was significantly reduced in diabetic rats. CONCLUSIONS: Preclinical studies show that evaluation of corneal sensitivity may be an alternative method for the early detection of PN and has potential for translation to clinical studies.

Effect of enriching the diet with menhaden oil or daily treatment with resolvin D1 on neuropathy in a mouse model of type 2 diabetes.

The purpose of this study was to determine the effect of supplementing the diet of a mouse model of type 2 diabetes with menhaden (fish) oil or daily treatment with resolvin D1 on diabetic neuropathy. The end points evaluated included motor and sensory nerve conduction velocity, thermal sensitivity, innervation of sensory nerves in the cornea and skin, and the retinal ganglion cell complex thickness. Menhaden oil is a natural source for n-3 polyunsaturated fatty acids, which have been shown to have beneficial effects in other diseases. Resolvin D1 is a metabolite of docosahexaenoic acid and is known to have anti-inflammatory and neuroprotective properties. To model type 2 diabetes, mice were fed a high-fat diet for 8 wk followed by a low dosage of streptozotocin. After 8 wk of hyperglycemia, mice in experimental groups were treated for 6 wk with menhaden oil in the diet or daily injections of 1 ng/g body wt resolvin D1. Our findings show that menhaden oil or resolvin D1 did not improve elevated blood glucose, HbA1C, or glucose utilization. Untreated diabetic mice were thermal hypoalgesic, had reduced motor and sensory nerve conduction velocities, had decreased innervation of the cornea and skin, and had thinner retinal ganglion cell complex. These end points were significantly improved with menhaden oil or resolvin D1 treatment. Exogenously, resolvin D1 stimulated neurite outgrowth from primary cultures of dorsal root ganglion neurons from normal mice. These studies suggest that n-3 polyunsaturated fatty acids derived from fish oil could be an effective treatment for diabetic neuropathy.

Effect of diet-induced obesity or type 1 or type 2 diabetes on corneal nerves and peripheral neuropathy in C57Bl/6J mice.

We determined the impact diet-induced obesity (DIO) and types 1 and 2 diabetes have on peripheral neuropathy with emphasis on corneal nerve structural changes in C57Bl/6J mice. Endpoints examined included nerve conduction velocity, response to thermal and mechanical stimuli and innervation of the skin and cornea. DIO mice and to a greater extent type 2 diabetic mice were insulin resistant. DIO and both types 1 and 2 diabetic mice developed motor and sensory nerve conduction deficits. In the cornea of DIO and type 2 diabetic mice there was a decrease in sub-epithelial corneal nerves, innervation of the corneal epithelium, and corneal sensitivity. Type 1 diabetic mice did not present with any significant changes in corneal nerve structure until after 20 weeks of hyperglycemia. DIO and type 2 diabetic mice developed corneal structural damage more rapidly than type 1 diabetic mice although hemoglobin A1 C values were significantly higher in type 1 diabetic mice. This suggests that DIO with or without hyperglycemia contributes to development and progression of peripheral neuropathy and nerve structural damage in the cornea.

Early detection of subclinical visual damage after blast-mediated TBI enables prevention of chronic visual deficit by treatment with P7C3-S243.

PURPOSE: Traumatic brain injury (TBI) frequently leads to chronic visual dysfunction. The purpose of this study was to investigate the effect of TBI on retinal ganglion cells (RGCs), and to test whether treatment with the novel neuroprotective compound P7C3-S243 could prevent in vivo functional deficits in the visual system. METHODS: Blast-mediated TBI was modeled using an enclosed over-pressure blast chamber. The RGC physiology was evaluated using a multielectrode array and pattern electroretinogram (PERG). Histological analysis of RGC dendritic field and cell number were evaluated at the end of the study. Visual outcome measures also were evaluated based on treatment of mice with P7C3-S243 or vehicle control. RESULTS: We show that deficits in neutral position PERG after blast-mediated TBI occur in a temporally bimodal fashion, with temporary recovery 4 weeks after injury followed by chronically persistent dysfunction 12 weeks later. This later time point is associated with development of dendritic abnormalities and irreversible death of RGCs. We also demonstrate that ongoing pathologic processes during the temporary recovery latent period (including abnormalities of RGC physiology) lead to future dysfunction of the visual system. We report that modification of PERG to provocative postural tilt testing elicits changes in PERG measurements that correlate with a key in vitro measures of damage: the spontaneous and light-evoked activity of RGCs. Treatment with P7C3-S243 immediately after injury and throughout the temporary recovery latent period protects mice from developing chronic visual system dysfunction. CONCLUSIONS: Provocative PERG testing serves as a noninvasive test in the living organism to identify early damage to the visual system, which may reflect corresponding damage in the brain that is not otherwise detectable by noninvasive means. This provides the basis for developing an earlier diagnostic test to identify patients at risk for developing chronic CNS and visual system damage after TBI at an earlier stage when treatments may be more effective in preventing these sequelae. In addition, treatment with the neuroprotective agent P7C3-S243 after TBI protects from visual system dysfunction after TBI.

Effect of glycemic control on corneal nerves and peripheral neuropathy in streptozotocin-induced diabetic C57Bl/6J mice.

We sought to determine the impact that duration of hyperglycemia and control has on corneal nerve fiber density in relation to standard diabetic neuropathy endpoints. Control and streptozotocin-diabetic C57Bl/6J mice were analyzed after 4, 8, 12, and 20 weeks. For the 20-week time point, five groups of mice were compared: control, untreated diabetic, and diabetic treated with insulin designated as having either poor glycemic control, good glycemic control, or poor glycemic control switched to good glycemic control. Hyperglycemia was regulated by use of insulin-releasing pellets. Loss of corneal nerves in the sub-epithelial nerve plexus or corneal epithelium progressed slowly in diabetic mice requiring 20 weeks to reach statistical significance. In comparison, slowing of motor and sensory nerve conduction velocity developed rapidly with significant difference compared with control mice observed after 4 and 8 weeks of hyperglycemia, respectively. In diabetic mice with good glycemic control, average blood glucose levels over the 20-week experimental period were lowered from 589 ± 2 to 251 ± 9 mg/dl. All diabetic neuropathy endpoints examined were improved in diabetic mice with good glycemic control compared with untreated diabetic mice. However, good control of blood glucose was not totally sufficient in preventing diabetic neuropathy.

Nociceptive sensitization by complement C5a and C3a in mouse.

Activation of the complement system by injury increases inflammation by producing complement fragments C5a and C3a which are able to recruit and activate immune cells. Complement activation may contribute to pain after inflammation and injury. In this study, we examined whether C5a and C3a elicit nociception when injected into mouse hind paws in vivo, and whether C5a and C3a activate and/or sensitize mechanosensitive nociceptors when applied on peripheral terminals in vitro. We also examined the dorsal root ganglia (DRG) for C5a receptor (C5aR) mRNA and effects of C5a and C3a on intracellular Ca(2+) concentration ([Ca(2+)](i)) using Ca(2+) imaging. Heat hyperalgesia was elicited by intraplantar injection of C5a, and mechanical hyperalgesia by C5a and C3a. After exposure to either C5a or C3a, C-nociceptors were sensitized to heat as evidenced by an increased proportion of heat responsive fibers, lowered response threshold to heat and increased action potentials during and after heat stimulation. A-nociceptors were activated by complement. However, no change was observed in mechanical responses of A- and C-nociceptors after C5a and C3a application. The presence of C5aR mRNA was detected in DRG. C5a and C3a application elevated [Ca(2+)](i) and facilitated capsaicin-induced [Ca(2+)](i) responses in DRG neurons. The results suggest a potential role for complement fragments C5a and C3a in nociception by activating and sensitizing cutaneous nociceptors.

Selective nicotinic acetylcholine receptor agonists: potential therapies for neuropsychiatric disorders with cognitive dysfunction.

Cognitive impairment is one of the most functionally debilitating aspects of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease, despite treatment with available pharmacotherapies. There is emerging evidence that nicotine improves cognitive function in humans and nonhuman species and this has sparked interest in the development of novel nicotinic treatments for cognitive dysfunction. The examination of selective alpha7 and alpha4beta2 nicotinic acetylcholine receptor (nAChR) agonists suggests that both receptor subtypes mediate improvement in attention, learning and working memory. When compared with available pharmacotherapies, specific nAChR agonists may represent unique targets for the treatment of neuropsychiatric and neurodegenerative disorders that feature cognitive impairment as a key symptom.