Innate immunity in peripheral tissues is differentially impaired under normal and endotoxic conditions in aging.

Chronic low-grade inflammation is a hallmark of aging, aka "inflammaging", which is linked to a wide range of age-associated diseases. Immune dysfunction increases disease susceptibility, and increases morbidity and mortality of aging. Innate immune cells, including monocytes, macrophages and neutrophils, are the first responders of host defense and the key mediators of various metabolic and inflammatory insults. Currently, the understanding of innate immune programming in aging is largely fragmented. Here we investigated the phenotypic and functional properties of innate immune cells in various peripheral tissues of young and aged mice under normal and endotoxic conditions. Under the steady state, aged mice showed elevated pro-inflammatory monocytes/macrophages in peripheral blood, adipose tissue, liver, and colon. Under lipopolysaccharide (LPS)-induced inflammatory state, the innate immune cells of aged mice showed a different response to LPS stimulus than that of young mice. LPS-induced immune responses displayed differential profiles in different tissues and cell types. In the peripheral blood, when responding to LPS, the aged mice showed higher neutrophils, but lower pro-inflammatory monocytes than that in young mice. In the peritoneal fluid, while young mice exhibited significantly elevated pro-inflammatory neutrophils and macrophages in response to LPS, aged mice exhibited decreased pro-inflammatory neutrophils and variable cytokine responses in macrophages. In the adipose tissue, LPS induced less infiltrated neutrophils but more infiltrated macrophages in old mice than young mice. In the liver, aged mice showed a more robust increase of pro-inflammatory macrophages compared to that in young mice under LPS stimulation. In colon, macrophages showed relatively mild response to LPS in both young and old mice. We have further tested bone-marrow derived macrophages (BMDM) from young and aged mice, we found that BMDM from aged mice have impaired polarization, displaying higher expression of pro-inflammatory markers than those from young mice. These data collectively suggest that innate immunity in peripheral tissues is impaired in aging, and the dysregulation of immunity is tissue- and cell-dependent. Our findings in the rodent model underscore the complexity of aging immunity. Further investigation is needed to determine whether the immune profile observed in aged mice is applicable in age-associated diseases in humans.

Nutrient-sensing growth hormone secretagogue receptor in macrophage programming and meta-inflammation.

OBJECTIVE: Obesity-associated chronic inflammation, aka meta-inflammation, is a key pathogenic driver for obesity-associated comorbidity. Growth hormone secretagogue receptor (GHSR) is known to mediate the effects of nutrient-sensing hormone ghrelin in food intake and fat deposition. We previously reported that global Ghsr ablation protects against diet-induced inflammation and insulin resistance, but the site(s) of action and mechanism are unknown. Macrophages are key drivers of meta-inflammation. To unravel the role of GHSR in macrophages, we generated myeloid-specific Ghsr knockout mice (LysM-Cre;Ghsrf/f). METHODS: LysM-Cre;Ghsrf/f and control Ghsrf/f mice were subjected to 5 months of high-fat diet (HFD) feeding to induce obesity. In vivo, metabolic profiling of food intake, physical activity, and energy expenditure, as well as glucose and insulin tolerance tests (GTT and ITT) were performed. At termination, peritoneal macrophages (PMs), epididymal white adipose tissue (eWAT), and liver were analyzed by flow cytometry and histology. For ex vivo studies, bone marrow-derived macrophages (BMDMs) were generated from the mice and treated with palmitic acid (PA) or lipopolysaccharide (LPS). For in vitro studies, macrophage RAW264.7 cells with Ghsr overexpression or Insulin receptor substrate 2 (Irs2) knockdown were studied. RESULTS: We found that Ghsr expression in PMs was increased under HFD feeding. In vivo, HFD-fed LysM-Cre;Ghsrf/f mice exhibited significantly attenuated systemic inflammation and insulin resistance without affecting food intake or body weight. Tissue analysis showed that HFD-fed LysM-Cre;Ghsrf/f mice have significantly decreased monocyte/macrophage infiltration, pro-inflammatory activation, and lipid accumulation, showing elevated lipid-associated macrophages (LAMs) in eWAT and liver. Ex vivo, Ghsr-deficient macrophages protected against PA- or LPS-induced pro-inflammatory polarization, showing reduced glycolysis, increased fatty acid oxidation, and decreased NF-κB nuclear translocation. At molecular level, GHSR metabolically programs macrophage polarization through PKA-CREB-IRS2-AKT2 signaling pathway. CONCLUSIONS: These novel results demonstrate that macrophage GHSR plays a key role in the pathogenesis of meta-inflammation, and macrophage GHSR promotes macrophage infiltration and induces pro-inflammatory polarization. These exciting findings suggest that GHSR may serve as a novel immunotherapeutic target for the treatment of obesity and its associated comorbidity.

Nutrient-Sensing Ghrelin Receptor in Macrophages Modulates Bisphenol A-Induced Intestinal Inflammation in Mice.

Bisphenols are environmental toxins with endocrine disruptor activity, yet bisphenol A (BPA) and its analogs are still widely used in manufacturing plastic products. There is evidence showing that BPA elicits inflammation in humans and animals, but the target cell types of BPA are not well understood. In this study, we sought to determine BPA's direct effect on macrophages and BPA immunotoxicity in mouse intestine. Ghrelin is an important nutrient-sensing hormone, acting through its receptor growth hormone secretagogue receptor (GHSR) to regulate metabolism and inflammation. We found that BPA promotes intestinal inflammation, showing increased infiltrating immune cells in colons and enhanced expression of Ghsr and pro-inflammatory cytokines and chemokines, such as Il6 and Ccl2, in colonic mucosa. Moreover, we found that both long- and short-term BPA exposure elevated pro-inflammatory monocytes and macrophages in mouse peripheral blood mononuclear cells (PBMC) and peritoneal macrophages (PM), respectively. To determine the role of GHSR in BPA-mediated inflammation, we generated Ghsr deletion mutation in murine macrophage RAW264.7 using CRISPR gene editing. In wild-type RAW264.7 cells, the BPA exposure promotes macrophage pro-inflammatory polarization and increases Ghsr and cytokine/chemokine Il6 and Ccl2 expression. Interestingly, Ghsr deletion mutants showed a marked reduction in pro-inflammatory cytokine/chemokine expression in response to BPA, suggesting that GHSR is required for the BPA-induced pro-inflammatory response. Further understanding how nutrient-sensing GHSR signaling regulates BPA intestinal immunotoxicity will help design new strategies to mitigate BPA immunotoxicity and provide policy guidance for BPA biosafety.

A monocyte-leptin-angiogenesis pathway critical for repair post-infection.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

ZAG Regulates the Skin Barrier and Immunity in Atopic Dermatitis.

Adipokines modulate immune responses and lipid metabolism in allergic disease; however, little is known about their role in the skin barrier and atopic dermatitis (AD). We identified ZAG, an adipokine that regulates lipid mobilization, as a biomarker for AD. ZAG levels were consistently decreased in sera, T cells, and skin in human AD patients compared with healthy controls. ZAG was primarily detected in the stratum corneum along with FLG and LOR. Knockdown of ZAG with short hairpin RNA resulted in decreased FLG and increased TSLP. Topical ZAG treatment in AD mice recovered ZAG expression in the skin and improved AD-like symptoms, transepidermal water loss, and ceramide levels. Furthermore, topical ZAG treatment induced immunoregulatory effects, including reduction of IL-4, IL-17, and IFN-γ and increased Foxp3 in the skin and lymphoid organs. Interestingly, ZAG treatment also recovered decreased levels of ADAM17, an important player in skin barrier function and immune response in AD. Thus, ZAG deficiency is closely related to skin barrier function and the immune abnormalities of AD, and we suggest that restoration of ZAG may be a promising therapeutic option for the treatment of AD.

Thymic stromal lymphopoietin regulates eosinophil migration via phosphorylation of l-plastin in atopic dermatitis.

Infiltration of eosinophils in atopic dermatitis (AD), which contains inflammatory molecules and cytokines, recruits more inflammatory cells and causes further skin damage. Thymic stromal lymphopoietin (TSLP) is an epithelial cytokine that induces the proinflammatory Th2 immune response and plays an important role in allergic disease. In this study, we aimed to identify a novel protein that regulates TSLP in eosinophils to further understand the role of eosinophils in atopic dermatitis. Using a proteomics approach, we identified the TSLP-inducible protein l-plastin and confirmed upregulation of l-plastin and p-l-plastin in TSLP-treated human eosinophilic leukaemic (EoL-1) cells and in eosinophils from AD patients. Migration assays showed that migration of eosinophils increased when cells were treated with TSLP and when cells were treated with TSLP and an additional cytokine such as interleukin (IL)-3, IL-4, IL-5 or IL-13, when compared to migration of untreated eosinophils. We also confirmed a positive correlation between phosphorylation of l-plastin and an increase in migration of TSLP and cytokine-treated eosinophils. In addition, phosphorylation of l-plastin was sensitive to PKCßII inhibition. Our results suggest that TSLP-induced phosphorylation of l-plastin affects eosinophil migration, which may be mediated by the protein kinase C signalling pathway in atopic dermatitis, thus suggesting p-l-plastin as a potential drug target for eosinophil-targeted allergy therapy.

In vivo relative quantitative proteomics reveals HMGB1 as a downstream mediator of oestrogen-stimulated keratinocyte migration.

It is known that oestrogen influences skin wound healing by modulating the inflammatory response, cytokine expression and extracellular matrix deposition; accelerating re-epithelialization; and stimulating angiogenesis. To identify novel proteins associated with effects of oestrogen on keratinocyte, stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry was performed. Using SILAC, quantification of 1085 proteins was achieved. Among these proteins, 60 proteins were upregulated and 32 proteins were downregulated. Among significantly upregulated proteins, high-mobility group protein B1 (HMGB1) has been further evaluated for its role in the effect of oestrogen on keratinocytes. HMGB1 expression was strongly induced in oestrogen-treated keratinocytes in dose- and time-dependent manner. Further, HMGB1 was able to significantly accelerate the rate of HaCaT cell migration. To determine whether HMGB1 is involved in E2-induced HaCaT cell migration, cells were transfected with HMGB1 siRNA. Knockdown of HMGB1 blocked oestrogen-induced keratinocyte migration. Collectively, these experiments demonstrate that HMGB1 is a novel downstream mediator of oestrogen-stimulated keratinocyte migration.

Positive reactions to nickel on a patch test do not predict clinical outcome of nickel alloy-based atrial septal defect occluder implantation.

BACKGROUND: Patch testing is thought to be necessary prior to metal device implantation to rule out metal allergy-related complications; however, there are controversies over the effects of nickel allergy on the outcome of nickel alloy-based device implantation. OBJECTIVE: This study aimed to evaluate the adverse events in a Korean population of nickel allergy patients who underwent atrial septal defect (ASD) closure with a nickel-titanium alloy-based device. METHODS: We retrospectively reviewed the medical records of patients who underwent ASD closure with a nitinol device. RESULTS: Among 38 patients who had ASD closure, 4 of 5 nickel-allergic patients and 10 of the 33 non-nickel-allergic patients had post-closure complications. All patients fared well, without device failure culminating in device removal. CONCLUSION: In this study, positive reactions to nickel in a patch test were not associated with adverse early or late outcomes following ASD closure with a nickel alloy-based device.

DAMP molecules S100A9 and S100A8 activated by IL-17A and house-dust mites are increased in atopic dermatitis.

S100A9 and S100A8 are called damage-associated molecular pattern (DAMP) molecules because of their pro-inflammatory properties. Few studies have evaluated S100A9 and S100A8 function as DAMP molecules in atopic dermatitis (AD). We investigated how house-dust mites affect S100A9 and S100A8 expression in Th2 cytokine- and Th17 cytokine-treated keratinocytes, and how secretion of these molecules affects keratinocyte-derived cytokines. Finally, we evaluated expression of these DAMP molecules in AD patients. S100A9 expression and S100A8 expression were strongly induced in IL-17A- and Dermatophagoides (D.) farinae-treated keratinocytes, respectively. Furthermore, co-treatment with D. farinae and IL-17A strongly increased expression of S100A9 and S100A8 compared with D. farinae-Th2 cytokine co-treatment. The IL-33 mRNA level increased in a dose-dependent manner in S100A9-treated keratinocytes, but TSLP expression did not change. S100A8/A9 levels were also higher in the lesional skin and serum of AD patients, and correlated with disease severity. Taken together, S100A9 and S100A8 may be involved in inducing DAMP-mediated inflammation in AD triggered by IL-17A and house-dust mites.

PTEN regulation by the Akt/GSK-3ß axis during RANKL signaling.

Phosphatase and tensin homolog (PTEN) negatively regulates phosphoinositide 3-kinase (PI3K)/Akt signaling as a lipid phosphatase for the second messenger phosphatidylinositol 3,4,5-triphosphate. We discovered recently that inactivating glycogen synthase kinase-3ß (GSK-3ß) via Akt plays an important role in receptor activator of nuclear factor κb ligand (RANKL)-induced osteoclastogenesis. However, the signaling link between GSK-3ß and PTEN in RANKL signaling has not been revealed. Downregulating PTEN by RNA interference increases Akt and GSK-3ß phosphorylation levels by RANKL, thereby promoting the formation of osteoclasts. PTEN phosphorylation at threonine 366 (T366) decreased gradually during RANKL-induced osteoclastogenesis, whereas PTEN protein levels were unaffected. Interestingly, the PTEN phosphorylation defective mutant (T366A) showed increased osteoclastogenesis, which is consistent with its lower phosphatase activity, compared to that of wild-type PTEN. Moreover, treatment with the GSK-3 inhibitor SB216763 suppressed PTEN phosphorylation levels and phosphatase activity and enhanced Akt phosphorylation. These data suggest that inhibiting GSK-3ß during RANKL-induced osteoclastogenesis decreases PTEN phosphorylation, leading to enhanced osteoclast differentiation through Akt activation.

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells.

Skin is one of the most commonly studied tissues for microcirculation research owing to its close correlation of cutaneous vascular function, ageing and age-related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and ageing in the vascular environment of the skin, we performed a proteomic analysis of plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Among identified proteins, paraoxonase 1 (PON1) was selected in this study. To elucidate the role of PON1 on skin ageing and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked-down by small interfering RNA (siRNA) targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence-associated ß-galactosidase stained cells and enlarged and flattened cell morphology. At 48 h post-transfection, the protein expression of p16 in PON1 siRNA-treated HDMECs was higher than that in scrambled siRNA-treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age-related candidate biomarkers, were decreased by PON1 knock-down in HDMECs. In conclusion, these results suggest that PON1 functions as an ageing-related protein and plays an important role in the cellular senescence of HDMECs.

Association of stress with symptoms of atopic dermatitis.

Psychological stress and atopic dermatitis (AD) symptoms appear to form a vicious cycle. This study compared the degree of stress and impairment of dermatology life quality between patients with AD and healthy controls, and examined for neuropeptides and neurotrophins associated with stress in AD. Questionnaires, comprising five tests evaluating depression, anxiety, interaction anxiousness, private body consciousness, and dermatology life quality, were examined in age- and sex-matched patients with AD (n = 28) and healthy controls (n = 28). Immunohistochemical staining of nerve growth factor, substance P, corticotrophin-releasing factor receptor and neuropeptide Y was performed in the AD-involved and normal skin. Patients with AD showed high scores on all of the questionnaires, including Beck Depression Inventory, state anxiety, trait anxiety, Interaction Anxiousness Scale, Private Body Consciousness subscale, and Dermatology Life Quality Index. All of the parameters, except for Beck Depression Inventory, showed higher values in AD than healthy controls (p < 0.001). Statistically significant correlations were observed between each psychological parameter and Dermatology Life Quality Index. Among the clinical parameters, only pruritus was positively correlated with state anxiety (R = 0.573, p < 0.05) and trait anxiety (R = 0.525, p < 0.05). The Eczema Area and Severity Index score did not show any significant correlations with psychological parameters. Nerve growth factor-reactive cells were observed more abundantly and intensely in both epidermis and dermis of AD involved skin (n = 4) than in healthy controls (n = 3) (p = 0.022 and 0.029, respectively). Also, the number and intensity of neuropeptide Y-positive cells was significantly greater in the entire epidermis of patients with AD than in healthy controls (n = 3) (p = 0.029 and 0.026, respectively). We conclude that anxiety may be associated with the induction of pruritus through neuro-peptide Y and nerve growth factor.

Can blood components with age-related changes influence the ageing of endothelial cells?

Research on vascular endothelial cell ageing helps elucidate the pathogenesis of diseases associated with cell ageing. To investigate endothelial senescence, we used 2-DE coupled with MS to perform a proteomic analysis of: (i) peripheral blood mononuclear cells (PBMCs) from donors in their 20 s ('young') or 60 s ('old') and (ii) human dermal microvascular endothelial cells (HDMECs) treated with sera from young and old donors. Identified proteins could be classified into several functional categories: (i) cytoskeletal regulators: CapG and cofilin 1; (ii) stress response and signal pathway proteins: TXNDC5 and RSU-1; and (iii) apoptosis proteins: Annexin V. We confirmed by Western blot a decrease of RSU-1, CapG and TXNDC5 in PBMCs from old donors. RSU-1, which regulates signal transduction of the downstream Ras, showed decreased mRNA and protein levels in PBMCs from old donors and decreased mRNA levels in HDMECs treated with sera from old donors. In addition, Ras protein levels were increased in PBMCs from old donors. These data indicate that reduced RSU-1 might induce Ras expression, which subsequently could provoke Ras-induced senescence. In conclusion, our data suggest that blood components that exhibit age-related changes, such as alterations in cytoskeletal regulators and stress proteins, may be associated with endothelial cell ageing.