Myeloid-Cell-Derived VEGF Maintains Brain Glucose Uptake and Limits Cognitive Impairment in Obesity.

High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity.

Diabetes Impedes the Epigenetic Switch of Macrophages into Repair Mode.

In this issue of Immunity, Kimball et al. (2019) show that restoring expression of the chromatin modifying enzyme Setdb2 in macrophages rescues impaired wound healing associated with type 2 diabetes. Their findings reveal epigenetic regulation as central to the resolution of macrophage-mediated inflammation in tissue repair and have therapeutic implications for the treatment of diabetic wounds.

Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFß1 and IL-27.

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Interleukin-4 Receptor α Signaling in Myeloid Cells Controls Collagen Fibril Assembly in Skin Repair.

Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-ß induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.

Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing.

Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1ß, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort.

Epidermal mammalian target of rapamycin complex 2 controls lipid synthesis and filaggrin processing in epidermal barrier formation.

BACKGROUND: Perturbation of epidermal barrier formation will profoundly compromise overall skin function, leading to a dry and scaly, ichthyosis-like skin phenotype that is the hallmark of a broad range of skin diseases, including ichthyosis, atopic dermatitis, and a multitude of clinical eczema variants. An overarching molecular mechanism that orchestrates the multitude of factors controlling epidermal barrier formation and homeostasis remains to be elucidated. OBJECTIVE: Here we highlight a specific role of mammalian target of rapamycin complex 2 (mTORC2) signaling in epidermal barrier formation. METHODS: Epidermal mTORC2 signaling was specifically disrupted by deleting rapamycin-insensitive companion of target of rapamycin (Rictor), encoding an essential subunit of mTORC2 in mouse epidermis (epidermis-specific homozygous Rictor deletion [RicEKO] mice). Epidermal structure and barrier function were investigated through a combination of gene expression, biochemical, morphological and functional analysis in RicEKO and control mice. RESULTS: RicEKO newborns displayed an ichthyosis-like phenotype characterized by dysregulated epidermal de novo lipid synthesis, altered lipid lamellae structure, and aberrant filaggrin (FLG) processing. Despite a compensatory transcriptional epidermal repair response, the protective epidermal function was impaired in RicEKO mice, as revealed by increased transepidermal water loss, enhanced corneocyte fragility, decreased dendritic epidermal T cells, and an exaggerated percutaneous immune response. Restoration of Akt-Ser473 phosphorylation in mTORC2-deficient keratinocytes through expression of constitutive Akt rescued FLG processing. CONCLUSION: Our findings reveal a critical metabolic signaling relay of barrier formation in which epidermal mTORC2 activity controls FLG processing and de novo epidermal lipid synthesis during cornification. Our findings provide novel mechanistic insights into epidermal barrier formation and could open up new therapeutic opportunities to restore defective epidermal barrier conditions.

Myeloid Cell-Restricted STAT3 Signaling Controls a Cell-Autonomous Antifibrotic Repair Program.

Myeloid cells can be beneficial as well as harmful in tissue regenerative responses. The molecular mechanisms by which myeloid cells control this critical decision of the immune system are not well understood. Using two different models of physiological acute or pathological chronic skin damage, in this study we identified myeloid cell-restricted STAT3 signaling as important and an injury context-dependent regulator of skin fibrosis. Targeted disruption of STAT3 signaling in myeloid cells significantly accelerated development of pathological skin fibrosis in a model of chronic bleomycin-induced tissue injury, whereas the impact on wound closure dynamics and quality of healing after acute excision skin injury was minor. Chronic bleomycin-mediated tissue damage in control mice provoked an antifibrotic gene signature in macrophages that was characterized by upregulated expression of IL-10, SOCS3, and decorin. In contrast, in STAT3-deficient macrophages this antifibrotic repair program was abolished whereas TGF-ß1 expression was increased. Notably, TGF-ß1 synthesis in cultured control bone marrow-derived macrophages (BMDMs) was suppressed after IL-10 exposure, and this suppressive effect was alleviated by STAT3 deficiency. Accordingly, coculture of IL-10-stimulated control BMDMs with fibroblasts suppressed expression of the TGF-ß1 downstream target connective tissue growth factor in fibroblasts, whereas this suppressive effect was lost by STAT3 deficiency in BMDMs. Our findings highlight a previously unrecognized protective role of myeloid cell-specific STAT3 signaling in immune cell-mediated skin fibrosis, and its regulatory pathway could be a potential target for therapy.

Tight Junction Proteins Claudin-1 and Occludin Are Important for Cutaneous Wound Healing.

Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance.

Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells.

Anti-VEGF-directed therapies have been a milestone for treating retinal vascular diseases. Depletion of monocyte lineage cells suppresses pathological neovascularization in the oxygen-induced retinopathy mouse model. However, the question whether myeloid-derived VEGF-A expression is responsible for the pathogenesis in oxygen-induced retinopathy remained unknown. We analyzed LysMCre-driven myeloid cell-specific VEGF-A knockout mice as well as mice with complete depletion of circulating macrophages through clodronate-liposome treatment in the oxygen-induced retinopathy model by immunohistochemistry, qPCR, and flow cytometry. Furthermore, we analyzed VEGF-A mRNA expression in MIO-M1 cells alone and in co-culture with BV-2 cells in vitro. The myeloid cell-specific VEGF-A knockout did not change relative retinal VEGF-A mRNA levels, the relative avascular area or macrophage/granulocyte numbers in oxygen-induced retinopathy and under normoxic conditions. We observed an insignificantly attenuated pathology in systemically clodronate-liposome treated knockouts but evident VEGF-A expression in activated Müller cells on immunohistochemically stained sections. MIO-M1 cells had significantly higher expression levels of VEGF-A in co-culture with BV-2 cells compared to cultivating MIO-M1 cells alone. Our data show that myeloid-derived cells contribute to pathological neovascularization in oxygen-induced retinopathy through activation of VEGF-A expression in Müller cells.

Clinical assessment of a foam dressing containing growth factor-enhancing hydrated polyurethanes.

OBJECTIVE: This study assesses a novel dressing concept in venous leg ulcer (VLU) patients. It is based on boosting endogenous growth factor activities synthesised by functional granulation tissue. METHODS: Patients received treatment for eight weeks with a hydrated polyurethane-containing foam dressing plus concomitant compression therapy. Wound area reduction (WAR), percentage of wounds achieving a relative WAR of ≥40% and ≥60%, wound pain ratings for the last 24 hours and at dressing changes, EQ-5D Quality of Life questionnaire data, dressing handling and safety parameters were recorded. RESULTS: There were 128 patients who received treatment and data for 123 wound treatment courses were documented. Wound area size decreased from 13.3±9.8cm2 to 10.5±12.2cm2 at week eight and median relative WAR was 48.8%. At week eight, a relative WAR ≥40% was reached by 54.5% of the wounds, 41.5% reached a relative WAR of ≥60% and complete healing was observed in 13.5% of wounds. Median wound pain ratings (last 24 hours before dressing change) declined significantly from 30 to 15.5 (100 visual analogue scale [VAS], p=0.0001) and pain at dressing changes from 30 to 12.5 (p≤0.0001). The EQ-5D VAS rating increased from 58.4±19.2mm to 63.1±19.1mm (p=0.0059). CONCLUSION: This clinical assessment shows that the concept of boosting endogenous growth factors through hydrated polyurethanes has the potential to accelerate WAR in VLU patients while decreasing pain levels and improving quality of life parameters.

IL-10 Indirectly Regulates Corneal Lymphangiogenesis and Resolution of Inflammation via Macrophages.

The role of IL-10, a primarily anti-inflammatory cytokine, in the regulation of inflammatory lymphangiogenesis is undetermined. Herein, we show that IL-10 modulates corneal lymphangiogenesis and resolution of inflammation. IL-10 was not expressed in healthy corneas but was up-regulated in inflamed corneas by infiltrating macrophages. Macrophages up-regulated the expression of prolymphangiogenic vascular endothelial growth factor-C upon stimulation with IL-10. Consistently, corneal inflammation resulted in reduced expression of vascular endothelial growth factor-C and decreased corneal lymphangiogenesis in IL-10-deficient mice (IL-10(-/-)). The effect of IL-10 on lymphangiogenesis was indirect via macrophages, because IL-10 did not directly affect lymphatic endothelial cells. The expression of proinflammatory cytokines and the numbers of infiltrating macrophages increased and remained elevated in inflamed corneas of IL-10(-/-) mice, indicating that IL-10 deficiency led to more severe and prolonged inflammation. The corneal phenotype of IL-10 deficient mice was mimicked in mice with conditional deletion of Stat3 in myeloid cells (lysozyme M Cre mice Stat3(fl/fl) mice), corroborating the critical role of macrophages in the regulation of lymphangiogenesis. Furthermore, local treatment with IL-10 promoted lymphangiogenesis and faster egress of macrophages from inflamed corneas. Taken together, we demonstrate that IL-10 indirectly regulates inflammatory corneal lymphangiogenesis via macrophages. Reduced lymphangiogenesis in IL-10(-/-) and lysozyme M Cre Stat3(fl/fl) mice is associated with more severe inflammatory responses, whereas IL-10 treatment results in faster resolution of inflammation. IL-10 might be used therapeutically to terminate pathological inflammation.

Myeloid Cell-Restricted Insulin/IGF-1 Receptor Deficiency Protects against Skin Inflammation.

Myeloid cells are key regulators of tissue homeostasis and disease. Alterations in cell-autonomous insulin/IGF-1 signaling in myeloid cells have recently been implicated in the development of systemic inflammation and insulin-resistant diabetes mellitus type 2 (DM). Impaired wound healing and inflammatory skin diseases are frequent DM-associated skin pathologies, yet the underlying mechanisms are elusive. In this study, we investigated whether myeloid cell-restricted IR/IGF-1R signaling provides a pathophysiologic link between systemic insulin resistance and the development of cutaneous inflammation. Therefore, we generated mice lacking both the insulin and IGF-1 receptor in myeloid cells (IR/IGF-1R(MKO)). Whereas the kinetics of wound closure following acute skin injury was similar in control and IR/IGF-1R(MKO) mice, in two different conditions of dermatitis either induced by repetitive topical applications of the detergent SDS or by high-dose UV B radiation, IR/IGF-1R(MKO) mice were protected from inflammation, whereas controls developed severe skin dermatitis. Notably, whereas during the early phase in both inflammatory conditions the induction of epidermal proinflammatory cytokine expression was similar in control and IR/IGF-1R(MKO) mice, during the late stage, epidermal cytokine expression was sustained in controls but virtually abrogated in IR/IGF-1R(MKO) mice. This distinct kinetic of epidermal cytokine expression was paralleled by proinflammatory macrophage activation in controls and a noninflammatory phenotype in mutants. Collectively, our findings provide evidence for a proinflammatory IR/IGF-1R-dependent pathway in myeloid cells that plays a critical role in the dynamics of an epidermal-dermal cross-talk in cutaneous inflammatory responses, and may add to the mechanistic understanding of diseases associated with disturbances in myeloid cell IR/IGF-1R signaling, including DM.

Wound-Healing Studies in Cornea and Skin: Parallels, Differences and Opportunities.

The cornea and the skin are both organs that provide the outer barrier of the body. Both tissues have developed intrinsic mechanisms that protect the organism from a wide range of external threats, but at the same time also enable rapid restoration of tissue integrity and organ-specific function. The easy accessibility makes the skin an attractive model system to study tissue damage and repair. Findings from skin research have contributed to unravelling novel fundamental principles in regenerative biology and the repair of other epithelial-mesenchymal tissues, such as the cornea. Following barrier disruption, the influx of inflammatory cells, myofibroblast differentiation, extracellular matrix synthesis and scar formation present parallel repair mechanisms in cornea and skin wound healing. Yet, capillary sprouting, while pivotal in proper skin wound healing, is a process that is rather associated with pathological repair of the cornea. Understanding the parallels and differences of the cellular and molecular networks that coordinate the wound healing response in skin and cornea are likely of mutual importance for both organs with regard to the development of regenerative therapies and understanding of the disease pathologies that affect epithelial-mesenchymal interactions. Here, we review the principal events in corneal wound healing and the mechanisms to restore corneal transparency and barrier function. We also refer to skin repair mechanisms and their potential implications for regenerative processes in the cornea.

Bug or no bug: challenges in diagnosing cutaneous mycobacterial infections.

Cutaneous mycobacterioses are rare in Germany. Nevertheless, early diagnosis and subsequent effective treatment requires awareness of these conditions. Moreover, mycobacterial infections are on the differential diagnosis list of many skin diseases. Diagnoses of cutaneous mycobacterioses are based on clinical features, but also on laboratory investigations, including bacterial culture, histopathology and PCR-based methods. Knowledge about the opportunities and limitations of theses laboratory tests is pivotal to reasonable clinical decision-making. In this paper, we review the current diagnostic options when suspecting a case of cutaneous mycobacterial infection.

Keim oder kein Keim: Herausforderungen bei der Diagnose mykobakterieller Infektionen der Haut.

Kutane Mykobakteriosen sind in Deutschland selten. Dennoch ist es für eine frühzeitige Diagnose und anschließende wirksame Behandlung erforderlich, dass diese Krankheitsbilder im ärztlichen Bewusstsein verankert sind. Darüber hinaus stehen Infektionen mit Mykobakterien auf der Liste der Differentialdiagnosen vieler Hautkrankheiten. Diagnosen kutaner Mykobakteriosen beruhen auf klinischen Merkmalen und auf Laboruntersuchungen, einschließlich bakterieller Kulturen, histopathologischer Untersuchungen und PCR-basierten Verfahren. Das Wissen um Möglichkeiten und Grenzen dieser Laboruntersuchungen ist von zentraler Bedeutung, um eine angemessene klinische Entscheidung zu treffen. In diesem Beitrag diskutieren wir die aktuellen diagnostischen Möglichkeiten, die in Verdachtsfällen kutaner Mykobakteriosen zur Verfügung stehen.

Tropism-modified AAV vectors overcome barriers to successful cutaneous therapy.

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvß8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.

Proteolytic processing regulates placental growth factor activities.

Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth.

CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair.

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2(+)Ly6C(+) macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.

Repair or regenerate--how can we tip the balance?

The fourth EMBO conference on 'The Molecular and Cellular Basis of Regeneration and Repair', held in September 2012, brought together researchers from both the regeneration and wound-healing fields. The meeting spanned a wide range of research topics from basic science to clinical application, and a veritable melting pot of model organisms and approaches resulted in an excellent fourth conference in this series.