FADD- and RIPK3-Mediated Cell Death Ensures Clearance of Ly6Chigh Wound Macrophages from Damaged Tissue.

Cells of the monocyte/macrophage lineage are an integral component of the body's innate ability to restore tissue function after injury. In parallel to mounting an inflammatory response, clearance of monocytes/macrophages from the wound site is critical to re-establish tissue functionality and integrity during the course of healing. The role of regulated cell death in macrophage clearance from damaged tissue and its implications for the outcome of the healing response is little understood. In this study, we explored the role of macrophage-specific FADD-mediated cell death on Ripk3-/- background in a mechanical skin injury model in mice. We found that combined inhibition of RIPK3-mediated necroptosis and FADD-caspase-8-mediated apoptosis in macrophages profoundly delayed wound healing. Importantly, RIPK3 deficiency alone did not considerably alter the wound healing process and macrophage population dynamics, arguing that inhibition of FADD-caspase-8-dependent death of macrophages is primarily responsible for delayed wound closure. Notably, TNF blockade reversed the accumulation of Ly6Chigh macrophages induced by combined deficiency of FADD and RIPK3, indicating a critical dual role of TNF-mediated prosurvival and cell death signaling, particularly in this highly proinflammatory macrophage subset. Our findings reveal a previously uncharacterized cross-talk of inflammatory and cell death signaling in macrophages in regulating repair processes in the skin.

Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair.

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.

Role of Macrophages in Wound Healing.

Monocytes/macrophages are key components of the body's innate ability to restore tissue function after injury. In most tissues, both embryo-derived tissue-resident macrophages and recruited blood monocyte-derived macrophages contribute to the injury response. The developmental origin of injury-associated macrophages has a major impact on the outcome of the healing process. Macrophages are abundant at all stages of repair and coordinate the progression through the different phases of healing. They are highly plastic cells that continuously adapt to their environment and acquire phase-specific activation phenotypes. Advanced omics methodologies have revealed a vast heterogeneity of macrophage activation phenotypes and metabolic status at injury sites in different organs. In this review, we highlight the role of the developmental origin, the link between the wound phase-specific activation state and metabolic reprogramming as well as the fate of macrophages during the resolution of the wounding response.

Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing.

Understanding macrophage heterogeneity in tissue repair is a major challenge. Here, we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing. We use a modified version of the original Smart-seq2 protocol to increase speed and accuracy. This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation. For complete details on the use and execution of this protocol, please refer to Willenborg et al. (2021).

A common framework of monocyte-derived macrophage activation.

Macrophages populate every organ during homeostasis and disease, displaying features of tissue imprinting and heterogeneous activation. The disconnected picture of macrophage biology that has emerged from these observations is a barrier for integration across models or with in vitro macrophage activation paradigms. We set out to contextualize macrophage heterogeneity across mouse tissues and inflammatory conditions, specifically aiming to define a common framework of macrophage activation. We built a predictive model with which we mapped the activation of macrophages across 12 tissues and 25 biological conditions, finding a notable commonality and finite number of transcriptional profiles, in particular among infiltrating macrophages, which we modeled as defined stages along four conserved activation paths. These activation paths include a "phagocytic" regulatory path, an "inflammatory" cytokine-producing path, an "oxidative stress" antimicrobial path, or a "remodeling" extracellular matrix deposition path. We verified this model with adoptive cell transfer experiments and identified transient RELMɑ expression as a feature of monocyte-derived macrophage tissue engraftment. We propose that this integrative approach of macrophage classification allows the establishment of a common predictive framework of monocyte-derived macrophage activation in inflammation and homeostasis.

Gene-selective transcription promotes the inhibition of tissue reparative macrophages by TNF.

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn's Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF's anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.

Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing.

Wound healing is a coordinated process that initially relies on pro-inflammatory macrophages, followed by a pro-resolution function of these cells. Changes in cellular metabolism likely dictate these distinct activities, but the nature of these changes has been unclear. Here, we profiled early- versus late-stage skin wound macrophages in mice at both the transcriptional and functional levels. We found that glycolytic metabolism in the early phase is not sufficient to ensure productive repair. Instead, by combining conditional disruption of the electron transport chain with deletion of mitochondrial aspartyl-tRNA synthetase, followed by single-cell sequencing analysis, we found that a subpopulation of early-stage wound macrophages are marked by mitochondrial ROS (mtROS) production and HIF1α stabilization, which ultimately drives a pro-angiogenic program essential for timely healing. In contrast, late-phase, pro-resolving wound macrophages are marked by IL-4Rα-mediated mitochondrial respiration and mitohormesis. Collectively, we identify changes in mitochondrial metabolism as a critical control mechanism for macrophage effector functions during wound healing.

Macrophage-Mediated Tissue Vascularization: Similarities and Differences Between Cornea and Skin.

Macrophages are critical mediators of tissue vascularization both in health and disease. In multiple tissues, macrophages have been identified as important regulators of both blood and lymphatic vessel growth, specifically following tissue injury and in pathological inflammatory responses. In development, macrophages have also been implicated in limiting vascular growth. Hence, macrophages provide an important therapeutic target to modulate tissue vascularization in the clinic. However, the molecular mechanisms how macrophages mediate tissue vascularization are still not entirely resolved. Furthermore, mechanisms might also vary among different tissues. Here we review the role of macrophages in tissue vascularization with a focus on their role in blood and lymphatic vessel formation in the barrier tissues cornea and skin. Comparing mechanisms of macrophage-mediated hem- and lymphangiogenesis in the angiogenically privileged cornea and the physiologically vascularized skin provides an opportunity to highlight similarities but also tissue-specific differences, and to understand how macrophage-mediated hem- and lymphangiogenesis can be exploited for the treatment of disease, including corneal wound healing after injury, graft rejection after corneal transplantation or pathological vascularization of the skin.

Role of collagen XII in skin homeostasis and repair.

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFß, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.

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.

Role of LTBP4 in alveolarization, angiogenesis, and fibrosis in lungs.

Deficiency of the extracellular matrix protein latent transforming growth factor-ß (TGF-ß)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-ß, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-ß activity resulting in profibrotic processes in the lung.

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.

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.

Pathogenic IgG antibodies against desmoglein 3 in pemphigus vulgaris are regulated by HLA-DRB1*04:02-restricted T cells.

Pemphigus vulgaris (PV) is considered as a model for an autoantibody-mediated organ-specific autoimmune disorder. IgG autoantibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3), the major autoantigen in PV, cause loss of epidermal keratinocyte adhesion, resulting in blisters and erosions of the skin and mucous membranes. The association of human autoimmune diseases with distinct HLA alleles is a well-known phenomenon, such as the association with HLA-DRB1*04:02 in PV. However, direct evidence that HLA-DRB1*04:02-restricted autoreactive CD4(+) T cells recognizing immunodominant epitopes of Dsg3 initiate the production of Dsg3-reactive IgG autoantibodies is still missing. In this study, we show in a humanized HLA-DRB1*04:02-transgenic mouse model that HLA-DRB1*04:02-restricted T cell recognition of human Dsg3 epitopes leads to the induction of pathogenic IgG Abs that induce loss of epidermal adhesion, a hallmark in the immune pathogenesis of PV. Activation of Dsg3-reactive CD4(+) T cells by distinct human Dsg3 peptides that bind to HLA-DRß1*04:02 is tightly regulated by the HLA-DRB1*04:02 allele and leads, via CD40-CD40L-dependent T cell-B cell interaction, to the production of IgG Abs that recognize both N- and COOH-terminal epitopes of the human Dsg3 ectodomain. These findings demonstrate key cellular and humoral immune events in the autoimmune cascade of PV in a humanized HLA-transgenic mouse model. We show that CD4(+) T cells recognizing immunodominant Dsg3 epitopes in the context of the PV-associated HLA-DRB1*04:02 induce the secretion of Dsg3-specific IgG in vivo. Finally, these results identify Dsg3-reactive CD4(+) T cells as potential therapeutic targets in the future.

Macrophages - sensors and effectors coordinating skin damage and repair.

Restoration of skin integrity and homeostasis following injury is a vital process. Wound healing disorders, including chronic skin ulcers and pathological scarring, are of major clinical impact. The current therapeutic approaches are often not sufficient. The development of novel efficient therapies requires a thorough understanding of the underlying molecular mechanisms. A cardinal feature of non-healing skin ulcers and excessive scarring is a prolonged inflammatory response at the wound site, which aborts the healing response. Modulation of the local immune response may be an effective therapeutic strategy to correct impaired healing conditions. Yet, the specific mechanisms of inflammation, particularly the role of the diverse leukocyte lineages attracted to the site of tissue damage, have not been resolved. Recent findings in diverse experimental model systems and clinical studies have refined the understanding of monocyte/macrophage biology and the role of cells of the monocytic lineage in tissue regeneration. Thus, monocytes/macrophages are emerging as novel and interesting therapeutic targets to interfere in wound healing pathologies. In this article we will review the role of monocytes/macrophages in skin repair in the light of the recent literature and findings from our own group. This article will provide a rationale for monocyte/macrophage-based therapies to facilitate the healing response.

Genetic ablation of mast cells redefines the role of mast cells in skin wound healing and bleomycin-induced fibrosis.

Conclusive evidence for the impact of mast cells (MCs) in skin repair is still lacking. Studies in mice examining the role of MC function in the physiology and pathology of skin regenerative processes have obtained contradictory results. To clarify the specific role of MCs in regenerative conditions, here we used a recently developed genetic mouse model that allows conditional MC ablation to examine MC-specific functions in skin. This mouse model is based on the cell type-specific expression of Cre recombinase in connective tissue-type MCs under control of the Mcpt5 promoter and the Cre-inducible diphtheria toxin receptor-mediated cell lineage ablation by diphtheria toxin. In response to excisional skin injury, genetic ablation of MCs did not affect the kinetics of reepithelialization, the formation of vascularized granulation tissue, or scar formation. Furthermore, genetic ablation of MCs failed to prevent the development of skin fibrosis upon bleomycin challenge. The amount of deposited collagen and the biochemistry of collagen fibril crosslinks within fibrotic lesions were comparable in MC-depleted and control mice. Collectively, our findings strongly suggest that significant reduction of MC numbers does not affect skin wound healing and bleomycin-induced fibrosis in mice, and provide to our knowledge previously unreported insight in the long-debated contribution of MCs in skin regenerative processes.

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.

Vascular endothelial insulin/IGF-1 signaling controls skin wound vascularization.

Type 2 diabetes mellitus affects 6% of western populations and represents a major risk factor for the development of skin complications, of which impaired wound healing, manifested in e.g. "diabetic foot ulcer", is most prominent. Impaired angiogenesis is considered a major contributing factor to these non-healing wounds. At present it is still unclear whether diabetes-associated wound healing and skin vascular dysfunction are direct consequences of impaired insulin/IGF-1 signaling, or secondary due to e.g. hyperglycemia. To directly test the role of vascular endothelial insulin signaling in the development of diabetes-associated skin complications and vascular function, we inactivated the insulin receptor and its highly related receptor, the IGF-1 receptor, specifically in the endothelial compartment of postnatal mice, using the inducible Tie-2CreERT (DKO(IVE)) deleter. Impaired endothelial insulin/IGF-1 signaling did not have a significant impact on endothelial homeostasis in the skin, as judged by number of vessels, vessel basement membrane staining intensity and barrier function. In contrast, challenging the skin through wounding strongly reduced neo-angiogenesis in DKO(IVE) mice, accompanied by reduced granulation tissue formation reduced. These results show that endothelial insulin/IGF signaling is essential for neo-angiogenesis upon wounding, and imply that reduced endothelial insulin/IGF signaling directly contributes to diabetes-associated impaired healing.