CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

A superior extracellular matrix binding motif to enhance the regenerative activity and safety of therapeutic proteins.

Among therapeutic proteins, cytokines and growth factors have great potential for regenerative medicine applications. However, these molecules have encountered limited clinical success due to low effectiveness and major safety concerns, highlighting the need to develop better approaches that increase efficacy and safety. Promising approaches leverage how the extracellular matrix (ECM) controls the activity of these molecules during tissue healing. Using a protein motif screening strategy, we discovered that amphiregulin possesses an exceptionally strong binding motif for ECM components. We used this motif to confer the pro-regenerative therapeutics platelet-derived growth factor-BB (PDGF-BB) and interleukin-1 receptor antagonist (IL-1Ra) a very high affinity to the ECM. In mouse models, the approach considerably extended tissue retention of the engineered therapeutics and reduced leakage in the circulation. Prolonged retention and minimal systemic diffusion of engineered PDGF-BB abolished the tumour growth-promoting adverse effect that was observed with wild-type PDGF-BB. Moreover, engineered PDGF-BB was substantially more effective at promoting diabetic wound healing and regeneration after volumetric muscle loss, compared to wild-type PDGF-BB. Finally, while local or systemic delivery of wild-type IL-1Ra showed minor effects, intramyocardial delivery of engineered IL-1Ra enhanced cardiac repair after myocardial infarction by limiting cardiomyocyte death and fibrosis. This engineering strategy highlights the key importance of exploiting interactions between ECM and therapeutic proteins for developing effective and safer regenerative therapies.

Growth Factor and Cytokine Delivery Systems for Wound Healing.

Skin wound healing is a highly coordinated process involving multiple tissue-resident and recruited cell types. Cells within the wound microenvironment respond to key secreted factors such as pro-proliferative growth factors and immunomodulatory cytokines to repair the skin and promptly restore its essential barrier role. Therefore, recombinant growth factors and cytokines are promising therapeutics for skin wounds, in particular for large acute wounds such as burns, or wounds associated with underlying pathologies such as nonhealing chronic and diabetic wounds. However, translation of growth factors and cytokines into clinically effective treatments has been limited. Short half-life, poor stability, rapid diffusion, uncontrolled signaling, and systemic side effects are currently the key challenges to developing efficient growth factor- and cytokine-based therapies. To overcome these limitations, novel delivery systems have been developed to improve the regenerative potential of recombinant growth factors and cytokines. In this review, we discuss biomaterial and protein engineering strategies used to optimize the delivery of growth factor and cytokine therapeutics for skin wound treatment.

Modulation of the Activity of Stem and Progenitor Cells by Immune Cells.

Numerous components of the immune system, including inflammatory mediators, immune cells and cytokines, have a profound modulatory effect on the homeostatic regulation and regenerative activity of endogenous stem cells and progenitor cells. Thus, understanding how the immune system interacts with stem/progenitor cells could build the foundation to design novel and more effective regenerative therapies. Indeed, utilizing and controlling immune system components may be one of the most effective approaches to promote tissue regeneration. In this review, we first summarize the effects of various immune cell types on endogenous stem/progenitor cells, focusing on the tissue healing context. Then, we present interesting regenerative strategies that control or mimic the effect of immune components on stem/progenitor cells, in order to enhance the regenerative capacity of endogenous and transplanted stem cells. We highlight the potential clinical translation of such approaches for multiple tissues and organ systems, as these novel regenerative strategies could considerably improve or eventually substitute stem cell-based therapies. Overall, harnessing the power of the cross-talk between the immune system and stem/progenitor cells holds great potential for the development of novel and effective regenerative therapies.

Nociceptor-derived Reg3γ prevents endotoxic death by targeting kynurenine pathway in microglia.

Nociceptors can fine-tune local or systemic immunity, but the mechanisms of nociceptive modulation in endotoxic death remain largely unknown. Here, we identified C-type lectin Reg3γ as a nociceptor-enriched hormone that protects the host from endotoxic death. During endotoxemia, nociceptor-derived Reg3γ penetrates the brain and suppresses the expression of microglial indoleamine dioxygenase 1, a critical enzyme of the kynurenine pathway, via the Extl3-Bcl10 axis. Endotoxin-administered nociceptor-null mice and nociceptor-specific Reg3γ-deficient mice exhibit a high mortality rate accompanied by decreased brain HK1 phosphorylation and ATP production despite normal peripheral inflammation. Such metabolic arrest is only observed in the brain, and aberrant production of brain quinolinic acid, a neurotoxic metabolite of the kynurenine pathway, causes HK1 suppression. Strikingly, the central administration of Reg3γ protects mice from endotoxic death by enhancing brain ATP production. By identifying nociceptor-derived Reg3γ as a microglia-targeted hormone, this study provides insights into the understanding of tolerance to endotoxic death.

Restoration of the healing microenvironment in diabetic wounds with matrix-binding IL-1 receptor antagonist.

Chronic wounds are a major clinical problem where wound closure is prevented by pathologic factors, including immune dysregulation. To design efficient immunotherapies, an understanding of the key molecular pathways by which immunity impairs wound healing is needed. Interleukin-1 (IL-1) plays a central role in regulating the immune response to tissue injury through IL-1 receptor (IL-1R1). Generating a knockout mouse model, we demonstrate that the IL-1-IL-1R1 axis delays wound closure in diabetic conditions. We used a protein engineering approach to deliver IL-1 receptor antagonist (IL-1Ra) in a localised and sustained manner through binding extracellular matrix components. We demonstrate that matrix-binding IL-1Ra improves wound healing in diabetic mice by re-establishing a pro-healing microenvironment characterised by lower levels of pro-inflammatory cells, cytokines and senescent fibroblasts, and higher levels of anti-inflammatory cytokines and growth factors. Engineered IL-1Ra has translational potential for chronic wounds and other inflammatory conditions where IL-1R1 signalling should be dampened.

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Zinc Finger Protein St18 Protects against Septic Death by Inhibiting VEGF-A from Macrophages.

Zinc finger protein St18 was initially reported as candidate tumor suppressor gene, and also suggested that fibroblast St18 positively regulates NF-κB activation. Despite the pleiotropic functions of St18, little is known about its roles in macrophages. Here, we report that myeloid St18 is a potent inhibitor of VEGF-A. Mice lacking St18 in myeloid lineages exhibit increased retinal vasculature with enhanced serum VEGF-A concentrations. Despite the normal activation of NF-κB target genes, these mice are highly susceptible to LPS-induced shock, polymicrobial sepsis, and experimental colitis, accompanied by enhanced vascular and intestinal leakage. Pharmacological inhibition of VEGF signaling rescued the high mortality rate of myeloid-specific St18-deficient mice in response to inflammation. Mechanistically, St18 directly binds to Sp1 and attenuates its activity, leading to the suppression of Sp1 target gene VEGF-A. Using mouse genetic and pharmacological models, we reveal myeloid St18 as a critical septic death protector.

Enhancing the regenerative effectiveness of growth factors by local inhibition of interleukin-1 receptor signaling.

Although growth factors (GFs) are key molecules for regenerative medicine, their use has been limited by issues associated with suboptimal delivery systems and incomplete understanding of their signaling dynamics. Here, we explored how proinflammatory signals affect GF regenerative potential. Using bone regeneration in mouse, we found that the regenerative capacity of two clinically relevant GFs (BMP-2 and PDGF-BB) is impaired by interleukin-1 receptor (IL-1R1). Mechanistically, IL-1R1 activation in bone-forming cells desensitizes them to GFs and accelerates senescence. Moreover, administration of the GFs triggers IL-1 release by macrophages. To provide localized and sustained IL-1R1 inhibition, we engineered IL-1R antagonist (IL-1Ra) to bind the extracellular matrix (ECM) very strongly and demonstrate that codelivering GFs with ECM-binding IL-1Ra induces superior regeneration. Thus, we highlight that GF regenerative activity is hindered by proinflammatory signals, and GF-based therapies should integrate immunomodulation. Particularly, ECM-binding IL-1Ra holds clinical translational potential by enhancing efficacy of GF therapies.

Growth factors with enhanced syndecan binding generate tonic signalling and promote tissue healing.

Growth factors can stimulate tissue regeneration, but the side effects and low effectiveness associated with suboptimal delivery systems have impeded their use in translational regenerative medicine. Physiologically, growth factor interactions with the extracellular matrix control their bioavailability and spatiotemporal cellular signalling. Growth factor signalling is also controlled at the cell surface level via binding to heparan sulfate proteoglycans, such as syndecans. Here we show that vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) that were engineered to have a syndecan-binding sequence trigger sustained low-intensity signalling (tonic signalling) and reduce the desensitization of growth factor receptors. We also show in mouse models that tonic signalling leads to superior morphogenetic activity, with syndecan-binding growth factors inducing greater bone regeneration and wound repair than wild-type growth factors, as well as reduced tumour growth (associated with PDGF-BB delivery) and vascular permeability (triggered by VEGF-A). Tonic signalling via syndecan binding may also enhance the regenerative capacity of other growth factors.

Growth Factor Engineering Strategies for Regenerative Medicine Applications.

Growth factors are critical molecules for tissue repair and regeneration. Therefore, recombinant growth factors have raised a lot of hope for regenerative medicine applications. While using growth factors to promote tissue healing has widely shown promising results in pre-clinical settings, their success in the clinic is not a forgone conclusion. Indeed, translation of growth factors is often limited by their short half-life, rapid diffusion from the delivery site, and low cost-effectiveness. Trying to circumvent those limitations by the use of supraphysiological doses has led to serious side-effects in many cases and therefore innovative technologies are required to improve growth factor-based regenerative strategies. In this review, we present protein engineering approaches seeking to improve growth factor delivery and efficacy while reducing doses and side effects. We focus on engineering strategies seeking to improve affinity of growth factors for biomaterials or the endogenous extracellular matrix. Then, we discuss some examples of increasing growth factor stability and bioactivity, and propose new lines of research that the field of growth factor engineering for regenerative medicine may adopt in the future.

The ATP Transporter VNUT Mediates Induction of Dectin-1-Triggered Candida Nociception.

Candida albicans infection can cause skin, vulvar, or oral pain. Despite the obvious algesic activity of C. albicans, the molecular mechanisms of fungal nociception remain largely unknown. Here we show that the C. albicans-specific signaling pathway led to severe mechanical allodynia. We discovered that C. albicans-derived ß-glucan stimulated nociceptors depending on Dectin-1, and two pathways in inflammatory pain. The major pathway operates via the Dectin-1-mediated ATP-P2X3/P2X2/3 axis through intercellular relationships between keratinocytes and primary sensory neurons, which depends on the ATP transporter vesicular nucleotide transporter (VNUT). The other pathway operates via the Dectin-1-mediated PLC-TRPV1/TRPA1 axis in primary sensory neurons. Intriguingly, C. albicans-derived ß-glucan has the ability to enhance histamine-independent pruritus, and VNUT inhibitor clodronate can be used to treat unpleasant feelings induced by ß-glucan. Collectively, this is the first report to indicate that Dectin-1 and VNUT mediated innate sensory mechanisms that detect fungal infection.

Immune Regulation of Skin Wound Healing: Mechanisms and Novel Therapeutic Targets.

Significance: The immune system plays a central role in orchestrating the tissue healing process. Hence, controlling the immune system to promote tissue repair and regeneration is an attractive approach when designing regenerative strategies. This review discusses the pathophysiology of both acute and chronic wounds and possible strategies to control the immune system to accelerate chronic wound closure and promote skin regeneration (scar-less healing) of acute wounds. Recent Advances: Recent studies have revealed the key roles of various immune cells and immune mediators in skin repair. Thus, immune components have been targeted to promote chronic wound repair or skin regeneration and several growth factors, cytokines, and biomaterials have shown promising results in animal models. However, these novel strategies are often struggling to meet efficacy standards in clinical trials, partly due to inadequate drug delivery systems and safety concerns. Critical Issues: Excess inflammation is a major culprit in the dysregulation of normal wound healing, and further limiting inflammation effectively reduces scarring. However, current knowledge is insufficient to efficiently control inflammation and specific immune cells. This is further complicated by inadequate drug delivery methods. Future Directions: Improving our understanding of the molecular pathways through which the immune system controls the wound healing process could facilitate the design of novel regenerative therapies. Additionally, better delivery systems may make current and future therapies more effective. To promote the entry of current regenerative strategies into clinical trials, more evidence on their safety, efficacy, and cost-effectiveness is also needed.

Immune Regulation of Tissue Repair and Regeneration via miRNAs-New Therapeutic Target.

The importance of immunity in tissue repair and regeneration is now evident. Thus, promoting tissue healing through immune modulation is a growing and promising field. Targeting microRNAs (miRNAs) is an appealing option since they regulate immunity through post-transcriptional gene fine-tuning in immune cells. Indeed, miRNAs are involved in inflammation as well as in its resolution by controlling immune cell phenotypes and functions. In this review, we first discuss the immunoregulatory role of miRNAs during the restoration of tissue homeostasis after injury, focusing mainly on neutrophils, macrophages and T lymphocytes. As tissue examples, we present the immunoregulatory function of miRNAs during the repair and regeneration of the heart, skeletal muscles, skin and liver. Secondly, we discuss recent technological advances for designing therapeutic strategies which target miRNAs. Specifically, we highlight the possible use of miRNAs and anti-miRNAs for promoting tissue regeneration via modulation of the immune system.

Regulatory T-Cells: Potential Regulator of Tissue Repair and Regeneration.

The identification of stem cells and growth factors as well as the development of biomaterials hold great promise for regenerative medicine applications. However, the therapeutic efficacy of regenerative therapies can be greatly influenced by the host immune system, which plays a pivotal role during tissue repair and regeneration. Therefore, understanding how the immune system modulates tissue healing is critical to design efficient regenerative strategies. While the innate immune system is well known to be involved in the tissue healing process, the adaptive immune system has recently emerged as a key player. T-cells, in particular, regulatory T-cells (Treg), have been shown to promote repair and regeneration of various organ systems. In this review, we discuss the mechanisms by which Treg participate in the repair and regeneration of skeletal and heart muscle, skin, lung, bone, and the central nervous system.

Nociceptors Boost the Resolution of Fungal Osteoinflammation via the TRP Channel-CGRP-Jdp2 Axis.

Candida albicans can enter skeletal tissue through a skin wound in an immunocompromised host or by contamination during orthopedic surgery. Such Candida osteomyelitis is accompanied by severe pain and bone destruction. It is established that nociceptor innervation occurs in skin and bone, but the mechanisms of nociceptive modulation in fungal inflammation remain unclear. In this study, we show that C. albicans stimulates Nav1.8-positive nociceptors via the ß-glucan receptor Dectin-1 to induce calcitonin gene-related peptide (CGRP). This induction of CGRP is independent of Bcl-10 or Malt-1 but dependent on transient receptor potential cation channel subfamily V member 1 (TRPV1)/transient receptor potential cation channel subfamily A member 1 (TRPA1) ion channels. Hindpaw ß-glucan injection after Nav1.8-positive nociceptor ablation or in TRPV1/TRPA1 deficiency showed dramatically increased osteoinflammation accompanied by impaired CGRP production. Strikingly, CGRP suppressed ß-glucan-induced inflammation and osteoclast multinucleation via direct suppression of nuclear factor-κB (NF-κB) p65 by the transcriptional repressor Jdp2 and inhibition of actin polymerization, respectively. These findings clearly suggest a role for Dectin-1-mediated sensocrine pathways in the resolution of fungal osteoinflammation.

Netrins as prophylactic targets in skeletal diseases: A double-edged sword?

The netrin family of proteins are involved in axon guidance during central nervous system development. In vertebrates, two membrane bound forms and five secreted forms of netrin have been reported. In addition to their critical role in neural morphogenesis, a growing number of reports suggest that netrin family proteins also play a role in inflammatory conditions, angiogenesis, and tumorigenesis. In these processes, Unc5 and DCC family proteins serve as receptors of netrin proteins. Recently, it was reported that some netrin family proteins may be involved in the pathogenesis of skeletal diseases including osteoporosis and arthritis. For example, administration of secreted netrin family proteins such as netrin 1 and netrin 4 has prophylactic potential in pathogenic bone degradation in mice. However, netrin 1 blocking antibody also protects mice from inflammatory bone destruction. Therefore, netrin family proteins are involved in the regulation of bone homeostasis, but their bona fide roles in the skeletal system remain controversial. In this review, we discuss the osteo-innate-immune functions of the netrin family of proteins, and summarize their therapeutic potential.

Cytoprotection, proliferation and epidermal differentiation of adipose tissue-derived stem cells on emu oil based electrospun nanofibrous mat.

Electrospun nanofibrous scaffolds containing natural substances with wound healing properties such as Emu oil (EO) may have a great potential for increasing the efficiency of stem cell-based skin bioengineering. For this purpose, EO blended PCL/PEG electrospun nanofibrous mats were successfully fabricated and characterized using FE-SEM, FTIR and Universal Testing Machine. The efficiency of the scaffolds in supporting the adherence, cytoprotection, proliferation and differentiation of adipose tissue-derived stem cells (ADSCs) to keratinocyte was evaluated. GC/MS and HPLC were used to determine the composition of pure EO, which revealed to be mainly fatty acids and carotenoids. FE-SEM and cell proliferation assays showed that adhesion and proliferation of ADSCs on EO-PCL/PEG nanofibers was significantly higher than on PCL/PEG nanofibers. Additionally, EO-PCL/PEG nanofibers with free radical scavenging properties conferred a cytoprotective effect against cell-damaging free radicals, while the ability to support cell adhesion and growth was maintained or even improved. Immunostaining of ADSCs on EO-PCL/PEG nanofibers confirmed the change in morphology of ADSCs from spindle to polygonal shape suggesting their differentiation toward an epidermal linage. Moreover, the expression levels of the keratin 10, filaggrin, and involucrin that are involved in epidermal differentiation were upregulated in a stage-specific manner. This preliminary study shows that EO-PCL/PEG nanofibers could be a good candidate for the fabrication of wound dressings and skin bioengineered substitutes with ADSCs.

Promoting tissue regeneration by modulating the immune system.

The immune system plays a central role in tissue repair and regeneration. Indeed, the immune response to tissue injury is crucial in determining the speed and the outcome of the healing process, including the extent of scarring and the restoration of organ function. Therefore, controlling immune components via biomaterials and drug delivery systems is becoming an attractive approach in regenerative medicine, since therapies based on stem cells and growth factors have not yet proven to be broadly effective in the clinic. To integrate the immune system into regenerative strategies, one of the first challenges is to understand the precise functions of the different immune components during the tissue healing process. While remarkable progress has been made, the immune mechanisms involved are still elusive, and there is indication for both negative and positive roles depending on the tissue type or organ and life stage. It is well recognized that the innate immune response comprising danger signals, neutrophils and macrophages modulates tissue healing. In addition, it is becoming evident that the adaptive immune response, in particular T cell subset activities, plays a critical role. In this review, we first present an overview of the basic immune mechanisms involved in tissue repair and regeneration. Then, we highlight various approaches based on biomaterials and drug delivery systems that aim at modulating these mechanisms to limit fibrosis and promote regeneration. We propose that the next generation of regenerative therapies may evolve from typical biomaterial-, stem cell-, or growth factor-centric approaches to an immune-centric approach. STATEMENT OF SIGNIFICANCE: Most regenerative strategies have not yet proven to be safe or reasonably efficient in the clinic. In addition to stem cells and growth factors, the immune system plays a crucial role in the tissue healing process. Here, we propose that controlling the immune-mediated mechanisms of tissue repair and regeneration may support existing regenerative strategies or could be an alternative to using stem cells and growth factors. The first part of this review we highlight key immune mechanisms involved in the tissue healing process and marks them as potential target for designing regenerative strategies. In the second part, we discuss various approaches using biomaterials and drug delivery systems that aim at modulating the components of the immune system to promote tissue regeneration.