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.

Plasmodium vivax liver stage assay platforms using Indian clinical isolates.

BACKGROUND: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates. METHODS: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites. RESULTS: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low. CONCLUSIONS: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Tregs delivered post-myocardial infarction adopt an injury-specific phenotype promoting cardiac repair via macrophages in mice.

Regulatory T cells (Tregs) are key immune regulators that have shown promise in enhancing cardiac repair post-MI, although the mechanisms remain elusive. Here, we show that rapidly increasing Treg number in the circulation post-MI via systemic administration of exogenous Tregs improves cardiac function in male mice, by limiting cardiomyocyte death and reducing fibrosis. Mechanistically, exogenous Tregs quickly home to the infarcted heart and adopt an injury-specific transcriptome that mediates repair by modulating monocytes/macrophages. Specially, Tregs lead to a reduction in pro-inflammatory Ly6CHi CCR2+ monocytes/macrophages accompanied by a rapid shift of macrophages towards a pro-repair phenotype. Additionally, exogenous Treg-derived factors, including nidogen-1 and IL-10, along with a decrease in cardiac CD8+ T cell number, mediate the reduction of the pro-inflammatory monocyte/macrophage subset in the heart. Supporting the pivotal role of IL-10, exogenous Tregs knocked out for IL-10 lose their pro-repair capabilities. Together, this study highlights the beneficial use of a Treg-based therapeutic approach for cardiac repair with important mechanistic insights that could facilitate the development of novel immunotherapies for MI.

Local administration of regulatory T cells promotes tissue healing.

Regulatory T cells (Tregs) are crucial immune cells for tissue repair and regeneration. However, their potential as a cell-based regenerative therapy is not yet fully understood. Here, we show that local delivery of exogenous Tregs into injured mouse bone, muscle, and skin greatly enhances tissue healing. Mechanistically, exogenous Tregs rapidly adopt an injury-specific phenotype in response to the damaged tissue microenvironment, upregulating genes involved in immunomodulation and tissue healing. We demonstrate that exogenous Tregs exert their regenerative effect by directly and indirectly modulating monocytes/macrophages (Mo/MΦ) in injured tissues, promoting their switch to an anti-inflammatory and pro-healing state via factors such as interleukin (IL)-10. Validating the key role of IL-10 in exogenous Treg-mediated repair and regeneration, the pro-healing capacity of these cells is lost when Il10 is knocked out. Additionally, exogenous Tregs reduce neutrophil and cytotoxic T cell accumulation and IFN-γ production in damaged tissues, further dampening the pro-inflammatory Mo/MΦ phenotype. Highlighting the potential of this approach, we demonstrate that allogeneic and human Tregs also promote tissue healing. Together, this study establishes exogenous Tregs as a possible universal cell-based therapy for regenerative medicine and provides key mechanistic insights that could be harnessed to develop immune cell-based therapies to enhance tissue healing.

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.

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.

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.

Antibodies to a CA 19-9 Related Antigen Complex Identify SOX9 Expressing Progenitor Cells In Human Foetal Pancreas and Pancreatic Adenocarcinoma.

The Sialyl Lewis A antigen, or CA 19-9, is the prototype serum biomarker for adenocarcinoma of the pancreas. Despite extensive clinical study of CA 19-9 in gastrointestinal malignancies, surprisingly little is known concerning the specific cell types that express this marker during development, tissue regeneration and neoplasia. SOX9 is a transcription factor that plays a key role in these processes in foregut tissues. We report the biochemistry and tissue expression of the GCTM-5 antigen, a pancreatic cancer marker related to, but distinct from, CA19-9. This antigen, defined by two monoclonal antibodies recognising separate epitopes on a large glycoconjugate protein complex, is co-expressed with SOX9 by foregut ductal progenitors in the developing human liver and pancreas, and in pancreatic adenocarcinoma. These progenitors are distinct from cell populations identified by DCLK1, LGR5, or canonical markers of liver and pancreatic progenitor cells. Co-expression of this antigen complex and SOX9 also characterises the ductal metaplasia of submucosal glands that occurs during the development of Barrett's oesophagus. The GCTM-5 antigen complex can be detected in the sera of patients with pancreatic adenocarcinoma. The GCTM-5 epitope shows a much more restricted pattern of expression in the normal adult pancreas relative to CA19-9. Our findings will aid in the identification, characterisation, and monitoring of ductal progenitor cells during development and progression of pancreatic adenocarcinoma in man.

Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement.

Over the past two decades, human embryonic stem cells (hESCs) have gained attention due to their pluripotent and proliferative ability which enables production of almost all cell types in the human body in vitro and makes them an excellent tool to study human embryogenesis and disease, as well as for drug discovery and cell transplantation therapies. Discovery of human-induced pluripotent stem cells (hiPSCs) further expanded therapeutic applications of human pluripotent stem cells (PSCs). hPSCs provide a stable and unlimited original cell source for producing suitable cells and tissues for downstream applications. Therefore, engineering the environment in which these cells are grown, for stable and quality-controlled hPSC maintenance and production, is one of the key factors governing the success of these applications. hPSCs are maintained in a particular niche using specific cell culture components. Ideally, the culture should be free of xenobiotic components to render hPSCs suitable for therapeutic applications. Substantial efforts have been put to identify effective components, and develop culture conditions and protocols, for their large-scale expansion without compromising on quality. In this review, we discuss different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs. Understanding the role of culture components would enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of hPSCs thereby increasing their potential applications.

Chemically defined and growth-factor-free culture system for the expansion and derivation of human pluripotent stem cells.

The large-scale and cost-effective production of quality-controlled human pluripotent stem cells (hPSCs) for use in cell therapy and drug discovery would ideally require a chemically defined xenobiotic-free culture system. Towards the development of such a system, costs associated with the use of recombinant proteins as supplements in basal culture media need to be reduced. Here, we describe a growth-factor-free culture medium that uses just three chemical compounds and a lower number of recombinant proteins than used in commercially available media. We show that the culture medium supports the long-term propagation of hPSCs, as confirmed by karyotype, the expression of pluripotency markers and the capacity to differentiate into cell types derived from the three embryonic germ layers. hPSCs growing in the medium were less dependent on glycolytic pathways than cells grown in medium containing growth factors. Moreover, the medium supported the generation of induced pluripotent stem cells derived from either human dermal fibroblasts or peripheral blood mononuclear cells. Our findings should facilitate the ongoing development of a completely xeno-free, chemically defined, synthetic culture system for hPSCs.