Optimizing a five-factor cocktail to prepare reparative macrophages for wound healing.

The treatment of non-healing wounds, such as diabetic ulcers, remains a critical clinical challenge. Recent breakthroughs in cell therapy have shown great promise, with one primary focus on preparing cells with comprehensive reparative functions and foreseeable safety. In our previous study, we recapitulated the pro-regenerative and immunosuppressive functions of tumor-associated macrophages (TAMs) in non-tumor-derived macrophages, endowing the latter with characteristics for promoting diabetic wound healing - termed TAMs-educated macrophages (TAMEMs). To eliminate the use of tumor-derived sources and devise a more controllable method to prepare TAMEM-like cells, in this study, we identify a cocktail comprising five recombinant proteins as an essential condition to induce non-polarized macrophages (termed TAMEMs5) into therapeutic cells with pro-healing functions. The screened five factors are osteopontin (OPN), macrophage inflammatory protein (MIP)-2, chemokine (C-C motif) ligand 8 (CCL8), vascular endothelial growth factor (VEGF)-B, and macrophage colony-stimulating factor (M-CSF). We demonstrate the rationale for screening these factors and the phenotype of TAMEMs5 prepared from murine bone marrow-derived macrophages, which exhibit angiogenic and immunomodulatory effects in vitro. Then, we induce primary human monocytes from periphery blood into TAMEMs5, which show pro-healing effects in a human primary cell-based ex vivo model (T-SkinTM). Our study demonstrates a simple, effective, and controllable approach to induce primary macrophages to possess repairing activities, which may provide insights for developing cell-based therapeutics for non-healing wounds clinically.

Carbohydrates as putative pattern recognition receptor agonists in vaccine development.

Adjuvants are essential components of modern vaccines. One general mechanism underlying their immunostimulatory functions is the activation of pattern recognition receptors (PRRs) of innate immune cells. Carbohydrates - as essential signaling molecules on microbial surfaces - are potent PRR agonists and candidate materials for adjuvant design. Here, we summarize the latest trends in developing carbohydrate-containing adjuvants, with fresh opinions on how the physicochemical characteristics of the glycans (e.g., molecular size, assembly status, monosaccharide components, and functional group patterns) affect their adjuvant activities in aiding antigen transport, regulating antigen processing, and enhancing adaptive immune responses. From a translational perspective, we also discuss potential technologies for solving long-lasting challenges in carbohydrate adjuvant design.

Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing.

Nonhealing diabetic wounds, with persistent inflammation and damaged vasculature, have failed conventional treatments and require comprehensive interference. Here, inspired by tumor-associated macrophages (TAMs) that produce abundant immunosuppressive and proliferative factors in tumor development, we generate macrophages to recapitulate TAMs' reparative functions, by culturing normal macrophages with TAMs' conditional medium (TAMs-CM). These TAMs-educated macrophages (TAMEMs) outperform major macrophage phenotypes (M0, M1, or M2) in suppressing inflammation, stimulating angiogenesis, and activating fibroblasts in vitro. When delivered to skin wounds in diabetic mice, TAMEMs efficiently promote healing. Based on TAMs-CM's composition, we further reconstitute a nine-factor cocktail to train human primary monocytes into TAMEMsC-h , which fully resemble TAMEMs' functions without using tumor components, thereby having increased safety and enabling the preparation of autologous cells. Our study demonstrates that recapitulating TAMs' unique reparative activities in nontumor cells can lead to an effective cell therapeutic approach with high translational potential for regenerative medicine.

A Toll-like Receptor-Activating, Self-Adjuvant Glycan Nanocarrier.

The global pandemic of COVID-19 highlights the importance of vaccination, which remains the most efficient measure against many diseases. Despite the progress in vaccine design, concerns with suboptimal antigen immunogenicity and delivery efficiency prevail. Self-adjuvant carriers-vehicles that can simultaneously deliver antigens and act as adjuvants-may improve efficacies in these aspects. Here, we developed a self-adjuvant carrier based on an acetyl glucomannan (acGM), which can activate toll-like receptor 2 (TLR2) and encapsulate the model antigen ovalbumin (OVA) via a double-emulsion process. In vitro tests showed that these OVA@acGM-8k nanoparticles (NPs) enhanced cellular uptake and activated TLR2 on the surface of dendritic cells (DCs), with increased expression of co-stimulatory molecules (e.g. CD80 and CD86) and pro-inflammatory cytokines (e.g. TNF-α and IL12p70). In vivo experiments in mice demonstrated that OVA@acGM-8k NPs accumulated in the lymph nodes and promoted DCs' maturation. The immunization also boosted the humoral and cellular immune responses. Our findings suggest that this self-adjuvant polysaccharide carrier could be a promising approach for vaccine development.

Reprograming the immune niche for skin tissue regeneration - From cellular mechanisms to biomaterials applications.

Despite the rapid development of therapeutic approaches for skin repair, chronic wounds such as diabetic foot ulcers remain an unaddressed problem that affects millions of people worldwide. Increasing evidence has revealed the crucial and diverse roles of the immune cells in the development and repair of the skin tissue, prompting new research to focus on further understanding and modulating the local immune niche for comprehensive, 'perfect' regeneration. In this review, we first introduce how different immunocytes and certain stromal cells involved in innate and adaptive immunity coordinate to maintain the immune niche and tissue homeostasis, with emphasis on their specific roles in normal and pathological wound healing. We then discuss novel engineering approaches - particularly biomaterials systems and cellular therapies - to target different players of the immune niche, with three major aims to i) overcome 'under-healing', ii) avoid 'over-healing', and iii) promote functional restoration, including appendage development. Finally, we highlight how these strategies strive to manage chronic wounds and achieve full structural and functional skin recovery by creating desirable 'soil' through modulating the immune microenvironment.

A "Bridge-Building" Glycan Scaffold Mimicking Microbial Invasion for In Situ Endothelialization.

The globally high prevalence of peripheral artery diseases poses a pressing need for biomaterials grafts to rebuild vasculature. When implanted, they should promote endothelial cells (ECs) adhesion both profoundly and selectively-but the latter expectation remains unfulfilled. Here, this work is inspired by fungi that invade blood vessels via the "bridge" of galectins that, secreted by ECs, can simultaneously bind carbohydrates on fungal surface and integrin receptors on ECs. A glucomannan decanoate (GMDE) substrate mimicking fungal carbohydrates that highly and preferentially supports ECs adhesion while rejecting several other cell types is designed. Electrospun GMDE scaffolds efficiently sequester endogenous galectin-1-which bridges ECs to the scaffolds as it functions in fungal invasions-and promote blood perfusion in a murine limb ischemic model. Meanwhile, the application of GMDE requires no exogenous pro-angiogenic agents and causes no organ toxicity or adverse inflammation in mice, highlighting its high safety of potential translation. This glycan material, uniquely mimicking a microbial action and harnessing a secreted protein as a "bridge," represents an effective, safe, and different strategy for ischemic vascular therapy.

Smectite promotes probiotic biofilm formation in the gut for cancer immunotherapy.

Administration of probiotics to regulate the immune system is a potential anti-tumor strategy. However, oral administration of probiotics is ineffective because of the poor inhabitation of exogenous bacteria in host intestines. Here we report that smectite, a type of mineral clay and established anti-diarrhea drug, promotes expansion of probiotics (especially Lactobacillus) in the murine gut and subsequently elicits anti-tumor immune responses. The ion-exchangeable microstructure of smectite preferentially promotes lactic acid bacteria (LABs) to form biofilms on smectite in vitro and in vivo. In mouse models, smectite laden with LAB biofilms (Lactobacillus and Bifidobacterium) inhibits tumor growth (when used alone) and enhances the efficacy of chemotherapy or immunotherapy (when used in combination with either of them) by activating dendritic cells (DCs) via Toll-like receptor 2 (TLR2) signaling. Our findings suggest oral administration of smectite as a promising strategy to enrich probiotics in vivo for cancer immunotherapy.

High-salt diet inhibits tumour growth in mice via regulating myeloid-derived suppressor cell differentiation.

High-salt diets are associated with an elevated risk of autoimmune diseases, and immune dysregulation plays a key role in cancer development. However, the correlation between high-salt diets (HSD) and cancer development remains unclear. Here, we report that HSD increases the local concentration of sodium chloride in tumour tissue, inducing high osmotic stress that decreases both the production of cytokines required for myeloid-derived suppressor cells (MDSCs) expansion and MDSCs accumulation in the blood, spleen, and tumour. Consequently, the two major types of MDSCs change their phenotypes: monocytic-MDSCs differentiate into antitumour macrophages, and granulocytic-MDSCs adopt pro-inflammatory functions, thereby reactivating the antitumour actions of T cells. In addition, the expression of p38 mitogen-activated protein kinase-dependent nuclear factor of activated T cells 5 is enhanced in HSD-induced M-MDSC differentiation. Collectively, our study indicates that high-salt intake inhibits tumour growth in mice by activating antitumour immune surveillance through modulating the activities of MDSCs.

A pocket-escaping design to prevent the common interference with near-infrared fluorescent probes in vivo.

Near-infrared (NIR) fluorescent probes are among the most attractive chemical tools for biomedical imaging. However, their in vivo applications are hindered by albumin binding, generating unspecific fluorescence that masks the specific signal from the analyte. Here, combining experimental and docking methods, we elucidate that the reason for this problem is an acceptor (A) group-mediated capture of the dyes into hydrophobic pockets of albumin. This pocket-capturing phenomenon commonly applies to dyes designed under the twisted intramolecular charge-transfer (TICT) principle and, therefore, represents a generic but previously unidentified backdoor problem. Accordingly, we create a new A group that avoids being trapped into the albumin pockets (pocket-escaping) and thereby construct a NIR probe, BNLBN, which effectively prevents this backdoor problem with increased imaging accuracy for liver fibrosis in vivo. Overall, our study explains and overcomes a fundamental problem for the in vivo application of a broad class of bioimaging tools.

A toll-like receptor agonist mimicking microbial signal to generate tumor-suppressive macrophages.

Switching macrophages from a pro-tumor type to an anti-tumor state is a promising strategy for cancer immunotherapy. Existing agents, many derived from bacterial components, have safety or specificity concerns. Here, we postulate that the structures of the bacterial signals can be mimicked by using non-toxic biomolecules of simple design. Based on bioactivity screening, we devise a glucomannan polysaccharide with acetyl modification at a degree of 1.8 (acGM-1.8), which specifically activates toll-like receptor 2 (TLR2) signaling and consequently induces macrophages into an anti-tumor phenotype. For acGM-1.8, the degree of acetyl modification, glucomannan pattern, and acetylation-induced assembly are three crucial factors for its bioactivity. In mice, intratumoral injection of acGM-1.8 suppresses the growth of two tumor models, and this polysaccharide demonstrates higher safety than four classical TLR agonists. In summary, we report the design of a new, safe, and specific TLR2 agonist that can generate macrophages with strong anti-tumor potential in mice.

A Jak2-selective inhibitor potently reverses the immune suppression by modulating the tumor microenvironment for cancer immunotherapy.

Small molecule therapeutics can be potent tools for cancer immunotherapy. They may be devised to target the tumor associated macrophages (TAMs) and regulatory T cells (Treg), which are major immunosuppressive cells in the tumor microenvironment. The infiltration and functionalization of these cells, which essentially promote tumor development, are mediated by the hyper-activation of the Jak-STAT3 signaling pathway. Here, we demonstrated that compound 9#, a novel inhibitor of Jak2, could suppress Jak2-STAT3 signaling in macrophages (peritoneal macrophages and THP-1 cells) and direct the macrophages toward the pro-inflammatory (M1-like) phenotype. When tested in ex vivo TAM culture and in vivo tumor models, compound 9# could reverse the phenotype of TAM from M2- to M1-type by promoting IL-12 expression. Further study suggested that compound 9# also inhibited the induction of Treg both in vitro and in vivo via blockage of Jak2 signaling. Finally, compound 9# potently increased the frequency and anti-tumor activity of CD4+ and CD8+ T lymphocytes, leading to effective suppression of tumor growth. Taken together, our findings indicated that compound 9# could be a potential candidate of small molecule therapeutics for cancer immunotherapy.