Since the Edmonton protocol was proposed in 2000, islet transplantation has developed rapidly as a new technology. This protocol proposed a glucocorticoid-free immunosuppressive regimen consisting of sirolimus, tacrolimus, and daclizumab and a scheme of sequential transplantation of islets from at least two donor pancreases. During the follow-up period (with a median follow-up time of 1 year), all seven patients who received this protocol achieved insulin independence (1). Subsequent clinical trials verified the protocol’s effectiveness and noted the necessity of minimizing the interval between 2 islet infusions (2). The outcomes of islet transplantation over the past two decades were assessed in a large cohort study conducted by the Edmonton group. Among 255 patients, the 5-year insulin independence rate after operation was 32%, and 8% still had insulin independence 20 years later (3). Islet transplantation has the advantages of minimal trauma, high safety, a short hospital stay, less patient suffering, and repeatability. It is especially suitable for “brittle diabetes” type I diabetes patients, type II diabetes patients with pancreatic islet dysfunction, diabetes patients after liver and kidney transplantation, and patients with nonmalignant pancreatic resection for the prevention of postoperative diabetes. Compared with traditional drug and insulin therapy, animal experiments have proven that islet transplantation can increase the body and muscle weight of diabetic rats, more effectively reduce proteinuria, significantly improve the conduction velocity of the tail nerve, restore thermal and ameliorate mechanical nociceptive thresholds, and improve the residual β-cell state in the recipient pancreas (4). In a clinical control experiment, islet transplantation slowed the progression of diabetic microvascular complications, such as a declining renal glomerular filtration rate and retinal changes (5); compared with no transplantation, islet transplantation resulted in near-normal platelet activation and prothrombotic factor levels, cerebral metabolism and function, and neuropsychological test results (6).

The most common and ideal transplantation route is through the transhepatic portal vein (7). Nevertheless, islet cells implanted in the portal vein are often lost within three days due to hypoxia, nutritional deficiency, the mechanical pressure of the portal vein, and the immediate blood-mediated inflammatory response (IBMIR). The early loss of implanted islets is an essential factor in the failure to achieve insulin independence in single transplant patients. The IBMIR was first proposed by the W Bennet team in 1999 and was validated in in vivo and in vitro experiments. The reaction is defined as a rapid, strong, and nonspecific immune inflammatory response induced by tissue factors exposed on the surfaces of islets, with characteristics such as platelet activation, aggregation, coagulation, and complement system activation, as well as an infiltration of neutrophils, monocytes and macrophages and release of inflammatory factors. Usually, within 15 minutes after transplantation, the islets are encircled by a thrombus; after 1 hour, the islets undergo massive apoptosis due to the infiltration of white blood cells (8). This reaction also leads to early apoptosis of autologous islets in patients who undergo total pancreatectomy and autologous islet transplantation (9). At present, the standard clinical therapy relies on anticoagulants such as heparin, soluble complement receptor 1, and protease inhibitors such as ulinastatin. However, the IBMIR is a complex reaction involving multiple factors, and 50-70% of transplanted islets still undergo early apoptosis due to the IBMIR in existing therapeutic schemes. Therefore, the inhibition of the IBMIR to avoid early apoptosis of transplanted islets is an urgent issue in clinical work (10).

Because macrophages are essential in the IBMIR and subsequent adaptive immunity and are plastic and heterogeneous in inflammatory reactions (11, 12), This article reviews the strategy of innate immune regulation with macrophages as the primary target in recent years in islet transplantation to provide references for subsequent basic research and improve the survival rate of islets in clinical islet transplantation ( Tables 1 , 2 ).

Clinical islet transplantation requires four steps: perfusion of the donor pancreas, digestion of the pancreas to separate the islets from exocrine glands, purification of the islets, and transplantation into recipients through the portal vein (7). When the prepared islets are infused into patients via the portal vein, it will trigger IBMIR.

IBMIR is initiated by a strong activation of the coagulation cascade. After contact with blood in the portal vein, islet tissue factor expression induces the extrinsic coagulation pathway. The negative charge on the islet surface triggers the intrinsic coagulation pathway. At the same time, islets secrete inflammatory factors such as IL-8 and MCP-1, which have chemotactic and proinflammatory effects on macrophages and neutrophils (46, 47).

Activated platelets may attach through binding to extracellular matrix (ECM) and collagen on the surface of the islet. Meanwhile, owing to the fast and transient expression of p-selectin on the membrane of activated platelet alpha granules and vascular endothelial Weibel-Palade bodies, the p-selectin lectin-like domain binds to sialyl Lewis x and the p-selectin glycoprotein ligand 1 in the neutrophils and mononuclear cells, thus mediating the rolling of neutrophils and mononuclear cells on the endothelial cell surface and the adhesion of neutrophils and mononuclear cells to platelets (48, 49). On the other hand, vascular endothelial cells secrete IL-6 and IL-8 to promote the aggregation of neutrophils and macrophages (46).

Complement activation is triggered by the natural immunoglobulins IgG and IgM. When isolated islets are exposed to blood, the complement system is quickly activated, leading to the lysis of islet cells. At the same time, the production of the allergic toxins C3a and C5a further induces the aggregation of macrophages and neutrophils and promotes mononuclear cells to release cytokines such as IL-1, IL-6, IL-8, and TNF-α (50).

Granulocytes appear 8 hours after islet transplantation and extensively infiltrate the transplants after 12 hours. Neutrophils are predominant members of the granulocyte family and the first line of defense of innate immunity. They contain many cytokines, which are released when activated and have destructive effects on islets; neutrophils make significant contributions to the activation and recruitment of macrophages in acute inflammation sites. After activation, they produce various chemokines to attract mononuclear cells and macrophages; in addition, the infiltration of neutrophils leads to the release of cytokines from T cells and macrophages, such as tumor necrosis factor α (TNF-α) and macrophage inflammatory protein 1α. The mobilization of this immune effector could not only expand IBMIR but also induce subsequent adaptive immunity, inducing and enhancing cellular rejection responses (50, 51) ( Figure 1 ).

The gathered macrophages continue to secrete cytokines such as IL-6 and IL-8 to maintain the inflammatory response and release proinflammatory factors such as IL-1β, IFN-γ, and TNF-α. The IL-1β secreted by macrophages and neutrophils binds to the IL-1β receptor on the surface of islet cells, activating the IL-1 receptor-associated kinase to activate TNF receptor-associated factor 6, which leads to the phosphorylation and degradation of IκB, releasing NF-κB from the inhibitory IκB, and entering the nucleus of cells to regulate the transcription of various genes, including IL-1, IL-6, TNF-α and iNOS. The TNF-α produced by macrophages and islet cells binds to the TNF receptor, activating the NF-kB and MAPK pathways and inducing cell apoptosis. Apoptosis is activated by the activation of caspase-3, mediated by the MAPK pathway, or by activating effector caspases, including caspase-3, which FADD mediates. The interferon-γ (IFN-γ) produced by macrophages binds to IFN-γ receptors on the surface of the islets, activating JAK1 and JAK2. Activated JAK2 activates signal transducers and activators of transcription 1 (STAT1). Then, STAT1 is transferred to the nucleus for gene regulation, eventually leading to islet apoptosis. The apoptosis-promoting effect of STAT1 may be partially mediated by the activation of caspase-2, caspase-3 and caspase-7 (52). Under the combined effects of the cytokines IL-1β, TNF-α, and IFN-γ, overexpression of iNOS in β cells and macrophages leads to excessive NO synthesis. Subsequently, NO loses an electron to combine with superoxide free radicals, forming a highly active free radical peroxynitrite (ONOO-). The cytotoxicity of ONOO- then induces apoptosis in islet cells. On the other hand, macrophages play an antigen-presenting role, promoting the activation of T cells into CD8+ T cells and CD4+ T cells. Activated T cells produce cytokines such as IFN-γ, TNF-α, and lymphotoxin, thereby inducing β-cell apoptosis. T cells also express Fas receptor-associated ligands and TNF-related apoptosis-inducing ligands, activating effector caspases to cause cellular apoptosis. In addition, CD8+ T cells directly contact and promote the release of granzyme B into the cytoplasm of target cells through perforin, thereby activating nucleases and caspases to kill target cells (11, 50, 53).

Macrophages play crucial roles in the IBMIR after islet transplantation. Generally, they aggregate around the islets at 8 hours and infiltrate them at 12 hours posttransplantation. By 24 hours posttransplantation, the transplanted islets are entirely infiltrated (51). In islet transplantation, macrophages usually come from three sources: Kupffer cells (KCs) in the recipient’s liver, mononuclear macrophages from the recipient’s bone marrow, and macrophages resident in the donor’s islets.

For donor-derived macrophages, on the one hand, islet-resident macrophages mediate the production of islet interleukin (IL)-1β and impair the function of beta cells induced by islet amyloid-like polypeptides (54). On the other hand, some studies have shown that during islet compensation in the early stage of diabetes, islet-resident macrophages contribute to angiogenesis by supporting islet vascular endothelial growth factor A (VEGF-A) secretion during islet remodeling, suggesting their critical role in supporting islet compensation during diabetes (55). Further animal experiments confirmed that in a mouse autoimmune diabetes model, islet resident macrophages showed different phenotypes, such as maturation, self-replication, proinflammation, and immune tolerance, throughout the disease course, demonstrating heterogeneity in the inflammatory process (56). Although the number of donor-derived macrophages is usually small, their diverse functions in islet cells during the diabetes stage prove their importance. However, relevant research on donor-derived macrophages in islet transplantation is still lacking.

KCs are resident macrophages in the liver. KCs in the sinusoids can phagocytose pathogens from the arterial and venous systems, playing an essential role in innate immunity. Single-cell RNA sequencing analysis of freshly isolated human liver demonstrates the presence of two distinct intrahepatic CD68+ macrophage subsets in the steady state: one is an inflammatory macrophage subset enriched in the expression of LYZ, CSTA, and CD74; the other is a tolerogenic macrophage subset distinguished by high expression of CD5L, MARCO, and VSIG4. Two distinct cell populations under the same lipopolysaccharide (LPS)/IFN-γ stimulation conditions showed that the tolerogenic function subset secreted less TNF-α (57). In another study, F4/80+ KCs in the mouse liver were divided into CD68+ subsets with phagocytic activity and CD11b+ subsets with cytokine generation capability (58). KCs preferentially induce tolerogenic immunity under noninflammatory conditions and perceive the condition of liver tissue, and their response to environmental changes plays an essential role in the pathogenesis of liver diseases (59). In the development of alcohol-related liver disease, KCs induce oxidative stress and inflammation in the liver and promote the progression of alcohol-related liver disease by participating in reactive oxygen species (ROS) production and activating pathways leading to cytokine and chemokine production. The liver stellate cells activated by KCs contribute to the progression of liver fibrosis. In islet transplantation, implanted islet cells, acinar cells, and secreted soluble factors activate KCs to secrete proinflammatory cytokines, such as IL-1β and TNF-α. The inhibition of KCs may extend the survival of implanted islets (60, 61). In summary, KCs exhibit significant heterogeneity and strong plasticity under physiological and inflammatory conditions.

Monocytes are generally thought to be derived from myeloid progenitors derived from pluripotent hematopoietic stem cells in the bone marrow. Monocytes further differentiate into dendritic cells, macrophages, and osteoclasts. In humans, monocytes can be divided into the CD14hiCD16- classic subtype, which is mainly responsible for innate perception, immune response, migration, and differentiation into macrophages at the injury site, and CD14loCD16+ nonclassic monocytes, which are mainly responsible for vessel system monitoring and tissue repair. The two subtypes are analogous to the CX3CR1intCCR2+CD62L+CD43loLy6Chi inflammatory subtype and CX3CR1hiCCR2-CD62L-CD43hiLy6Clo patrolling monocytes found in mouse tissues. In addition, a small number of CD14+CD16+ “intermediate” subtype monocytes are mainly responsible for antigen presentation and cytokine secretion during the immune response and play an essential role in the inflammatory cascade, also known as transitional inflammatory monocytes (62–64). Under inflammatory conditions, such as islet transplantation within the portal vein, islets and endothelial cells release inflammatory factors such as CCR2, recruiting “classic” monocytes out of the bone marrow to the inflammatory sites and differentiating into dendritic cells and inflammatory macrophages, producing TNF, iNOS and ROS to trigger and expand the inflammatory response; “nonclassic” monocytes usually differentiate into M2 immunomodulatory phenotype macrophages while suppressing inflammation, thus promoting the vascularization of the transplanted islets and allowing the graft to colonize the hepatic sinuses and survive for a long time to perform islet functions (41). As essential participants in the innate immune response, monocyte-derived macrophages are also highly plastic and show “cross-differentiation” under the influence of different environments, which has been confirmed in the inflammatory environment of different diseases (62). In islet transplantation, depleting dendritic cells derived from recipient monocytes can enhance the early graft function (65). Another study demonstrated that bone marrow-derived mononuclear cells can be cultured into spheroids, with CXCR4+CD31+ myeloid cells being the main cell components. In the islet transplantation model under the renal capsule of syngeneic mice, cotransplantation of bone marrow–derived spheroids improves the blood supply reconstruction and graft function (66). Although research on macrophages from different sources in innate immune response in the field of islet transplantation remains limited, existing studies have provided initial insights into the influence of monocytes with different functions on islet transplantation.

Due to the importance of macrophages in the immune response to islet transplants, research on macrophages has always been a popular topic in the field of islet transplantation, and considerable progress and achievements have been made. Most of them focus on traditional drug therapy, interstitial cell coculture or cotransplantation, the optimization of islet isolation and culture methods, islet modification and engineering before transplantation, etc. The most common and effective strategy is to directly regulate the innate immune response of the recipient using traditional drugs to reduce damage to the graft caused by immune cells, including macrophages.

Islet transplantation, an emerging organ transplantation technology, solves the problem of insufficient insulin secretion and dramatically improves the quality of life of diabetic patients. Inhibiting the proinflammatory effect of macrophages in the IBMIR directly or indirectly through different therapeutic means can significantly improve the survival rate of early grafts. However, macrophages have a “double-edged sword” role in the inflammatory response. M2 macrophages have the potential to promote the proliferation of β cells and, at the same time, play an active role in tissue repair and blood supply reconstruction of the graft (104). Simply inhibiting the infiltration of macrophages into grafts is not conducive to graft repair and the vascularization process. Regulating the M1/M2 phenotype of macrophages is the most prevalent treatment strategy currently employed, which can mitigate the detrimental effects of inflammatory responses on grafts without compromising the functional capacity of M2 macrophages. However, on the one hand, the potential inhibitory effects of this approach on subsets of cells that are beneficial to graft function remain unclear. On the other hand, many studies have confirmed the role of M2 macrophages in renal and pulmonary fibrosis. M2 macrophages promote the proliferation and activation of fibroblasts by secreting cytokines and differentiate into αSMA+ myofibroblasts through a process called macrophage-to-myofibrolast transition mediated by TGFβ1–Smad3 signaling. Therefore, it is uncertain whether regulating macrophage polarization will lead to fibrosis of transplanted islets (105–107). In the latest research, mice’s kidney macrophages were divided into monocyte-derived macrophages and kidney resident macrophages by single‐cell RNA sequencing technology after renal ischemia-reperfusion. More importantly, S100a9hiLy6chi monocyte subsets were successfully defined by analyzing tissues and performing RNA velocity analysis during disease progression after renal ischemia-reperfusion, which played a role in initiating and amplifying inflammatory injury throughout the acute phase of acute kidney injury, and were verified in tissue samples from clinical patients. In the mouse model, targeting this subgroup to block S100a8/a9 signaling can effectively prevent acute kidney injury caused by ischemia-reperfusion (108). Further exploration of the heterogeneity of macrophages in inflammation and the targeting of different functional subsets to obtain the most ideal immune tolerance or modulation strategies are promising research directions.

Intraportal islet transplantation is the most common route of islet transplantation in clinical practice. However, in animal experiments, there are other sites that could be used, such as the renal capsule, peritoneal cavity and anterior chamber. These sites may mitigate the IBMIR by reducing islet contact with blood. For example, for patients with T1DM and End-Stage Renal Disease, a new transplantation strategy is to transplant prevascularized islets under the renal capsule of the donor’s kidney. In the non-human primate model, this strategy will not affect the renal function of the donor’s kidney, and it results in better islet function compared to Intraportal transplantation and renal capsule transplantation without prevascularization. Maintaining proper islet function benefits the long-term survival of the donor kidney (109). Compared with the cotransplantation of islets and MSCs, another popular area of research in recent years is the transplantation of islets into MSC-rich tissues. Brown adipose tissue (BAT) is densely vascularized and innervated and is rich in MSCs, M2 macrophages, and immunosuppressive regulatory T cells. BAT is a potential efficacious site for islet transplantation (110, 111). Bone marrow is associated with advantages similar to those of BAT (112). Spleen, an organ rich in MSC, has unique advantages as a transplantation site. On the one hand, insulin produced by transplanted islets can flow into the portal vein through the splenic vein, which is closer to physiological insulin release profiles. On the other hand, spleen has the potential to promote islet regeneration. Although some clinical transplantation centers have performed intrasplenic islet transplantation, there is a risk of arteriovenous thrombosis and subcapsular hematoma (113). A more ideal transplantation site and microenvironment are conducive to the survival and function of the graft. Although these sites have different advantages, none can meet the characteristics of minimal trauma, rich blood supply, and an immune-tolerant environment; at the same time, their safety and effectiveness still need to be verified. Additionally, they are associated with problems such as poor oxygenation and inconsistent outcomes in rodent and large animal islet transplantation models (114). Therefore, exploring new transplantation sites still requires a significant amount of time.

The strategy of combining medical and materials chemistry/engineering techniques can obtain outstanding research results in the field. Islet microencapsulation technology has been a popular research direction in recent years, and the ability of systems created with this technology to carry drugs and release drugs locally highlights its superiority, especially in the field of xenogeneic islet transplantation. Such systems can significantly reduce macrophage-mediated islet destruction and protect graft function. However, pericapsular fibrotic overgrowth is the main problem in this field, and proinflammatory and anti-inflammatory macrophages may mediate this adverse reaction. Only by clarifying the action mechanism of macrophages can we further promote the application of islet microencapsulation technology in clinical work (115). Another advantage of materials chemistry as a delivery system lies in its ability to enhance drug delivery and tissue absorption, while its specific absorption by target tissues solves the side effects of some drugs when administered systemically (116). The strategy of utilizing nano-materials to target liver tissues and regulate the maturation, activation, and polarization of macrophages within the liver has lately garnered significant attention in academic circles.

With the progression of single-cell sequencing and spatial transcriptomics technologies, the identification of previously unknown cell subsets with distinct functions has become feasible. In the future, through a deeper understanding of the role of macrophages in the inflammatory response and the continuous optimization of corresponding immunomodulatory techniques, a more suitable microenvironment for graft survival and function can be constructed, and the prognosis of patients with islet transplants can be gradually improved.

KD: Writing – original draft. JL: Writing – original draft. JZ: Writing – review & editing. TC: Writing – original draft. HL: Writing – original draft. FL: Writing – original draft. ZL: Writing – review & editing. BG: Writing – original draft. SW: Writing – review & editing. FW: Writing – review & editing.