Immune checkpoint inhibitors (ICIs) are successful in treating many cancers but may cause immune-related adverse events. ICI-mediated myocarditis has a high fatality rate with severe cardiovascular consequences. Targeted therapies for ICI myocarditis are currently limited.

We used a genetic mouse model of PD1 deletion (MRL/Pdcd1-/-) along with a novel drug-treated ICI myocarditis mouse model to recapitulate the disease phenotype. We performed single-cell RNA-sequencing, single-cell T-cell receptor sequencing, and cellular indexing of transcriptomes and epitopes on immune cells isolated from MRL and MRL/Pdcd1-/- mice at serial time points. We assessed the impact of macrophage deletion in MRL/Pdcd1-/- mice, then inhibited CXCR3 (C-X-C motif chemokine receptor 3) in ICI-treated mice to assess the therapeutic effect on myocarditis phenotype. Furthermore, we delineated the functional and mechanistic effects of CXCR3 blockade on T-cell and macrophage interactions. We then correlated the results in human single-cell multiomics data from blood and heart biopsy data from patients with ICI myocarditis.

Single-cell multiomics demonstrated expansion of CXCL (C-X-C motif chemokine ligand) 9/10+CCR2+ macrophages and CXCR3hi (C-X-C motif chemokine receptor 3 high-expressing) CD8+ (cluster of differentiation) effector T lymphocytes in the hearts of MRL/Pdcd1-/- mice correlating with onset of myocarditis development. Both depletion of CXCL9/10+CCR2+ (C-C motif chemokine receptor) macrophages and CXCR3 blockade, respectively, led to decreased CXCR3hi CD8+ T-cell infiltration into the heart and significantly improved survival. Transwell migration assays demonstrated that the selective blockade of CXCR3 and its ligand, CXCL10, reduced CXCR3+CD8+ T-cell migration toward macrophages, implicating this interaction in T-cell cardiotropism toward cardiac macrophages. Furthermore, cardiomyocyte apoptosis was induced by CXCR3hi CD8+ T cells. Cardiac biopsies from patients with confirmed ICI myocarditis demonstrated infiltrating CXCR3+ T cells and CXCL9+/CXCL10+ macrophages. Both mouse cardiac immune cells and patient peripheral blood immune cells revealed expanded TCRs (T-cell receptors) correlating with CXCR3hi CD8+ T cells in ICI myocarditis samples.

These findings bring forth the CXCR3-CXCL9/10 axis as an attractive therapeutic target for ICI myocarditis treatment, and more broadly as a druggable pathway in cardiac inflammation.

For successful transmission, the malaria parasite must traverse tissue epithelia and survive attack from the insect's innate immune system. Hemocytes play a multitude of roles in mosquitoes, including defense against invading pathogens. Here, we show that hemocytes of the major malaria vector Anopheles gambiae promote Plasmodium falciparum infection by maintaining midgut epithelial integrity by controlling cell proliferation upon blood feeding. The mosquito's hemocytes also control the midgut microbiota and immune gene expression. Our study unveils novel hemocyte functions that are exploited by the human malaria parasite to evade the mosquito's immune system.

Macrophages play an important role in disease progression of pediatric cholestatic liver disease, particularly biliary atresia (BA); however, the restorative versus pathogenic role for precise macrophage subsets remains poorly defined. We aimed to distinguish the transcriptional profiles and roles of defined macrophage subset(s) in murine BA.

We used multiparameter flow cytometry and RNA-sequencing analysis to profile recruited CD11bhiCD64+ hepatic macrophages by cell surface expression of MHCII and Ly6c in the Rhesus rotavirus (RRV)-induced murine model of BA versus saline controls. Modulation of macrophage numbers via intra-peritoneal injections of clodronate-loaded liposomes was performed to determine the association between macrophage numbers and histologic injury (Ishak score).

Ly6c+ macrophages demonstrated the greatest increase in numbers and percent of total macrophages in murine BA versus saline controls whereas MHCII+ macrophages decreased. Transcriptional changes in murine BA MHCII+ macrophages included reduced expression of the Kupffer cell gene signature, lower expression of genes involved in homeostatic processes, and increased expression of genes involved in inflammatory processes. Ly6c+ macrophages in murine BA showed increased expression for Hif1a and other genes involved in the cellular response to hypoxia. Among all subsets, the number of Ly6c+ macrophages exhibited the strongest correlation with severity of histologic liver injury by Ishak score.

Our data identify specific pathways upregulated in Ly6c vs MHCII+ macrophage subsets in murine BA. Transcriptional similarities between murine BA and human cholestatic macrophages may enable translation of future mechanistic studies to new macrophage subset-specific therapies.

Targeted delivery of therapeutic agents is a persistent challenge in modern medicine. Recent efforts in this area have highlighted the utility of extracellular vesicles (EVs) as drug carriers, given that they naturally occur in bloodstream and tissues, and can be loaded with a wide range of therapeutic molecules. However, biodistribution and tissue tropism of EVs remain difficult to study systematically. Here, a multiplexed approach is developed for simultaneous tracking of EVs from various cell lines within a single in vivo experiment. EVs are used from 16 different cell lines, and through controlled fusion with lipid nanoparticles (LNPs) carrying single-stranded DNA barcodes, uniquely barcoded hybrid EV particle (hEV) library is generated. These hEVs are combined for a multiplexed in vivo biodistribution profiling in mice, and discovered that HAP1-derived hEVs demonstrated lung tropism, suggesting that these hEVs may be used for targeted drug delivery into lung tissue. To examine this possibility further, it is shown that HAP1 hEV loaded with Cre mRNA displayed functional delivery to the lungs. Overall, the barcoded hEV technology enables rapid profiling of biodistribution across EV cell sources, which is poised to improve throughput and extent of EV studies, while reducing the number of animals required for research.

During viral infection, phagocytic cells participate in numerous immunological processes. A common approach to elucidate the specific contributions of phagocytic cells is through direct comparison of viral pathogenesis of phagocyte-depleted and phagocyte-intact animals. Clodronate liposomes are a versatile and simple means of depleting phagocytes in any animal model. Selection of clodronate liposome route of delivery can yield systemic or local phagocyte depletion as needed. Here we describe the application of clodronate liposome-mediated depletion of blood-contacting phagocytes to investigate their interactions with La Crosse virus (or other bunyaviruses) in a reductionist model of viremia.

Oleuropein (OLP) holds promise as a therapeutic candidate for both Plasmodium falciparum infection and cancer. It modulates the phosphoinositide 3-kinase (PI3K)-Akt1 signaling pathway to regulate inflammation and restore immune homeostasis. Moreover, it influences the cell death/autophagy axis, along with increasing the antimalarial efficacy of artemisinin. Our findings indicate that the anti-breast-cancer effect of OLP could be mediated by regulating the balance of T helper 17 and regulatory T cells. Additionally, we discuss the use of hematopoietic-stem-cell-transplanted immunodeficient mice with a humanized immune system for validating the antimalarial activity, autophagy and anticancer activity of OLP.

Microbial molecules translocated from the intestinal lumen into the host's internal environment play a role in various physiological functions. Previously, we identified that butyrate, a short-chain fatty acid produced by intestinal bacteria, lipoteichoic acid, a cell wall component of gram-positive bacteria, and lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, induce sleep when their naturally occurring translocation is mimicked by direct delivery into the portal vein. Our findings suggested that these microbial molecules exert their sleep-promoting effects within the hepatoportal region. In the present experiments, we tested the hypothesis that resident liver macrophages, known as Kupffer cells, play a crucial role in the LPS-responsive, sleep-promoting mechanisms within the hepatoportal region. Intraportal administration of LPS induced increased sleep and fever in control rats. Remarkably, in Kupffer cell-depleted animals, both of these responses were significantly suppressed. These findings highlight the potential role of Kupffer cells in mediating the non-rapid-eye movement sleep-promoting and febrile effects of LPS translocated from the intestinal microbiota into the portal circulation. The strategic location of Kupffer cells within the hepatoportal region, coupled with their ability to rapidly take up LPS and other microbial molecules, together with their high secretory activity of multiple signaling molecules, underlie their key role in the communication between the intestinal microbiota and the brain.

Pulmonary surfactant is a lipoprotein complex lining the alveolar surface to decrease the surface tension and facilitate inspiration. Surfactant deficiency is often seen in premature infants and in children and adults with respiratory distress syndrome. Mechanical stretch of alveolar type 2 epithelial (AT2) cells during lung expansion is the primary physiological factor that stimulates surfactant secretion; however, it is unclear whether there is a mechanosensor dedicated to this process. Here, we show that loss of the mechanosensitive channels TMEM63A and TMEM63B (TMEM63A/B) resulted in atelectasis and respiratory failure in mice due to a deficit of surfactant secretion. TMEM63A/B were predominantly localized at the limiting membrane of the lamellar body (LB), a lysosome-related organelle that stores pulmonary surfactant and ATP in AT2 cells. Activation of TMEM63A/B channels during cell stretch facilitated the release of surfactant and ATP from LBs fused with the plasma membrane. The released ATP evoked Ca2+ signaling in AT2 cells and potentiated exocytic fusion of more LBs. Our study uncovered a vital physiological function of TMEM63 mechanosensitive channels in preparing the lungs for the first breath at birth and maintaining respiration throughout life.

Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by severe inflammation and pulmonary dysfunction. Despite advancements in critical care, effective pharmacological interventions for ARDS remain elusive. While Janus kinase 2 (JAK2) inhibitors have emerged as an innovative treatment for numerous autoinflammatory diseases, their therapeutic potential in ARDS remains unexplored. In this study, we investigated the contribution of JAK2 and its underlying mechanisms in ARDS utilizing myeloid-specific JAK2 knockout murine models alongside a pharmacological JAK2 inhibitor. Notably, myeloid-specific JAK2 knockout led to a notable attenuation of ARDS induced by intratracheal administration of LPS, accompanied by reduced levels of neutrophils and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and lung tissue. Intriguingly, the ameliorative effects were abolished upon the depletion of monocyte-derived alveolar macrophages (Mo-AMs) rather than tissue-resident alveolar macrophages (TR-AMs). JAK2 deficiency markedly reversed LPS-induced activation of STAT5 in macrophages. Remarkably, pharmacological JAK2 inhibition using baricitinib failed to substantially alleviate neutrophils infiltration, implying that specific inhibition of JAK2 in Mo-AMs is imperative for ARDS amelioration. Collectively, our data suggest that JAK2 may mitigate ARDS progression through the JAK2 pathway in Mo-AMs, underscoring JAK2 in alveolar macrophages, particularly Mo-AMs, as a promising therapeutic target for ARDS treatment.

Recently approved adeno-associated viral (AAV) vectors for liver monogenic diseases haemophilia A and B are exemplifying the success of liver-directed viral gene therapy. In parallel, additional gene therapy strategies are rapidly emerging to overcome some inherent AAV limitations, such as the non-persistence of the episomal transgene in the rapidly growing liver and immune response. Viral integrating vectors such as in vivo lentiviral gene therapy and non-viral vectors such as lipid nanoparticles encapsulating mRNA (LNP-mRNA) are rapidly being developed, currently at the preclinical and clinical stages, respectively. Macrophages are the first effector cells of the innate immune response triggered by gene therapy vectors. Macrophage uptake and activation following administration of viral gene therapy and LNP have been reported. In this study, we assessed the biodistribution of AAV, lentiviral, and LNP-mRNA gene therapy following the depletion of tissue macrophages by clodronate pre-treatment in neonatal and juvenile mice. Both neonatal and adult clodronate-treated mice showed a significant increase in lentiviral-transduced hepatocytes. In contrast, clodronate pre-treatment did not modify hepatocyte transduction mediated by hepatotropic AAV8 but reduced LNP-mRNA transfection in neonatal and juvenile animals. These results highlight the importance of age-specific responses in the liver and will have translational applications for gene therapy programs.

Pancreatic islets are 3D micro-organs that maintain β-cell functionality through cell-cell and cell-matrix communication. While primary islets, the gold standard for in vitro models, have a short culture life of approximately 1-2 weeks, we developed a novel protocol that employs reformed islets following dispersion coupled with a fine-tuned culture environment. Reformed islets exhibit physiological characteristics similar to primary islets, enabling high-resolution imaging and repeated functional assessment. Unlike other in vitro platforms, reformed islets retain an immune population, allowing the study of interactions between β cells and resident and infiltrating immune cells. Analyses showed that reformed islets have a similar composition and cytoarchitecture to primary islets, including macrophages and T cells, and can secrete insulin in response to glucose. Reformed islets exhibited partial dedifferentiation compared to native islets but were otherwise transcriptionally similar. The reformed islets offer a useful platform for studying diabetes pathology and can recapitulate both T1DM and T2DM disease milieus, providing an advantage over other models, such as mouse and human β-cell lines, which lack the input of non-β-endocrine cells and immune cell crosstalk.

Breast disseminated cancer cells (DCCs) can remain dormant in the lungs for extended periods, but the mechanisms limiting their expansion are not well understood. Research indicates that tissue-resident alveolar macrophages suppress breast cancer metastasis in lung alveoli by inducing dormancy. Through ligand-receptor mapping and intravital imaging, it was found that alveolar macrophages express transforming growth factor (TGF)-β2. This expression, along with persistent macrophage-cancer cell interactions via the TGF-βRIII receptor, maintains cancer cells in a dormant state. Depleting alveolar macrophages or losing the TGF-β2 receptor in cancer cells triggers metastatic awakening. Aggressive breast cancer cells are either suppressed by alveolar macrophages or evade this suppression by avoiding interaction and downregulating the TGF-β2 receptor. Restoring TGF-βRIII in aggressive cells reinstates TGF-β2-mediated macrophage growth suppression. Thus, alveolar macrophages act as a metastasis immune barrier, and downregulation of TGF-β2 signaling allows cancer cells to overcome macrophage-mediated growth suppression.

Regenerative medicine and transplantation science continuously seek methods to circumvent immune-mediated rejection and promote tissue regeneration. Sertoli cells, with their inherent immunoprotective properties, emerge as pivotal players in this quest. However, whether Sertoli cells can play immunomodulatory role in tadpole tail regeneration and can thus benefit the regeneration process are needed to be discovered.

Immature Sertoli cells from Xenopus tropicalis (XtiSCs) were transplanted into X. tropicalis tadpoles, followed by the amputation of the final third of their tails. We assessed the migration of XtiSCs, tail regeneration length, muscle degradation and growth, and macrophage counts across various regions including the entire tail, tail trunk, injection site, and regeneration site. The interactions between XtiSCs and macrophages were examined using a confocal microscope. To deplete macrophages, clodronate liposomes were administered prior to the transplantation of XtiSCs, while the administration of control liposomes acted as a negative control. Student's t-test was used to compare the effects of XtiSCs injection to those of a 2/3PBS injection across groups with no liposomes, control liposomes, and clodronate liposomes.

XtiSCs have excellent viability after transplantation to tadpole tail and remarkable homing capabilities to the regeneration site after tail amputation. XtiSCs injection increased macrophage numbers at 3 days post-amputation and 5 days post-amputation in the tail trunk, specifically at the injection site and at the regenerated tail, in a macrophage depleted environment (clodronate-liposome injection). What's more, XtiSCs injection decreased muscle fibers degradation significantly at 1 day post-amputation and facilitated new muscle growth significantly at 3 days post-amputation. In addition, whole-mount immunostaining showed that some XtiSCs co-localized with macrophages. And we observed potential mitochondria transport from XtiSCs to macrophages using MitoTracker staining in tadpole tail.

Our study delineates the novel role of XtiSCs in facilitating muscle regeneration post tadpole tail amputation, underscoring a unique interaction with macrophages that is crucial for regenerative success. This study not only highlights the therapeutic potential of Sertoli cells in regenerative medicine but also opens avenues for clinical translation, offering insights into immunoregulatory strategies that could enhance tissue regeneration and transplant acceptance.

Recurrent breast cancers often develop resistance to standard-of-care therapies. Identifying targetable factors contributing to cancer recurrence remains the rate-limiting step in improving long-term outcomes. In this study, we identify tumor cell-derived osteopontin as an autocrine and paracrine driver of tumor recurrence. Osteopontin promotes tumor cell proliferation, recruits macrophages, and synergizes with IL-4 to further polarize them into a pro-tumorigenic state. Macrophage depletion and osteopontin inhibition decrease recurrent tumor growth. Furthermore, targeting osteopontin in primary tumor-bearing female mice prevents metastasis, permits T cell infiltration and activation, and improves anti-PD-1 immunotherapy response. Clinically, osteopontin expression is higher in recurrent metastatic tumors versus female patient-matched primary breast tumors. Osteopontin positively correlates with macrophage infiltration, increases with higher tumor grade, and its elevated pathway activity is associated with poor prognosis and long-term recurrence. Our findings suggest clinical implications and an alternative therapeutic strategy based on osteopontin's multiaxial role in breast cancer progression and recurrence.

Dental implant osseointegration comprises two types of bone formation-contact and distance osteogenesis-which result in bone formation originating from the implant surface or bone edges, respectively. The physicochemical properties of the implant surface regulate initial contact osteogenesis by directly tuning the osteoprogenitor cells in the peri-implant environment. However, whether these implant surface properties can regulate osteoprogenitor cells distant from the implant remains unclear. Innate immune cells, including neutrophils and macrophages, govern bone metabolism, suggesting their involvement in osseointegration and distance osteogenesis. This narrative review discusses the role of innate immunity in osseointegration and the effects of implant surface properties on distant osteogenesis, focusing on innate immune regulation.

The role of innate immunity in bone formation and the effects of implant surface properties on innate immune function were reviewed based on clinical, animal, and in vitro studies.

Neutrophils and macrophages are responsible for bone formation during osseointegration, via inflammatory mediators. The microroughness and hydrophilic status of titanium implants have the potential to alleviate this inflammatory response of neutrophils, and induce an anti-inflammatory response in macrophages, to tune both contact and distance osteogenesis through the activation of osteoblasts. Thus, the surface micro-roughness and hydrophilicity of implants can regulate the function of distant osteoprogenitor cells through innate immune cells.

Surface modification of implants aimed at regulating innate immunity may be useful in promoting further osteogenesis and overcoming the limitations encountered in severe situations, such as early loading protocol application.

Anopheles mosquitoes are the sole vector of human malaria, the most burdensome vector-borne disease worldwide. Strategies aimed at reducing mosquito populations and limiting their ability to transmit disease show the most promise for disease control. Therefore, gaining an improved understanding of mosquito biology, and specifically that of the immune response, can aid efforts to develop new approaches that limit malaria transmission. Here, we use a genome-wide CRISPR screening approach for the first time in mosquito cells to identify essential genes in Anopheles and identify genes for which knockout confers resistance to clodronate liposomes, which have been widely used in mammals and arthropods to ablate immune cells. In the essential gene screen, we identified a set of 1280 Anopheles genes that are highly enriched for genes involved in fundamental cell processes. For the clodronate liposome screen, we identified several candidate resistance factors and confirm their roles in the uptake and processing of clodronate liposomes through in vivo validation in Anopheles gambiae, providing new mechanistic detail of phagolysosome formation and clodronate liposome function. In summary, we demonstrate the application of a genome-wide CRISPR knockout platform in a major malaria vector and the identification of genes that are important for fitness and immune-related processes.

Engineering nanovaccines capable of targeting dendritic cells (DCs) is desperately required to maximize antigen cross-presentation to effector immune cells, elicit strong immune responses, and avoid adverse reactions. Here, we showed that glucose transporter 1 (Glut-1) on DCs is a reliable target for delivering antigens to DCs, and thus, a versatile antigen delivery strategy using glucosylated nanovaccines was developed for DC-targeted antigen delivery and tumor immunotherapy. The developed glucosylated ovalbumin-loaded nanovaccines highly accumulated in lymph nodes and efficiently engaged with Glut-1 on DCs to accelerate intracellular antigen delivery and promote DC maturation and antigen presentation, which elicited potent antitumor immunity to prevent and inhibit ovalbumin-expressing melanoma. Moreover, immunotherapeutic experiments in DC- and macrophage-depleted animal models confirmed that the glucosylated nanovaccines functioned mainly through DCs. In addition, the neoantigen-delivering glucosylated nanovaccines were further engineered to elicit tumor-specific immune responses against MC38 tumors. This study offers a DC-targeted antigen delivery strategy for cancer immunotherapy.

Immune checkpoint blockade has led to breakthroughs in the treatment of advanced gastric cancer. However, the prominent heterogeneity in gastric cancer, notably the heterogeneity of the tumor microenvironment, highlights the idea that the antitumor response is a reflection of multifactorial interactions. Through transcriptomic analysis and dynamic plasma sample analysis, we identified a metabolic "face-off" mechanism within the tumor microenvironment, as shown by the dual prognostic significance of nicotinamide metabolism. Specifically, macrophages and fibroblasts expressing the rate-limiting enzymes nicotinamide phosphoribosyltransferase and nicotinamide N-methyltransferase, respectively, regulate the nicotinamide/1-methylnicotinamide ratio and CD8+ T cell function. Mechanistically, nicotinamide N-methyltransferase is transcriptionally activated by the NOTCH pathway transcription factor RBP-J and is further inhibited by macrophage-derived extracellular vesicles containing nicotinamide phosphoribosyltransferase via the SIRT1/NICD axis. Manipulating nicotinamide metabolism through autologous injection of extracellular vesicles restored CD8+ T cell cytotoxicity and the anti-PD-1 response in gastric cancer.

Palatal expansion is a common way of treating maxillary transverse deficiency. Under mechanical force, the midpalatal suture is expanded, causing local immune responses. This study aimed to determine whether macrophages participate in bone remodeling of the midpalatal suture during palatal expansion and the effects on bone remodeling.

Palatal expansion model and macrophage depletion model were established. Micro-CT, histological staining, and immunohistochemical staining were used to investigate the changes in the number and phenotype of macrophages during palatal expansion as well as the effects on bone remodeling of the midpalatal suture. Additionally, the effect of mechanically induced M2 macrophages on palatal osteoblasts was also elucidated in vitro.

The number of macrophages increased significantly and polarized toward M2 phenotype with the increase of the expansion time, which was consistent with the trend of bone remodeling. After macrophage depletion, the function of osteoblasts and bone formation at the midpalatal suture were impaired during palatal expansion. In vitro, conditioned medium derived from M2 macrophages facilitated osteogenic differentiation of osteoblasts and decreased the RANKL/OPG ratio.

Macrophages through polarizing toward M2 phenotype participated in midpalatal suture bone remodeling during palatal expansion, which may provide a new idea for promoting bone remodeling from the perspective of regulating macrophage polarization.

Immunotherapy has become an important part of the oncotherapy arsenal. Its applicability in various cancer types is impressive, as well as its use of endogenous mechanisms to achieve desired ends. However, off-target or on-target-off-tumor toxicity, limited activity, lack of control in combination treatments and, especially for solid tumors, low local accumulation, have collectively limited clinical use thereof. These limitations are partially alleviated by delivery systems. Lipid-based nanoparticles (NPs) have emerged as revolutionary carriers due to favorable physicochemical characteristics, with specific applications and strengths particularly useful in immunotherapeutic agent delivery. The aim of this review is to highlight the challenges faced by immunotherapy and how lipid-based NPs have been, and may be further utilized to address such challenges. We discuss recent fundamental and clinical applications of NPs in a range of areas and provide a detailed discussion of the main obstacles in immune checkpoint inhibition therapies, adoptive cellular therapies, and cytokine therapies. We highlight how lipid-based nanosystems could address these through either delivery, direct modulation of the immune system, or targeting of the immunosuppressive tumor microenvironment. We explore advanced and emerging liposomal and lipid nanoparticle (LNP) systems for nucleic acid delivery, intrinsic and extrinsic stimulus-responsive formulations, and biomimetic lipid-based nanosystems in immunotherapy. Finally, we discuss the key challenges relating to the clinical use of lipid-based NP immunotherapies, suggesting future research directions for the near term to realize the potential of these innovative lipid-based nanosystems, as they become the crucial steppingstone towards the necessary enhancement of the efficacy of immunotherapy.

Bacterial infection involves a complex interaction between the pathogen and host where the outcome of infection is not solely determined by pathogen eradication. To identify small molecules that promote host survival by altering the host-pathogen dynamic, we conducted an in vivo chemical screen using zebrafish embryos and found that treatment with 3-hydroxykynurenine (3-HK) protects from lethal bacterial infection. 3-HK, a metabolite produced through host tryptophan metabolism, has no direct antibacterial activity but enhances host survival by restricting bacterial expansion in macrophages through a systemic mechanism that targets kainate-sensitive glutamate receptors. These findings reveal a new pathway by which tryptophan metabolism and kainate-sensitive glutamate receptors function and interact to modulate immunity, with important implications for the coordination between the immune and nervous systems in pathological conditions.

Hepatocellular carcinoma (HCC) is one of the cancers that seriously threaten human health. Immunotherapy serves as the mainstay of treatment for HCC patients by targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) axis. However, the effectiveness of anti-PD-1/PD-L1 treatment is limited when HCC becomes drug-resistant. Tumor-associated macrophages (TAMs) are an important factor in the negative regulation of PD-1 antibody targeted therapy in the tumor microenvironment (TME). Therefore, as an emerging direction in cancer immunotherapy research for the treatment of HCC, it is crucial to elucidate the correlations and mechanisms between TAMs and PD-1/PD-L1-mediated immune tolerance. This paper summarizes the effects of TAMs on the pathogenesis and progression of HCC and their impact on HCC anti-PD-1/PD-L1 immunotherapy, and further explores current potential therapeutic strategies that target TAMs in HCC, including eliminating TAMs in the TME, inhibiting TAMs recruitment to tumors and functionally repolarizing M2-TAMs (tumor-supportive) to M1-TAMs (antitumor type).

Cancer stem cells (CSCs) are a distinct subpopulation of cancer cells with the capacity to constantly self-renew and differentiate, and they are the main driver in the progression of cancer resistance and relapse. The tumor microenvironment (TME) constructed by CSCs is the "soil" adapted to tumor growth, helping CSCs evade immune killing, enhance their chemical resistance, and promote cancer progression.

We aim to elaborate the tight connection between CSCs and immunosuppressive components of the TME. We attempt to summarize and provide a therapeutic strategy to eradicate CSCs based on the destruction of the tumor ecological niche.

This review is focused on three main key concepts. First, we highlight that CSCs recruit and transform normal cells to construct the TME, which further provides ecological niche support for CSCs. Second, we describe the main characteristics of the immunosuppressive components of the TME, targeting strategies and summarize the progress of corresponding drugs in clinical trials. Third, we explore the multilevel insights of the TME to serve as an ecological niche for CSCs.

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are widely expressed by many types of retinal neurons and are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin and FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases acting through retinal neurons and MG "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed.

Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining.

Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired.

Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.

Hepatic fibrosis is a complex chronic liver disease in which both macrophages and hepatic stellate cells (HSCs) play important roles. Many studies have shown that clodronate liposomes (CLD-lipos) effectively deplete macrophages. However, no liposomes have been developed that target both HSCs and macrophages. This study aimed to evaluate the therapeutic efficacy of lipopolysaccharide-coupled clodronate liposomes (LPS-CLD-lipos) and the effects of liposomes size on hepatic fibrosis. Three rat models of hepatic fibrosis were established in vivo; diethylnitrosamine (DEN), bile duct ligation (BDL), and carbon tetrachloride (CCl4). Hematoxylin and eosin staining and serological liver function indices were used to analyze pathological liver damage. Masson's trichrome and Sirius red staining were used to evaluate the effect of liposomes on liver collagen fibers. The hydroxyproline content in liver tissues was determined. In vitro cell counting kit-8 (CCK-8) and immunofluorescence assays were used to further explore the effects of LPS modification and liposomes size on the killing of macrophages and HSCs. Both in vitro and in vivo experiments showed that 200 nm LPS-CLD-lipos significantly inhibited hepatic fibrosis and the abnormal deposition of collagen fibers in the liver and improved the related indicators of liver function. Further results showed that 200 nm LPS-CLD-lipos increased the clearance of macrophages and induced apoptosis of hepatic stellate cells, significantly. The present study demonstrated that 200 nm LPS-CLD-lipos could significantly inhibit hepatic fibrosis and improve liver function-related indices and this study may provide novel ideas and directions for hepatic fibrosis treatment.

Rapid uptake of nanoparticles by mononuclear phagocyte system (MPS) significantly hampers their therapeutic efficacy. Temporal MPS blockade is one of the few ways to overcome this barrier - the approach rediscovered many times under different names but never extensively used in clinic. Using meta-analysis of the published data we prove the efficacy of this technique for enhancing particle circulation in blood and their delivery to tumours, describe a century of its evolution and potential combined mechanism behind it. Finally, we discuss future directions of the research focusing on the features essential for successful clinical translation of the method.

Previous studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated.

Here we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery.

Macrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process.

Together, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

Acute injury in the airways or the lung activates local progenitors and stimulates changes in cell-cell interactions to restore homeostasis, but it is not appreciated how more distant niches are impacted. We utilized mouse models of airway-specific epithelial injury to examine secondary tissue-wide alveolar, immune, and mesenchymal responses. Single-cell transcriptomics and in vivo validation revealed transient, tissue-wide proliferation of alveolar type 2 (AT2) progenitor cells after club cell-specific ablation. The AT2 cell proliferative response was reliant on alveolar macrophages (AMs) via upregulation of Spp1 which encodes the secreted factor Osteopontin. A previously uncharacterized mesenchymal population we termed Mesenchymal Airway/Adventitial Niche Cell 2 (MANC2) also exhibited dynamic changes in abundance and a pro-fibrotic transcriptional signature after club cell ablation in an AM-dependent manner. Overall, these results demonstrate that acute airway damage can trigger distal lung responses including altered cell-cell interactions that may contribute to potential vulnerabilities for further dysregulation and disease.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Skeletal muscles exert remarkable regenerative or adaptive capacities in response to injuries or mechanical loads. However, the cellular networks underlying muscle adaptation are poorly understood compared to those underlying muscle regeneration. We employed single-cell RNA sequencing to investigate the gene expression patterns and cellular networks activated in overloaded muscles and compared these results with those observed in regenerating muscles. The cellular composition of the 4-day overloaded muscle, when macrophage infiltration peaked, closely resembled that of the 10-day regenerating muscle. In addition to the mesenchymal progenitor-muscle satellite cell (MuSC) axis, interactome analyses or targeted depletion experiments revealed communications between mesenchymal progenitors-macrophages and macrophages-MuSCs. Furthermore, granulin, a macrophage-derived factor, inhibited MuSC differentiation, and Granulin-knockout mice exhibited blunted muscle hypertrophy due to the premature differentiation of overloaded MuSCs. These findings reveal the critical role of granulin through the relayed communications of mesenchymal progenitors, macrophages, and MuSCs in facilitating efficient muscle hypertrophy.

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya, eastern- (EEEV), and Venezuelan- (VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology.