Specific imaging of CD8 + T-Cell dynamics with a nanobody radiotracer against human CD8ß.

PURPOSE: While immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8+ T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8+ T-cell dynamics. METHODS: Nanobodies targeting human CD8ß were generated by llama immunizations and subsequent biopanning. The lead anti-human CD8ß nanobody was characterized on binding, specificity, stability and toxicity. The lead nanobody was labeled with technetium-99m, gallium-68 and copper-64 for non-invasive imaging of human T-cell lymphomas and CD8+ T cells in human CD8 transgenic mice and non-human primates by SPECT/CT or PET/CT. Repeated imaging of CD8+ T cells in MC38 tumor-bearing mice allowed visualization of CD8+ T-cell dynamics. RESULTS: The nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8+ T cells were non-invasively visualized by SPECT and PET imaging in naïve and tumor-bearing mice and in naïve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8+ T cells and/or faster in vivo pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8+ T-cell dynamics already at early timepoints. CONCLUSION: This study describes the development of a more specific human CD8+ T-cell-targeting immunotracer, allowing follow-up of immunotherapy responses by non-invasive imaging of human CD8+ T-cell dynamics.

The role of RNA modifications in disease-associated macrophages.

In recent years, the field of epitranscriptomics has witnessed significant breakthroughs with the identification of more than 150 different chemical modifications in different RNA species. It has become increasingly clear that these chemical modifications play an important role in the regulation of fundamental processes linked to cell fate and development. Further interest was sparked by the ability of the epitranscriptome to regulate pathogenesis. However, despite the involvement of macrophages in a multitude of diseases, a clear knowledge gap exists in the understanding of how RNA modifications regulate the phenotype of these cells. Here, we provide a comprehensive overview of the known roles of macrophage RNA modifications in the context of different diseases.

Infection history imprints prolonged changes to the epigenome, transcriptome and function of Kupffer cells.

BACKGROUND & AIMS: Liver macrophages fulfill various homeostatic functions and represent an essential line of defense against pathogenic insults. However, it remains unclear whether a history of infectious disease in the liver leads to long-term alterations to the liver macrophage compartment. METHODS: We utilized a curable model of parasitic infection invoked by the protozoan parasite Trypanosoma brucei brucei to investigate whether infection history can durably reshape hepatic macrophage identity and function. Employing a combination of fate mapping, single-cell CITE-sequencing, single-nuclei multiome analysis, epigenomic analysis, and functional assays, we studied the alterations to the liver macrophage compartment during and after the resolution of infection. RESULTS: We show that T. brucei brucei infection alters the composition of liver-resident macrophages, leading to the infiltration of monocytes that differentiate into various infection-associated macrophage populations with divergent transcriptomic profiles. Whereas infection-associated macrophages disappear post-resolution of infection, monocyte-derived macrophages engraft in the liver, assume a Kupffer cell (KC)-like profile and co-exist with embryonic KCs in the long-term. Remarkably, the prior exposure to infection imprinted an altered transcriptional program on post-resolution KCs that was underpinned by an epigenetic remodeling of KC chromatin landscapes and a shift in KC ontogeny, along with transcriptional and epigenetic alterations in their niche cells. This reprogramming altered KC functions and was associated with increased resilience to a subsequent bacterial infection. CONCLUSION: Our study demonstrates that a prior exposure to a parasitic infection induces trained immunity in KCs, reshaping their identity and function in the long-term. IMPACT AND IMPLICATIONS: Although the liver is frequently affected during infections, and despite housing a major population of resident macrophages known as Kupffer cells (KCs), it is currently unclear whether infections can durably alter KCs and their niche cells. Our study provides a comprehensive investigation into the long-term impact of a prior, cured parasitic infection, unveiling long-lasting ontogenic, epigenetic, transcriptomic and functional changes to KCs as well as KC niche cells, which may contribute to KC remodeling. Our data suggest that infection history may continuously reprogram KCs throughout life with potential implications for subsequent disease susceptibility in the liver, influencing preventive and therapeutic approaches.

Q586B2 is a crucial virulence factor during the early stages of Trypanosoma brucei infection that is conserved amongst trypanosomatids.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.

Preclinical Evaluation of a Radiotheranostic Single-Domain Antibody Against Fibroblast Activation Protein α.

Fibroblast activation protein α (FAP) is highly expressed on cancer-associated fibroblasts of epithelial-derived cancers. Breast, colon, and pancreatic tumors often show strong desmoplastic reactions, which result in a dominant presence of stromal cells. FAP has gained interest as a target for molecular imaging and targeted therapies. Single-domain antibodies (sdAbs) are the smallest antibody-derived fragments with beneficial pharmacokinetic properties for molecular imaging and targeted therapy. Methods: We describe the generation, selection, and characterization of a sdAb against FAP. In mice, we assessed its imaging and therapeutic potential after radiolabeling with tracer-dose 131I and 68Ga for SPECT and PET imaging, respectively, and with 131I and 225Ac for targeted radionuclide therapy. Results: The lead sdAb, 4AH29, exhibiting picomolar affinity for a distinct FAP epitope, recognized both purified and membrane-bound FAP protein. Radiolabeled versions, including [68Ga]Ga-DOTA-4AH29, [225Ac]Ac-DOTA-4AH29, and [131I]I-guanidinomethyl iodobenzoate (GMIB)-4AH29, displayed radiochemical purities exceeding 95% and effectively bound to recombinant human FAP protein and FAP-positive GM05389 human fibroblasts. These radiolabeled compounds exhibited rapid and specific accumulation in human FAP-positive U87-MG glioblastoma tumors, with low but specific uptake in lymph nodes, uterus, bone, and skin (∼2-3 percentage injected activity per gram of tissue [%IA/g]). Kidney clearance of unbound [131I]I-GMIB-4AH29 was fast (<1 %IA/g after 24 h), whereas [225Ac]Ac-DOTA-4AH29 exhibited slower clearance (8.07 ± 1.39 %IA/g after 24 h and 2.47 ± 0.18 %IA/g after 96 h). Mice treated with [225Ac]Ac-DOTA-4AH29 and [131I]I-GMIB-4AH29 demonstrated prolonged survival compared with those receiving vehicle solution. Conclusion: [68Ga]Ga-DOTA-4AH29 and [131I]I-GMIB-4AH29 enable precise FAP-positive tumor detection in mice. Therapeutic [225Ac]Ac-DOTA-4AH29 and [131I]I-GMIB-4AH29 exhibit strong and sustained tumor targeting, resulting in dose-dependent therapeutic effects in FAP-positive tumor-bearing mice, albeit with kidney toxicity observed later for [225Ac]Ac-DOTA-4AH29. This study confirms the potential of radiolabeled sdAb 4AH29 as a radiotheranostic agent for FAP-positive cancers, warranting clinical evaluation.

Lipocalin-2: A Nurturer of Tumor Progression and a Novel Candidate for Targeted Cancer Therapy.

Within the tumor microenvironment (TME) exists a complex signaling network between cancer cells and stromal cells, which determines the fate of tumor progression. Hence, interfering with this signaling network forms the basis for cancer therapy. Yet, many types of cancer, in particular, solid tumors, are refractory to the currently used treatments, so there is an urgent need for novel molecular targets that could improve current anti-cancer therapeutic strategies. Lipocalin-2 (Lcn-2), a secreted siderophore-binding glycoprotein that regulates iron homeostasis, is highly upregulated in various cancer types. Due to its pleiotropic role in the crosstalk between cancer cells and stromal cells, favoring tumor progression, it could be considered as a novel biomarker for prognostic and therapeutic purposes. However, the exact signaling route by which Lcn-2 promotes tumorigenesis remains unknown, and Lcn-2-targeting moieties are largely uninvestigated. This review will (i) provide an overview on the role of Lcn-2 in orchestrating the TME at the level of iron homeostasis, macrophage polarization, extracellular matrix remodeling, and cell migration and survival, and (ii) discuss the potential of Lcn-2 as a promising novel drug target that should be pursued in future translational research.

Ontogeny, functions and reprogramming of Kupffer cells upon infectious disease.

The liver is a vital metabolic organ that also performs important immune-regulatory functions. In the context of infections, the liver represents a target site for various pathogens, while also having an outstanding capacity to filter the blood from pathogens and to contain infections. Pathogen scavenging by the liver is primarily performed by its large and heterogeneous macrophage population. The major liver-resident macrophage population is located within the hepatic microcirculation and is known as Kupffer cells (KCs). Although other minor macrophages reside in the liver as well, KCs remain the best characterized and are the best well-known hepatic macrophage population to be functionally involved in the clearance of infections. The response of KCs to pathogenic insults often governs the overall severity and outcome of infections on the host. Moreover, infections also impart long-lasting, and rarely studied changes to the KC pool. In this review, we discuss current knowledge on the biology and the various roles of liver macrophages during infections. In addition, we reflect on the potential of infection history to imprint long-lasting effects on macrophages, in particular liver macrophages.

Current status and future expectations of nanobodies in oncology trials.

INTRODUCTION: Monoclonal antibodies have revolutionized personalized medicine for cancer in recent decades. Despite their broad application in oncology, their large size and complexity may interfere with successful tumor targeting for certain applications of cancer diagnosis and therapy. Nanobodies have unique structural and pharmacological features compared to monoclonal antibodies and have successfully been used as complementary anti-cancer diagnostic and/or therapeutic tools. AREAS COVERED: Here, an overview is given of the nanobody-based diagnostics and therapeutics that have been or are currently being tested in oncological clinical trials. Furthermore, preclinical developments, which are likely to be translated into the clinic in the near future, are highlighted. EXPERT OPINION: Overall, the presented studies show the application potential of nanobodies in the field of oncology, making it likely that more nanobodies will be clinically approved in the upcoming future.

Flt3L therapy increases the abundance of Treg-promoting CCR7+ cDCs in preclinical cancer models.

Conventional dendritic cells (cDCs) are at the forefront of activating the immune system to mount an anti-tumor immune response. Flt3L is a cytokine required for DC development that can increase DC abundance in the tumor when administered therapeutically. However, the impact of Flt3L on the phenotype of distinct cDC subsets in the tumor microenvironment is still largely undetermined. Here, using multi-omic single-cell analysis, we show that Flt3L therapy increases all cDC subsets in orthotopic E0771 and TS/A breast cancer and LLC lung cancer models, but this did not result in a reduction of tumor growth in any of the models. Interestingly, a CD81+migcDC1 population, likely developing from cDC1, was induced upon Flt3L treatment in E0771 tumors as well as in TS/A breast and LLC lung tumors. This CD81+migcDC1 subset is characterized by the expression of both canonical cDC1 markers as well as migratory cDC activation and regulatory markers and displayed a Treg-inducing potential. To shift the cDC phenotype towards a T-cell stimulatory phenotype, CD40 agonist therapy was administered to E0771 tumor-bearing mice in combination with Flt3L. However, while αCD40 reduced tumor growth, Flt3L failed to improve the therapeutic response to αCD40 therapy. Interestingly, Flt3L+αCD40 combination therapy increased the abundance of Treg-promoting CD81+migcDC1. Nonetheless, while Treg-depletion and αCD40 therapy were synergistic, the addition of Flt3L to this combination did not result in any added benefit. Overall, these results indicate that merely increasing cDCs in the tumor by Flt3L treatment cannot improve anti-tumor responses and therefore might not be beneficial for the treatment of cancer, though could still be of use to increase cDC numbers for autologous DC-therapy.

Phase I Study of [68Ga]Ga-Anti-CD206-sdAb for PET/CT Assessment of Protumorigenic Macrophage Presence in Solid Tumors (MMR Phase I).

Macrophages play an important role throughout the body. Antiinflammatory macrophages expressing the macrophage mannose receptor (MMR, CD206) are involved in disease development, ranging from oncology to atherosclerosis and rheumatoid arthritis. [68Ga]Ga-NOTA-anti-CD206 single-domain antibody (sdAb) is a PET tracer targeting CD206. This first-in-human study, as its primary objective, evaluated the safety, biodistribution, and dosimetry of this tracer. The secondary objective was to assess its tumor uptake. Methods: Seven patients with a solid tumor of at least 10 mm, an Eastern Cooperative Oncology Group score of 0 or 1, and good renal and hepatic function were included. Safety was evaluated using clinical examination and blood sampling before and after injection. For biodistribution and dosimetry, PET/CT was performed at 11, 90, and 150 min after injection; organs showing tracer uptake were delineated, and dosimetry was evaluated. Blood samples were obtained at selected time points for blood clearance. Metabolites in blood and urine were assessed. Results: Seven patients were injected with, on average, 191 MBq of [68Ga]Ga-NOTA-anti-CD206-sdAb. Only 1 transient adverse event of mild severity was considered to be possibly, although unlikely, related to the study drug (headache, Common Terminology Criteria for Adverse Events grade 1). The blood clearance was fast, with less than 20% of the injected activity remaining after 80 min. There was uptake in the liver, kidneys, spleen, adrenals, and red bone marrow. The average effective dose from the radiopharmaceutical was 4.2 mSv for males and 5.2 mSv for females. No metabolites were detected. Preliminary data of tumor uptake in cancer lesions showed higher uptake in the 3 patients who subsequently progressed than in the 3 patients without progression. One patient could not be evaluated because of technical failure. Conclusion: [68Ga]Ga-NOTA-anti-CD206-sdAb is safe and well tolerated. It shows rapid blood clearance and renal excretion, enabling high contrast-to-noise imaging at 90 min after injection. The radiation dose is comparable to that of routinely used PET tracers. These findings and the preliminary results in cancer patients warrant further investigation of this tracer in phase II clinical trials.

A few good reasons to use nanobodies for cancer treatment.

mAbs have been instrumental for targeted cancer therapies. However, their relatively large size and physicochemical properties result in a heterogenous distribution in the tumor microenvironment, usually restricted to the first cell layers surrounding blood vessels, and a limited ability to penetrate the brain. Nanobodies are tenfold smaller, resulting in a deeper tumor penetration and the ability to reach cells in poorly perfused tumor areas. Nanobodies are rapidly cleared from the circulation, which generates a fast target-to-background contrast that is ideally suited for molecular imaging purposes but may be less optimal for therapy. To circumvent this problem, nanobodies have been formatted to noncovalently bind albumin, increasing their serum half-life without majorly increasing their size. Finally, nanobodies have shown superior qualities to infiltrate brain tumors as compared to mAbs. In this review, we discuss why these features make nanobodies prime candidates for targeted therapy of cancer.

Hallmarks of Cancer Affected by the MIF Cytokine Family.

New diagnostic methods and treatments have significantly decreased the mortality rates of cancer patients, but further improvements are warranted based on the identification of novel tumor-promoting molecules that can serve as therapeutic targets. The macrophage migration inhibitory factor (MIF) family of cytokines, comprising MIF and DDT (also known as MIF2), are overexpressed in almost all cancer types, and their high expressions are related to a worse prognosis for the patients. MIF is involved in 9 of the 10 hallmarks of cancer, and its inhibition by antibodies, nanobodies, or small synthetic molecules has shown promising results. Even though DDT is also proposed to be involved in several of the hallmarks of cancer, the available information about its pro-tumoral role and mechanism of action is more limited. Here, we provide an overview of the involvement of both MIF and DDT in cancer, and we propose that blocking both cytokines is needed to obtain the maximum anti-tumor response.

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting.

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.

Junctional adhesion molecule-A is dispensable for myeloid cell recruitment and diversification in the tumor microenvironment.

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1ß-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming.

The Interface of Tumour-Associated Macrophages with Dying Cancer Cells in Immuno-Oncology.

Tumour-associated macrophages (TAMs) are essential players in the tumour microenvironment (TME) and modulate various pro-tumorigenic functions such as immunosuppression, angiogenesis, cancer cell proliferation, invasion and metastasis, along with resistance to anti-cancer therapies. TAMs also mediate important anti-tumour functions and can clear dying cancer cells via efferocytosis. Thus, not surprisingly, TAMs exhibit heterogeneous activities and functional plasticity depending on the type and context of cancer cell death that they are faced with. This ultimately governs both the pro-tumorigenic and anti-tumorigenic activity of TAMs, making the interface between TAMs and dying cancer cells very important for modulating cancer growth and the efficacy of chemo-radiotherapy or immunotherapy. In this review, we discuss the interface of TAMs with cancer cell death from the perspectives of cell death pathways, TME-driven variations, TAM heterogeneity and cell-death-inducing anti-cancer therapies. We believe that a better understanding of how dying cancer cells influence TAMs can lead to improved combinatorial anti-cancer therapies, especially in combination with TAM-targeting immunotherapies.

GBM tumors are heterogeneous in their fatty acid metabolism and modulating fatty acid metabolism sensitizes cancer cells derived from recurring GBM tumors to temozolomide.

Glioblastoma is a highly lethal grade of astrocytoma with very low median survival. Despite extensive efforts, there is still a lack of alternatives that might improve these prospects. We uncovered that the chemotherapeutic agent temozolomide impinges on fatty acid synthesis and desaturation in newly diagnosed glioblastoma. This response is, however, blunted in recurring glioblastoma from the same patient. Further, we describe that disrupting cellular fatty acid homeostasis in favor of accumulation of saturated fatty acids such as palmitate synergizes with temozolomide treatment. Pharmacological inhibition of SCD and/or FADS2 allows palmitate accumulation and thus greatly augments temozolomide efficacy. This effect was independent of common GBM prognostic factors and was effective against cancer cells from recurring glioblastoma. In summary, we provide evidence that intracellular accumulation of saturated fatty acids in conjunction with temozolomide based chemotherapy induces death in glioblastoma cells derived from patients.

The Heat Is On: 20-HETE Instructs an Immunosuppressive Phenotype in Cancer-Associated Fibroblasts.

Immunotherapy of cancer is a burgeoning field of research since the realization that our immune system intrinsically has the capacity to restrict tumor occurrence and progression. Though strategies to maximize antitumor T-cell activation are well established, the efficacy of these therapies is limited by an insufficient knowledge of the intricate tumor microenvironment and its capacity to thwart antitumor immunity. Chen and colleagues now uncover a novel immunosuppressive pathway in non-small cell lung carcinoma. Overexpression of cytochrome P450F2 in cancer cells increases production of 20-hydroxyeicosatetraenoic acid, which instructs the expression of immunosuppressive molecules in cancer-associated fibroblasts by binding the GPR75 receptor and activating STAT3/c-Jun signaling. This work proposes several innovative therapeutic anchor points that may improve the efficacy of existing immunotherapies. See related article by Chen et al., p. 4016.

Differential plasticity and fate of brain-resident and recruited macrophages during the onset and resolution of neuroinflammation.

Microglia and border-associated macrophages (BAMs) are brain-resident self-renewing cells. Here, we examined the fate of microglia, BAMs, and recruited macrophages upon neuroinflammation and through resolution. Upon infection, Trypanosoma brucei parasites invaded the brain via its border regions, triggering brain barrier disruption and monocyte infiltration. Fate mapping combined with single-cell sequencing revealed microglia accumulation around the ventricles and expansion of epiplexus cells. Depletion experiments using genetic targeting revealed that resident macrophages promoted initial parasite defense and subsequently facilitated monocyte infiltration across brain barriers. These recruited monocyte-derived macrophages outnumbered resident macrophages and exhibited more transcriptional plasticity, adopting antimicrobial gene expression profiles. Recruited macrophages were rapidly removed upon disease resolution, leaving no engrafted monocyte-derived cells in the parenchyma, while resident macrophages progressively reverted toward a homeostatic state. Long-term transcriptional alterations were limited for microglia but more pronounced in BAMs. Thus, brain-resident and recruited macrophages exhibit diverging responses and dynamics during infection and resolution.

Enteric glial cells favor accumulation of anti-inflammatory macrophages during the resolution of muscularis inflammation.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206+ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206+ Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.