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.

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.

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.

The Road to Personalized Myeloma Medicine: Patient-specific Single-domain Antibodies for Anti-idiotypic Radionuclide Therapy.

To this day, multiple myeloma remains an incurable cancer. For many patients, recurrence is unavoidably a result of lacking treatment options in the minimal residual disease stage. This is due to residual and treatment-resistant myeloma cells that can cause disease relapse. However, patient-specific membrane-expressed paraproteins could hold the key to target these residual cells responsible for disease recurrence. Here, we describe the therapeutic potential of radiolabeled, anti-idiotypic camelid single-domain antibody fragments (sdAbs) as tumor-restrictive vehicles against a membrane-bound paraprotein in the syngeneic mouse 5T33 myeloma model and analogously assess the feasibility of sdAb-based personalized medicine for patients with multiple myeloma. Llamas were immunized using extracts containing paraprotein from either murine or human sera, and selective sdAbs were retrieved using competitive phage display selections of immune libraries. An anti-5T33 idiotype sdAb was selected for targeted radionuclide therapy with the ß--particle emitter 177Lu and the α-particle emitter 225Ac. sdAb-based radionuclide therapy in syngeneic mice with a low 5T33 myeloma lesion load significantly delayed tumor progression. In five of seven patients with newly diagnosed myeloma, membrane expression of the paraprotein was confirmed. Starting from serum-isolated paraprotein, for two of three selected patients anti-idiotype sdAbs were successfully generated.

Macrophages are metabolically heterogeneous within the tumor microenvironment.

Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although metabolism influences macrophage function, studies on the metabolic characteristics of ex vivo tumor-associated macrophage (TAM) subsets are rather limited. Using transcriptomic and metabolic analyses, we now reveal that pro-inflammatory major histocompatibility complex (MHC)-IIhi TAMs display a hampered tricarboxylic acid (TCA) cycle, while reparative MHC-IIlo TAMs show higher oxidative and glycolytic metabolism. Although both TAM subsets rapidly exchange lactate in high-lactate conditions, only MHC-IIlo TAMs use lactate as an additional carbon source. Accordingly, lactate supports the oxidative metabolism in MHC-IIlo TAMs, while it decreases the metabolic activity of MHC-IIhi TAMs. Lactate subtly affects the transcriptome of MHC-IIlo TAMs, increases L-arginine metabolism, and enhances the T cell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as a carbon source and metabolic and functional regulator of TAMs.

Macrophage miR-210 induction and metabolic reprogramming in response to pathogen interaction boost life-threatening inflammation.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies.

Immunogenicity Risk Profile of Nanobodies.

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications. One Sentence Summary: Nanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.

Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33.

Nanobodies (Nbs) are the smallest antigen-binding, single domain fragments derived from heavy-chain-only antibodies from Camelidae. Among the several advantages over conventional monoclonal antibodies, their small size (12-15 kDa) allows them to extravasate rapidly, to show improved tissue penetration, and to clear rapidly from blood, which are important characteristics for cancer imaging and targeted radiotherapy. Herein, we identified Nbs against CD33, a marker for acute myeloid leukemia (AML). A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells. The equilibrium dissociation constants (KD) of these six Nbs and CD33 range from 4 to 270 nM, and their melting temperature (Tm) varies between 52.67 and 67.80 °C. None of these Nbs showed leukemogenicity activity in vitro. The selected six candidates were radiolabeled with 99mTc, and their biodistribution was evaluated in THP-1-tumor-bearing mice. The imaging results demonstrated the fast tumor-targeting capacity of the Nbs in vivo. Among the anti-CD33 Nbs, Nb_7 showed the highest tumor uptake (2.53 ± 0.69 % injected activity per gram (IA/g), with low background signal, except in the kidneys and bladder. Overall, Nb_7 exhibits the best characteristics to be used as an anti-CD33 targeting vehicle for future diagnostic or therapeutic applications.

Neutrophils enhance early Trypanosoma brucei infection onset.

In this study, Trypanosoma brucei was naturally transmitted to mice through the bites of infected Glossina morsitans tsetse flies. Neutrophils were recruited rapidly to the bite site, whereas monocytes were attracted more gradually. Expression of inflammatory cytokines (il1b, il6), il10 and neutrophil chemokines (cxcl1, cxcl5) was transiently up-regulated at the site of parasite inoculation. Then, a second influx of neutrophils occurred that coincided with the previously described parasite retention and expansion in the ear dermis. Congenital and experimental neutropenia models, combined with bioluminescent imaging, indicate that neutrophils do not significantly contribute to dermal parasite control and elicit higher systemic parasitemia levels during the infection onset. Engulfment of parasites by neutrophils in the skin was rarely observed and was restricted to parasites with reduced motility/viability, whereas live parasites escaped phagocytosis. To our knowledge, this study represents the first description of a trypanosome infection promoting role of early innate immunological reactions following an infective tsetse fly bite. Our data indicate that the trypanosome is not hindered in its early development and benefits from the host innate responses with the neutrophils being important regulators of the early infection, as already demonstrated for the sand fly transmitted Leishmania parasite.

The anuran skin peptide bradykinin mediates its own absorption across epithelial barriers of the digestive tract.

When faced with a potential predator, a wide range of frog species secrete a mixture of peptide toxins from their skin to defend themselves. We have recently shown that antimicrobial peptides (AMPs) in a frog's defensive poison enhance the uptake of these peptides across epithelia, thereby speeding up the process of predator intoxication. This study provides evidence that bradykinin, a widespread peptide toxin in anurans (frogs), is capable to pass through epithelial barriers independent of this delivery system. We quantified bradykinin peptides secreted by Bombina orientalis during acute stress, and found that at biologically relevant concentrations, bradykinin passage across model epithelia occurs even in the absence of AMPs. Monitoring of transepithelial electric resistance showed that bradykinin treatment caused a subtle yet prolonged reduction in barrier function, indicating that the peptide itself is capable to increase the permeability of epithelia. Yet, bradykinin does not cause cells to leak lactate dehydrogenase, suggesting that it does not damage cell membranes. Moreover, imaging of bradykinin-treated monolayers shows no endocytosis of fluorescent propidium iodide, indicating that the peptide does not perforate cell membranes at smaller scale and therefore is unlikely to cross epithelia via a transcellular passage. Together, these observations suggest that bradykinin, unlike other amphibian neuropeptide toxins, mediates its own passage across mucosal barriers, possibly through a paracellular route. This "self-administering" property, combined with the fact that bradykinins can potently disturb multiple physiological processes, could explain why these peptides are one of the most widespread antipredator peptides in the defensive secretions of frogs.

MIF inhibition interferes with the inflammatory and T cell-stimulatory capacity of NOD macrophages and delays autoimmune diabetes onset.

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression.

Reprint of: The non-mammalian MIF superfamily.

Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIF's evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance.

MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis.

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis.

M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

E-cadherin expression in macrophages dampens their inflammatory responsiveness in vitro, but does not modulate M2-regulated pathologies in vivo.

IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested.

Iron Homeostasis and Trypanosoma brucei Associated Immunopathogenicity Development: A Battle/Quest for Iron.

African trypanosomosis is a chronic debilitating disease affecting the health and economic well-being of developing countries. The immune response during African trypanosome infection consisting of a strong proinflammatory M1-type activation of the myeloid phagocyte system (MYPS) results in iron deprivation for these extracellular parasites. Yet, the persistence of M1-type MYPS activation causes the development of anemia (anemia of chronic disease, ACD) as a most prominent pathological parameter in the mammalian host, due to enhanced erythrophagocytosis and retention of iron within the MYPS thereby depriving iron for erythropoiesis. In this review we give an overview of how parasites acquire iron from the host and how iron modulation of the host MYPS affects trypanosomosis-associated anemia development. Finally, we also discuss different strategies at the level of both the host and the parasite that can/might be used to modulate iron availability during African trypanosome infections.

Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis.

Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing ß-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote ß-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to ß-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.