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 Transcription Factor ZEB2 Is Required to Maintain the Tissue-Specific Identities of Macrophages.

Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities.

Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

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.

Molecular profiling reveals a tumor-promoting phenotype of monocytes and macrophages in human cancer progression.

Monocytes and macrophages are major components of the tumor microenvironment, but their contributions to human cancer are poorly understood. We used molecular profiling combined with functional assays to investigate the role of these cells in human renal cell carcinoma (RCC). Blood monocytes from RCC patients displayed a tumor-promoting transcriptional profile that supported functions like angiogenesis and invasion. Induction of this protumor phenotype required an interleukin-1 receptor (IL-1R)-dependent mechanism. Indeed, targeting of IL-1-IL-1R axis in a human RCC xenograft model abrogated the protumor phenotype of tumor-associated macrophages (TAMs) and reduced tumor growth in vivo. Supporting this, meta-analysis of gene expression from human RCC tumors showed IL1B expression to correlate with myelomonocytic markers, protumor genes, and tumor staging. Analyzing RCC patient tumors confirmed the protumor phenotype of TAMs. These data provide direct evidence for a tumor-promoting role of monocytes and macrophages in human cancer and indicate IL-1-IL-1R as a possible therapeutic target.

Macrophage activation and polarization: nomenclature and experimental guidelines.

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.

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.

The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages.

RXR signaling is predicted to have a major impact in macrophages, but neither the biological consequence nor the genomic basis of its ligand activation is known. Comprehensive genome-wide studies were carried out to map liganded RXR-mediated transcriptional changes, active binding sites, and cistromic interactions in the context of the macrophage genome architecture. The macrophage RXR cistrome has 5200 genomic binding sites, which are not impacted by ligand. Active enhancers are characterized by PU.1 binding, an increase of enhancer RNA, and P300 recruitment. Using these features, 387 liganded RXR-bound enhancers were linked to 226 genes, which predominantly reside in CTCF/cohesin-limited functional domains. These findings were molecularly validated using chromosome conformation capture (3C) and 3C combined with sequencing (3C-seq), and we show that selected long-range enhancers communicate with promoters via stable or RXR-induced loops and that some of the enhancers interact with each other, forming an interchromosomal network. A set of angiogenic genes, including Vegfa, has liganded RXR-controlled enhancers and provides the macrophage with a novel inducible program.

Myeloid tumor necrosis factor and heme oxygenase-1 regulate the progression of colorectal liver metastases during hepatic ischemia-reperfusion.

The liver ischemia-reperfusion (IR) injury that occurs consequently to hepatic resection performed in patients with metastases can lead to tumor relapse for not fully understood reasons. We assessed the effects of liver IR on tumor growth and the innate immune response in a mouse model of colorectal (CR) liver metastasis. Mice subjected to liver ischemia 2 days after intrasplenic injection of CR carcinoma cells displayed a higher metastatic load in the liver, correlating with Kupffer cells (KC) death through the activation of receptor-interating protein 3 kinase (RIPK3) and caspase-1 and a recruitment of monocytes. Interestingly, the immunoregulatory mediators, tumor necrosis factor-α (TNF-α) and heme oxygenase-1 (HO-1) were strongly upregulated in recruited monocytes and were also expressed in the surviving KC following IR. Using TNFflox/flox LysMcre/wt mice, we showed that TNF deficiency in macrophages and monocytes favors tumor progression after IR. The antitumor effect of myeloid cell-derived TNF involved direct tumor cell apoptosis and a reduced expression of immunosuppressive molecules such as transforming growth factor-ß, interleukin (IL)-10, inducible nitric oxyde synthase (iNOS), IL-33 and HO-1. Conversely, a monocyte/macrophage-specific deficiency in HO-1 (HO-1flox/flox LysMcre/wt ) or the blockade of HO-1 function led to the control of tumor progression post-liver IR. Importantly, host cell RIPK3 deficiency maintains the KC number upon IR, inhibits the IR-induced innate cell recruitment, increases the TNF level, decreases the HO-1 level and suppresses the tumor outgrowth. In conclusion, tumor recurrence in host undergoing liver IR is associated with the death of antitumoral KC and the recruitment of monocytes endowed with immunosuppressive properties. In both of which HO-1 inhibition would reinforce their antitumoral activity.

Identifying the variables that drive tamoxifen-independent CreERT2 recombination: Implications for microglial fate mapping and gene deletions.

Ligand-dependent Cre recombinases such as the CreERT2 system allow for tamoxifen-inducible Cre recombination. Important examples are the Cx3cr1-CreERT2 and Sall1-CreERT2 lines that are widely used for fate mapping and gene deletion studies of brain macrophages. Our results now show that both CreERT2 lines can exhibit a high rate of tamoxifen-independent "leaky" excision with some reporter strains, while this is not observed with others. We suggest that this disparity is determined by the length of the floxed transcriptional STOP cassette that is incorporated in the various reporter lines. In addition, the rate of spontaneous recombination was also determined by the CreERT2 expression levels and the longevity of the CreERT2-expressing cells. The implications of these results are discussed in the context of fate mapping and inducible gene deletion studies in macrophages and microglia.

Monocytic myeloid-derived suppressor cells home to tumor-draining lymph nodes via CCR2 and locally modulate the immune response.

Efficient priming of anti-tumor T cells requires the uptake and presentation of tumor antigens by immunogenic dendritic cells (DCs) and occurs mainly in lymph nodes draining the tumor (tdLNs). However, tumors expand and activate myeloid-derived suppressor cells (MDSCs) that inhibit CTL functions by several mechanisms. While the immune-suppressive nature of the tumor microenvironment is largely documented, it is not known whether similar immune-suppressive mechanisms operate in the tdLNs. In this study, we analyzed MDSC characteristics within tdLNs. We show that, in a metastasis-free context, MO-MDSCs are the dominant MDSC population within tdLNs, that they are highly suppressive and that tumor proximity enhances their recruitment to tdLN via a CCR2/CCL2-dependent pathway. Altogether our results uncover a mechanism by which tumors evade the immune system that involves MDSC-mediated recruitment to the tdLN and the inhibition of T-cell activation even before reaching the highly immunosuppressive tumor microenvironment.

How to measure the immunosuppressive activity of MDSC: assays, problems and potential solutions.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of mononuclear and polymorphonuclear myeloid cells, which are present at very low numbers in healthy subjects, but can expand substantially under disease conditions. Depending on disease type and stage, MDSC comprise varying amounts of immature and mature differentiation stages of myeloid cells. Validated unique phenotypic markers for MDSC are still lacking. Therefore, the functional analysis of these cells is of central importance for their identification and characterization. Various disease-promoting and immunosuppressive functions of MDSC are reported in the literature. Among those, the capacity to modulate the activity of T cells is by far the most often used and best-established read-out system. In this review, we critically evaluate the assays available for the functional analysis of human and murine MDSC under in vitro and in vivo conditions. We also discuss critical issues and controls associated with those assays. We aim at providing suggestions and recommendations useful for the contemporary biological characterization of MDSC.

Interactions among myeloid regulatory cells in cancer.

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.

Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

Sepsis-leading to septic shock-is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity-matured nanobodies (Nbs; single-domain antigen-binding fragments derived from camelid heavy-chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIF's conformation and receptor binding), the TNF-inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half-life extended construct (NbE10-NbAlb8-NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory end-organ damage.-Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.-J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

Genetic ablation of IP3 receptor 2 increases cytokines and decreases survival of SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease characterized by the selective death of motor neurons. Disease pathophysiology is complex and not yet fully understood. Higher gene expression of the inositol 1,4,5-trisphosphate receptor 2 gene (ITPR2), encoding the IP3 receptor 2 (IP3R2), was detected in sporadic ALS patients. Here, we demonstrate that IP3R2 gene expression was also increased in spinal cords of ALS mice. Moreover, an increase of IP3R2 expression was observed in other models of chronic and acute neurodegeneration. Upregulation of IP3R2 gene expression could be induced by lipopolysaccharide (LPS) in murine astrocytes, murine macrophages and human fibroblasts indicating that it may be a compensatory response to inflammation. Preventing this response by genetic deletion of ITPR2 from SOD1G93A mice had a dose-dependent effect on disease duration, resulting in a significantly shorter lifespan of these mice. In addition, the absence of IP3R2 led to increased innate immunity, which may contribute to the decreased survival of the SOD1G93A mice. Besides systemic inflammation, IP3R2 knockout mice also had increased IFNγ, IL-6 and IL1α expression. Altogether, our data indicate that IP3R2 protects against the negative effects of inflammation, suggesting that the increase in IP3R2 expression in ALS patients is a protective response.

Paracrine interactions of cancer-associated fibroblasts, macrophages and endothelial cells: tumor allies and foes.

PURPOSE OF REVIEW: Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions. RECENT FINDINGS: Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells. SUMMARY: Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

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.

The role of hepatic macrophages in liver metastasis.

The liver is a major target organ for metastasis of both gastrointestinal and extra-gastrointestinal cancers. Due to its frequently inoperable nature, liver metastasis represents a leading cause of cancer-associated death worldwide. In the past years, the pivotal role of the immune system in this process is being increasingly recognised. In particular, the role of the hepatic macrophages, both recruited monocyte-derived macrophages (Mo-Mfs) and tissue-resident Kupffer cells (KCs), has been shown to be more versatile than initially imagined. However, the lack of tools to easily distinguish between these two macrophage populations has hampered the assignment of particular functionalities to specific hepatic macrophage subsets. In this Review, we highlight the most remarkable findings regarding the origin and functions of hepatic macrophage populations, and we provide a detailed description of their distinct roles in the different phases of the liver metastatic process.

Targeting Protumoral Tumor-Associated Macrophages with Nanobody-Functionalized Nanogels through Strain Promoted Azide Alkyne Cycloaddition Ligation.

Tumor-associated macrophages (TAMs) with high expression levels of the Macrophage Mannose Receptor (MMR, CD206) exhibit a strong angiogenic and immune suppressive activity. Thus, they are a highly attractive target in cancer immunotherapy, with the aim to modulate their protumoral behavior. Here, we introduce polymer nanogels as potential drug nanocarriers which were site-specifically decorated with a Nanobody (Nb) specific for the MMR. Using azide-functionalized RAFT chain transfer agents, they provide access to amphiphilic reactive ester block copolymers that self-assemble into micelles and are afterwards core-cross-linked toward fully hydrophilic nanogels with terminal azide groups on their surface. MMR-targeting Nb can site-selectively be functionalized with one single cyclooctyne moiety by maleimide-cysteine chemistry under mildly reducing conditions which enables successful chemoorthogonal conjugation to the nanogels. The resulting Nb-functionalized nanogels were highly efficient in targeting MMR-expressing cells and TAMs both in vitro and in vivo. We believe that these findings pave the road for targeted eradication or modulation of pro-tumoral MMRhigh TAMs.

RoMo: An efficient strategy for functional mosaic analysis via stochastic Cre recombination and gene targeting in the ROSA26 locus.

Functional mosaic analysis allows for the direct comparison of mutant cells with differentially marked control cells in the same organism. While this offers a powerful approach for elucidating the role of specific genes or signalling pathways in cell populations of interest, genetic strategies for generating functional mosaicism remain challenging. We describe a novel and streamlined approach for functional mosaic analysis, which combines stochastic Cre/lox recombination with gene targeting in the ROSA26 locus. With the RoMo strategy a cell population of interest is randomly split into a cyan fluorescent and red fluorescent subset, of which the latter overexpresses a chosen transgene. To integrate this approach into high-throughput gene targeting initiatives, we developed a procedure that utilizes Gateway cloning for the generation of new targeting vectors. RoMo can be used for gain-of-function experiments or for altering signaling pathways in a mosaic fashion. To demonstrate this, we developed RoMo-dnGs mice, in which Cre-recombined red fluorescent cells co-express a dominant-negative Gs protein. RoMo-dnGs mice allowed us to inhibit G protein-coupled receptor activation in a fraction of cells, which could then be directly compared to differentially marked control cells in the same animal. We demonstrate how RoMo-dnGs mice can be used to obtain mosaicism in the brain and in peripheral organs for various cell types. RoMo offers an efficient new approach for functional mosaic analysis that extends the current toolbox and may reveal important new insights into in vivo gene function.