Gut microbiota promotes pain chronicity in Myosin1A deficient male mice.

Chronic pain is a heavily debilitating condition and a huge socio-economic burden, with no efficient treatment. Over the past decade, the gut microbiota has emerged as an important regulator of nervous system's health and disease states. Yet, its contribution to the pathogenesis of chronic somatic pain remains poorly documented. Here, we report that male but not female mice lacking Myosin1a (KO) raised under single genotype housing conditions (KO-SGH) are predisposed to develop chronic pain in response to a peripheral tissue injury. We further underscore the potential of MYO1A loss-of-function to alter the composition of the gut microbiota and uncover a functional connection between the vulnerability to chronic pain and the dysbiotic gut microbiota of KO-SGH males. As such, parental antibiotic treatment modifies gut microbiota composition and completely rescues the injury-induced pain chronicity in male KO-SGH offspring. Furthermore, in KO-SGH males, this dysbiosis is accompanied by a transcriptomic activation signature in the dorsal root ganglia (DRG) macrophage compartment, in response to tissue injury. We identify CD206+CD163- and CD206+CD163+ as the main subsets of DRG resident macrophages and show that both are long-lived and self-maintained and exhibit the capacity to monitor the vasculature. Consistently, in vivo depletion of DRG macrophages rescues KO-SGH males from injury-induced chronic pain underscoring a deleterious role for DRG macrophages in a Myo1a-loss-of function context. Together, our findings reveal gene-sex-microbiota interactions in determining the predisposition to injury-induced chronic pain and point-out DRG macrophages as potential effector cells.

Proinflammatory polarization strongly reduces human macrophage in vitro phagocytosis of tumor cells in response to CD47 blockade.

Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds.

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis.

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

CD163+ macrophages monitor enhanced permeability at the blood-dorsal root ganglion barrier.

In dorsal root ganglia (DRG), macrophages reside close to sensory neurons and have largely been explored in the context of pain, nerve injury, and repair. However, we discovered that most DRG macrophages interact with and monitor the vasculature by sampling macromolecules from the blood. Characterization of the DRG vasculature revealed a specialized endothelial bed that transformed in molecular, structural, and permeability properties along the arteriovenous axis and was covered by macrophage-interacting pericytes and fibroblasts. Macrophage phagocytosis spatially aligned with peak endothelial permeability, a process regulated by enhanced caveolar transcytosis in endothelial cells. Profiling the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover, and function. CD163+ macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation, and were conserved in mouse and man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit.

Sex-dimorphic neuroprotective effect of CD163 in an α-synuclein mouse model of Parkinson's disease.

Alpha-synuclein (α-syn) aggregation and immune activation represent hallmark pathological events in Parkinson's disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such a response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine α-syn pre-formed fibrils in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to α-syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and α-syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a sex-dimorphic neuroprotective role for CD163 during α-syn-induced neurodegeneration.

T-cell derived extracellular vesicles prime macrophages for improved STING based cancer immunotherapy.

A key phenomenon in cancer is the establishment of a highly immunosuppressive tumour microenvironment (TME). Despite advances in immunotherapy, where the purpose is to induce tumour recognition and hence hereof tumour eradication, the majority of patients applicable for such treatment still fail to respond. It has been suggested that high immunological activity in the tumour is essential for achieving effective response to immunotherapy, which therefore have led to exploration of strategies that triggers inflammatory pathways. Here activation of the stimulator of interferon genes (STING) signalling pathway has been considered an attractive target, as it is a potent trigger of pro-inflammatory cytokines and types I and III interferons. However, immunotherapy combined with targeted STING agonists has not yielded sustained clinical remission in humans. This suggests a need for exploring novel adjuvants to improve the innate immunological efficacy. Here, we demonstrate that extracellular vesicles (EVs), derived from activated CD4+ T cells (T-EVs), sensitizes macrophages to elevate STING activation, mediated by IFNγ carried on the T-EVs. Our work support that T-EVs can disrupt the immune suppressive environment in the tumour by reprogramming macrophages to a pro-inflammatory phenotype, and priming them for a robust immune response towards STING activation.

Comparison of culture media reveals that non-essential amino acids strongly affect the phenotype of human monocyte-derived macrophages.

Macrophages are important innate immune cells with the ability to adapt their phenotype to environmental cues. Research on human macrophages often uses monocyte-derived macrophages cultured in vitro, but it is unclear if culture medium affects macrophage phenotype. The objective of this study was to determine the impact of culture medium composition on monocyte-derived macrophage phenotype. Monocyte-derived macrophages were generated in different formulations of culture media (RPMI 1640, DMEM, MEM, McCoy's 5a and IMDM). Viability, yield and cell size were monitored, and RT-qPCR, flow cytometry or ELISA was used to compare levels of phenotype markers (CD163, CD206, CD80, TNFα, IL-10, SIRPα, LILRB1 and Siglec-10). Yield, cell size, gene expression, membrane protein levels and release of soluble proteins were all affected by changes in culture medium composition. The most pronounced effects were observed after culture in DMEM, which lacks the non-essential amino acids asparagine, aspartic acid, glutamic acid and proline. Supplementation of DMEM with non-essential amino acids either fully or partly reversed most effects of DMEM on macrophage phenotype. The results suggest culture medium composition and amino acid availability affect the phenotype of human monocyte-derived macrophages cultured in vitro.

Invasive margin tissue-resident macrophages of high CD163 expression impede responses to T cell-based immunotherapy.

BACKGROUND: Primary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of pivotal importance to improve therapy outcome. METHOD: Here, two mouse models with resistance against therapeutic vaccine-induced tumor regression were studied. Exploration of the tumor microenvironment by high dimensional flow cytometry in combination with therapeutic in vivo settings allowed for the identification of immunological factors driving immunotherapy resistance. RESULTS: Comparison of the tumor immune infiltrate during early and late regression revealed a change from tumor-rejecting toward tumor-promoting macrophages. In concert, a rapid exhaustion of tumor-infiltrating T cells was observed. Perturbation studies identified a small but discernible CD163hi macrophage population, with high expression of several tumor-promoting macrophage markers and a functional anti-inflammatory transcriptome profile, but not other macrophages, to be responsible. In-depth analyses revealed that they localize at the tumor invasive margins and are more resistant to Csf1r inhibition when compared with other macrophages. In vivo studies validated the activity of heme oxygenase-1 as an underlying mechanism of immunotherapy resistance. The transcriptomic profile of CD163hi macrophages is highly similar to a human monocyte/macrophage population, indicating that they represent a target to improve immunotherapy efficacy. CONCLUSIONS: In this study, a small population of CD163hi tissue-resident macrophages is identified to be responsible for primary and secondary resistance against T-cell-based immunotherapies. While these CD163hi M2 macrophages are resistant to Csf1r-targeted therapies, in-depth characterization and identification of the underlying mechanisms driving immunotherapy resistance allows the specific targeting of this subset of macrophages, thereby creating new opportunities for therapeutic intervention with the aim to overcome immunotherapy resistance.

Sympathetic axonal sprouting induces changes in macrophage populations and protects against pancreatic cancer.

Neuronal nerve processes in the tumor microenvironment were highlighted recently. However, the origin of intra-tumoral nerves remains poorly known, in part because of technical difficulties in tracing nerve fibers via conventional histological preparations. Here, we employ three-dimensional (3D) imaging of cleared tissues for a comprehensive analysis of sympathetic innervation in a murine model of pancreatic ductal adenocarcinoma (PDAC). Our results support two independent, but coexisting, mechanisms: passive engulfment of pre-existing sympathetic nerves within tumors plus an active, localized sprouting of axon terminals into non-neoplastic lesions and tumor periphery. Ablation of the innervating sympathetic nerves increases tumor growth and spread. This effect is explained by the observation that sympathectomy increases intratumoral CD163+ macrophage numbers, which contribute to the worse outcome. Altogether, our findings provide insights into the mechanisms by which the sympathetic nervous system exerts cancer-protective properties in a mouse model of PDAC.

STAT3 is over-activated within CD163pos bone marrow macrophages in both Multiple Myeloma and the benign pre-condition MGUS.

Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163pos TAMs is associated with poor outcome in several human malignancies, including multiple myeloma (MM). Signal transducer and activator of transcription 3 (STAT3) is over-activated in human cancers, and specifically within TAMs activation of STAT3 may induce an immunosuppressive (M2-like) phenotype. Therefore, STAT3-inhibition in TAMs may be a future therapeutic strategy.We investigated TAM markers CD163, CD206, and activated STAT3 (pSTAT3) in patients with MGUS (n = 32) and MM (n = 45), as well as healthy controls (HCs, n = 13).Blood levels of the macrophage biomarkers sCD163 and sCD206, and circulating cytokines, as well as bone marrow mRNA expression of CD163 and CD206, were generally increased in MGUS and MM patients, compared to HCs, but to highly similar levels. By immunohistochemistry, bone marrow levels of pSTAT3 were increased specifically within CD163pos cells in both MGUS and MM patients.In conclusion, macrophage-related inflammatory changes, including activation of STAT3, were present already at the MGUS stage, at similar levels as in MM. Specific increase in pSTAT3 levels within CD163pos cells supports that the CD163 scavenger receptor may be a useful target for future delivery of STAT3-inhibitory drugs to TAMs in MM patients.

Therapeutic targeting of tumor-associated macrophages.

Tumor-associated macrophages are among the most abundant non-cancerous cells in the tumor microenvironment and in many cancers macrophage infiltration into the tumor is associated with poor prognosis. Macrophages contribute to tumor development by promoting angiogenesis and immune suppression, and display remarkable phenotypic heterogeneity in the tumor microenvironment. Therapeutic strategies targeting macrophages that currently are in clinical development are mainly focused on a general depletion of tumor-associated macrophages, either by targeting the CSF-1/CSF-1R axis or by inhibiting macrophage recruitment by blocking CCR2/CCL2 signaling. Despite good pre-clinical response rates the treatment strategies focusing on general macrophage targeting have only shown limited clinical success and new approaches that target specific subsets of tumo-associated macrophages are emerging. This chapter will briefly present the functions and heterogeneity of tumor-associated macrophages and provide an overview of the current state of clinical development for pan-targeting strategies as well as discuss new strategies for targeting specific macrophage subsets for future anti-tumor immunotherapies.

Soluble CD163 Changes Indicate Monocyte Association With Cognitive Deficits in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder with a significant immune component, as demonstrated by changes in immune biomarkers in patients' biofluids. However, which specific cells are responsible for those changes is unclear because most immune biomarkers can be produced by various cell types. OBJECTIVES: The aim of this study was to explore monocyte involvement in PD. METHODS: We investigated the monocyte-specific biomarker sCD163, the soluble form of the receptor CD163, in cerebrospinal fluid (CSF) and serum in two experiments, and compared it with other biomarkers and clinical data. Potential connections between CD163 and alpha-synuclein were studied in vitro. RESULTS: CSF-sCD163 increased in late-stage PD and correlated with the PD biomarkers alpha-synuclein, Tau, and phosphorylated Tau, whereas it inversely correlated with the patients' cognitive scores, supporting monocyte involvement in neurodegeneration and cognition in PD. Serum-sCD163 increased only in female patients, suggesting a sex-distinctive monocyte response. CSF-sCD163 also correlated with molecules associated with adaptive and innate immune system activation and with immune cell recruitment to the brain. Serum-sCD163 correlated with proinflammatory cytokines and acute-phase proteins, suggesting a relation to chronic systemic inflammation. Our in vitro study showed that alpha-synuclein activates macrophages and induces shedding of sCD163, which in turn enhances alpha-synuclein uptake by myeloid cells, potentially participating in its clearance. CONCLUSIONS: Our data present sCD163 as a potential cognition-related biomarker in PD and suggest a role for monocytes in both peripheral and brain immune responses. This may be directly related to alpha-synuclein's proinflammatory capacity but could also have consequences for alpha-synuclein processing. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Mouse CD163 deficiency strongly enhances experimental collagen-induced arthritis.

The scavenger receptor CD163 is highly expressed in macrophages in sites of chronic inflammation where it has a not yet defined role. Here we have investigated development of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) in CD163-deficient C57BL/6 mice. Compared to wild-type mice, the CIA in CD163-deficient mice had a several-fold higher arthritis score with early onset, prolonged disease and strongly enhanced progression. Further, the serum anti-collagen antibody isotypes as well as the cytokine profiles and T cell markers in the inflamed joints revealed that CD163-deficient mice after 52 days had a predominant Th2 response in opposition to a predominant Th1 response in CD163+/+ mice. Less difference in disease severity between the CD163+/+ and CD163-/- mice was seen in the CAIA model that to a large extent induces arthritis independently of T-cell response and endogenous Th1/Th2 balance. In conclusion, the present set of data points on a novel strong anti-inflammatory role of CD163.

Tissue-resident macrophages in omentum promote metastatic spread of ovarian cancer.

Experimental and clinical evidence suggests that tumor-associated macrophages (TAMs) play important roles in cancer progression. Here, we have characterized the ontogeny and function of TAM subsets in a mouse model of metastatic ovarian cancer that is representative for visceral peritoneal metastasis. We show that the omentum is a critical premetastatic niche for development of invasive disease in this model and define a unique subset of CD163+ Tim4+ resident omental macrophages responsible for metastatic spread of ovarian cancer cells. Transcriptomic analysis showed that resident CD163+ Tim4+ omental macrophages were phenotypically distinct and maintained their resident identity during tumor growth. Selective depletion of CD163+ Tim4+ macrophages in omentum using genetic and pharmacological tools prevented tumor progression and metastatic spread of disease. These studies describe a specific role for tissue-resident macrophages in the invasive progression of metastatic ovarian cancer. The molecular pathways of cross-talk between tissue-resident macrophages and disseminated cancer cells may represent new targets to prevent metastasis and disease recurrence.

Specific targeting of CD163+ TAMs mobilizes inflammatory monocytes and promotes T cell-mediated tumor regression.

Tumor-associated macrophages (TAMs) play critical roles in tumor progression but are also capable of contributing to antitumor immunity. Recent studies have revealed an unprecedented heterogeneity among TAMs in both human cancer and experimental models. Nevertheless, we still understand little about the contribution of different TAM subsets to tumor progression. Here, we demonstrate that CD163-expressing TAMs specifically maintain immune suppression in an experimental model of melanoma that is resistant to anti-PD-1 checkpoint therapy. Specific depletion of the CD163+ macrophages results in a massive infiltration of activated T cells and tumor regression. Importantly, the infiltration of cytotoxic T cells was accompanied by the mobilization of inflammatory monocytes that significantly contributed to tumor regression. Thus, the specific targeting of CD163+ TAMs reeducates the tumor immune microenvironment and promotes both myeloid and T cell-mediated antitumor immunity, illustrating the importance of selective targeting of tumor-associated myeloid cells in a therapeutic context.

The macrophage-related biomarkers sCD163 and sCD206 are released by different shedding mechanisms.

The hemoglobin receptor CD163 and the mannose receptor CD206 are both expressed on the surface of human macrophages. Upon inflammatory activation, the receptors are shed from the macrophage surface generating soluble products. The plasma concentration of both soluble CD163 (sCD163) and soluble CD206 (sCD206) are increased in several diseases, including inflammatory conditions and cancer. Here, we show that in contrast to CD163, LPS-mediated shedding of CD206 in humans is slow and a result of indirect signaling. Although both sCD163 and sCD206 were increased in response to LPS stimulation in vivo, only CD163 was shed from LPS-stimulated macrophages in vitro. Although both sCD163 and sCD206 were released from cultured macrophages stimulated with zymosan and PMA, shedding of CD206 was generally slower and less efficient and not reduced by inhibitors against the major protease classes. These data indicate that CD163 and CD206 are shed from the macrophages by very different mechanisms potentially involving distinctive inflammatory processes.

Targeted lipid nanoparticle delivery of calcitriol to human monocyte-derived macrophages in vitro and in vivo: investigation of the anti-inflammatory effects of calcitriol.

BACKGROUND: Vitamin D3 possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers. PURPOSE: The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo. METHODS: In this study we have designed and developed near-infrared calcitriol PEGylated nanoparticles (PEG-LNP(Cal)) using a microfluidic mixing technique and modified lipid nanoparticles (LNPs) to target the M specific endocytic receptor CD163. We have investigated LNP cellular uptake and anti-inflammatory effect in LPS-induced M in vitro by flow cytometry, confocal microscopy and gene expression analyses. LNP pharmacodynamics, bio-distribution and organ specific LNP accumulation was also investigated in mice in vivo. RESULTS: In vitro, we observed the specific uptake of PEG-LNP(Cal)-hCD163 in human M, which was significantly higher than the non-specific uptake of control PEG-LNP(Cal)-IgG(h) in M. Pretreatment with encapsulated calcitriol was able to attenuate intracellular TNF-expression, and M surface marker HLA-DR expression more efficiently than free calcitriol in LPS-induced M in vitro. Encapsulated calcitriol diminished mRNA gene levels of TNF-, NF-B, MCP-1 and IL-6, while upregulating IL-10. TNF- and IL-6 protein secretion also decreased. In mice, an in vivo pharmacodynamic study of PEG-LNP(Cal) showed a rapid clearance of IgG and CD163 modified LNPs compared to PEG-LNP(Cal). Antibody modified PEG-LNP(Cal) accumulated in the liver, spleen and kidney, whereas unmodified PEG-LNP(Cal) accumulation was only observed in the liver. CONCLUSION: Our results show that calcitriol can be effectively targeted to M. Our data confirms the anti-inflammatory properties of calcitriol and this may be a potential way to deliver high dose bioactive calcitriol to M during inflammation in vivo.

Membrane Cholesterol Efflux Drives Tumor-Associated Macrophage Reprogramming and Tumor Progression.

Macrophages possess intrinsic tumoricidal activity, yet tumor-associated macrophages (TAMs) rapidly adopt an alternative phenotype within the tumor microenvironment that is marked by tumor-promoting immunosuppressive and trophic functions. The mechanisms that promote such TAM polarization remain poorly understood, but once identified, they may represent important therapeutic targets to block the tumor-promoting functions of TAMs and restore their anti-tumor potential. Here, we have characterized TAMs in a mouse model of metastatic ovarian cancer. We show that ovarian cancer cells promote membrane-cholesterol efflux and depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4-mediated reprogramming, including inhibition of IFNγ-induced gene expression. Genetic deletion of ABC transporters, which mediate cholesterol efflux, reverts the tumor-promoting functions of TAMs and reduces tumor progression. These studies reveal an unexpected role for membrane-cholesterol efflux in driving TAM-mediated tumor progression while pointing to a potentially novel anti-tumor therapeutic strategy.