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.

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.

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.

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.

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.

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.

STAT3 inhibition specifically in human monocytes and macrophages by CD163-targeted corosolic acid-containing liposomes.

Tumor-associated macrophages (TAMs) are of major importance in cancer-related immune suppression, and tumor infiltration by CD163pos TAMs is associated with poor outcome in most human cancers. Therefore, therapeutic strategies for reprogramming TAMs from a tumor-supporting (M2-like) phenotype towards a tumoricidal (M1-like) phenotype are of great interest. Activation of the transcription factor STAT3 within the tumor microenvironment is associated with worse prognosis, and STAT3 activation promotes the immunosuppressive phenotype of TAMs. Therefore, we aimed to develop a drug for inhibition of STAT3 specifically within human TAMs by targeting the endocytic CD163 scavenger receptor, which is highly expressed on TAMs. Here, we report the first data on a CD163-targeted STAT3-inhibitory drug consisting of corosolic acid (CA) packaged within long-circulating liposomes (LCLs), which are CD163-targeted by modification with monoclonal anti-CD163 antibodies (αCD163)-CA-LCL-αCD163. We show, that activation of STAT3 (by phosphorylation) was inhibited by CA-LCL-αCD163 specifically within CD163pos cells, with minor effect on CD163neg cells. Furthermore, CA-LCL-αCD163 inhibited STAT3-regulated gene expression of IL-10, and increased expression of TNFα, thus indicating a pro-inflammatory effect of the drug on human macrophages. This M1-like reprogramming at the mRNA level was confirmed by significantly elevated levels of pro-inflammatory cytokines (IFNγ, IL-12, TNFα, IL-2) in the culture medium. Since liposomes are attractive vehicles for novel anti-cancer drugs, and since direct TAM-targeting may decrease adverse effects of systemic inhibition of STAT3, the present results encourage future investigation of CA-LCL-αCD163 in the in vivo setting.

Anti-Inflammatory Modulation of Microglia via CD163-Targeted Glucocorticoids Protects Dopaminergic Neurons in the 6-OHDA Parkinson's Disease Model.

UNLABELLED: Increasing evidence supports a decisive role for inflammation in the neurodegenerative process of Parkinson's disease (PD). The immune response in PD seems to involve, not only microglia, but also other immune cells infiltrated into the brain. Indeed, we observed here the infiltration of macrophages, specifically CD163+ macrophages, into the area of neurodegeneration in the 6-hydroxydopamine (6-OHDA) PD model. Therefore, we investigated the therapeutic potential of the infiltrated CD163+ macrophages to modulate local microglia in the brain to achieve neuroprotection. To do so, we designed liposomes targeted for the CD163 receptor to deliver dexamethasone (Dexa) into the CD163+ macrophages in the 6-OHDA PD model. Our data show that a fraction of the CD163-targeted liposomes were carried into the brain after peripheral intravenous injection. The 6-OHDA-lesioned rats that received repeated intravenous CD163-targeted liposomes with Dexa for 3 weeks exhibited better motor performance than the control groups and had minimal glucocorticoid-driven side effects. Furthermore, these animals showed better survival of dopaminergic neurons in substantia nigra and an increased number of microglia expressing major histocompatibility complex II. Therefore, rats receiving CD163-targeted liposomes with Dexa were partially protected against 6-OHDA-induced dopaminergic neurodegeneration, which correlated with a distinctive microglia response. Altogether, our data support the use of macrophages for the modulation of brain neurodegeneration and specifically highlight the potential of CD163-targeted liposomes as a therapeutic tool in PD. SIGNIFICANCE STATEMENT: The immune response now evident in the progression of Parkinson's disease comprises both local microglia and other immune cells. We provide evidence that CD163+ macrophages can be a target to modulate brain immune response to achieve neuroprotection in the 6-hydroxydopamine model. To do so, we targeted the CD163+ population, which to a low but significant extent infiltrated in the neurodegenerating area of the brain. Specially designed liposomes targeted for the CD163 receptor were loaded with glucocorticoids and injected peripherally to modify the infiltrated CD163 cells toward an anti-inflammatory profile. This modification of the CD163 population resulted in a distinctive microglial response that correlated with decreased dopaminergic cell death and better motor performance.

The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen.

Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd) system. However, the host has its own mechanism to recapture the free Hb via haptoglobin (Hp) binding and uptake of Hb-Hp by the CD163 receptor in macrophages. It has so far remained unclear how the Isd system competes with this host iron recycling system in situ to obtain the important nutrient. By binding and uptake studies, we now show that the IsdH protein, which serves as an Hb receptor in the Isd system, directly interferes with the CD163-mediated clearance by binding the Hb-Hp complex and inhibiting CD163 recognition. Analysis of truncated IsdH variants including one or more of three near iron transporter domains, IsdHN1, IsdHN2, and IsdHN3, revealed that Hb binding of IsdHN1 and IsdHN2 accounted for the high affinity for Hb-Hp complexes. The third near iron transporter domain, IsdHN3, exhibited redox-dependent heme extraction, when Hb in the Hb-Hp complex was in the oxidized met form but not in the reduced oxy form. IsdB, the other S. aureus Hb receptor, failed to extract heme from Hb-Hp, and it was a poor competitor for Hb-Hp binding to CD163. This indicates that Hb recognition by IsdH, but not by IsdB, sterically inhibits the receptor recognition of Hb-Hp. This function of IsdH may have an overall stimulatory effect on S. aureus heme acquisition and growth.

A disintegrin and metalloprotease-17 and galectin-9 are important regulators of local 4-1BB activity and disease outcome in rheumatoid arthritis.

OBJECTIVE: Co-stimulatory T cell cytokines are important in the progression of RA. This study investigates the interplay between 4-1BB, a disintegrin and metalloprotease-17 (ADAM17) and galectin-9 (Gal-9) in RA. METHODS: Stimulated mononuclear cells from patients with chronic RA (n = 12) were co-incubated with tissue inhibitor of metalloproteinase, 4-1BB ligand and Gal-9. Plasma samples were examined for soluble 4-1BB (s4-1BB) in newly diagnosed, treatment-naïve patients with RA (n = 97). The 28-joint DAS with CRP (28DAS-CRP), total Sharp score, erosion score and joint space narrowing were used to evaluate treatment outcome serially over a 2-year period. RESULTS: RA CD4(+) and CD8(+) synovial T cells express high levels of 4-1BB. The addition of TNF-α to cultured synovial mononuclear cells increased shedding of 4-1BB. 4-1BB ligand only increased TNF-α shedding in combination with Gal-9. RNA interference-mediated knockdown of ADAM17 or the addition of an ADAM17 inhibitor reduced the 4-1BB shedding. Shedding of 4-1BB was not influenced by Gal-9. Plasma levels of s4-1BB were increased in early RA and correlated with the number of swollen joints at baseline. After 3 months of treatment, the plasma levels of s4-1BB were equal to those of the controls. Baseline plasma levels of s4-1BB were inversely correlated with DAS28-CRP after 2 years of treatment, but not with total Sharp score, erosion score or joint space narrowing. CONCLUSION: ADAM17 induces 4-1BB shedding in RA. Gal-9 is pivotal for the function of 4-1BB and induction of TNF-α. Furthermore, high plasma levels of s4-1BB were associated with the number of swollen joints, but also with a low DAS28-CRP after 2 years treatment in early RA.

Structural basis for inflammation-driven shedding of CD163 ectodomain and tumor necrosis factor-α in macrophages.

The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primates.

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.

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.

Targeting dexamethasone to macrophages in a porcine endotoxemic model.

OBJECTIVES: Macrophages are important cells in immunity and the main producers of pro-inflammatory cytokines. The main objective was to evaluate if specific delivery of glucocorticoid to the macrophage receptor CD163 is superior to systemic glucocorticoid therapy in dampening the cytokine response to lipopolysaccharide infusion in pigs. DESIGN: Two randomized, placebo-controlled trials. SETTING: University hospital laboratory. SUBJECTS: Female farm-bred pigs (26-31 kg). DESIGN: A humanized antibody that binds to pig and human CD163 was produced, characterized, and conjugated with dexamethasone. In the first study (total n = 12), pigs were randomly assigned to four groups: 1) saline; 2) dexamethasone (1.0 mg/kg); 3) dexamethasone (0.02 mg/kg); and 4) anti-CD163-conjugated dexamethasone (0.02 mg/kg). In the second study (total n = 36), two additional groups were included in addition to the four original groups: 5) anti-CD163-conjugated dexamethasone (0.005 mg/kg); 6) unconjugated anti-CD163. Treatments were given 20 hours prior to infusion of lipopolysaccharide (1 µg × kg × h) for 5 hours. Blood samples were analyzed for cytokines, cortisol, and adrenocorticotropic hormone. RESULTS: In the saline group, lipopolysaccharide increased cytokine and plasma cortisol levels. In both studies, dexamethasone (1 mg/kg) and anti-CD163 dexamethasone (0.02 mg/kg) uniformly attenuated tumor necrosis factor-α peak levels (both p < 0.05) compared with low-dose dexamethasone (0.02 mg/kg). However, dexamethasone 1 mg/kg significantly suppressed plasma cortisol and adrenocorticotropic hormone levels compared with anti-CD163 dexamethasone (0.02 mg/kg; p < 0.05). No significant hemodynamic difference existed between groups. The anti-CD163 dexamethasone drug conjugate exhibited a fast plasma clearance, with a half-life of approximately 5-8 minutes. CONCLUSION: Targeted delivery of dexamethasone to macrophages using a humanized CD163 antibody as carrier exhibits anti-inflammatory effects comparable with 50 times higher concentrations of free dexamethasone and does not inhibit endogenous cortisol production. This antibody-drug complex showing similar affinity and specificity for human CD163 is, therefore, a promising drug candidate in this novel type of anti-inflammatory therapy.

Targeting the hemoglobin scavenger receptor CD163 in macrophages highly increases the anti-inflammatory potency of dexamethasone.

Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery.

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.

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.

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.

Tumor-promoting macrophages induce the expression of the macrophage-specific receptor CD163 in malignant cells.

Tumor-associated macrophages (TAMs) represent a distinct malignancy-promoting phenotype suggested to play a key role in tumor formation and metastasis. We aimed to investigate the expression of the monocyte/macrophage-restricted receptor CD163 in bladder tumor biopsies and assess the potential mechanism inducing the CD163 expression in tumor cells. A high CD163 mRNA expression (n = 87) was significantly associated with a poor 13-year overall survival (log-rank test, χ(2) = 8.931; p = 0.0028). Moreover, CD163 mRNA expression was significantly increased in muscle invasive (T2-T4), p = 0.017, and aggressive (grade III/IV) cancers (p = 0.015). The expression strongly correlated with local expression of IL-6 (r = 0.72; p <0.0001) and IL-10 (r = 0.75; p <0.0001), mediators known to induce CD163 expression in vitro. CD163 immunostaining (n = 46) confirmed the association between dense TAM infiltration and histologically advanced disease. In 39% of the biopsies, CD163 immunoreactivity was also observed in tumor cells, and CD163-expressing metastatic cells were identified in lymph node biopsies (n = 8). Bladder cancer cell lines did not express CD163; however, when cocultured with macrophages the bladder cancer cell expression of CD163 was significantly induced in an IL-6/IL-10 independent manner. In conclusion, we show a strong association between CD163 mRNA expression in bladder cancer biopsies and poor patient outcome. CD163 expression was not confined to the infiltrating TAMs, but was also expressed by a significant portion of the malignant cells in both tumors and lymph nodes. CD163 expressing tumor cells may constitute a subpopulation of tumor cells with a phenotypic shift associated with epithelial-to-mesenchymal transition (EMT) and increased metastatic activity induced by TAMs.