Canonical and atypical chemokine receptors in the neutrophil life cycle.

Chemokines are mainly studied for their local function in the control of leukocyte extravasation in homeostatic and inflammatory conditions. However, they have additional roles at the systemic level including the regulation of the hematopoietic process and leukocyte differentiation. Due to the redundancy and pleiotropicity of the chemokine system, chemokines have often multiple and complex roles in neutrophil differentiation ranging from retention and control of proliferation of progenitors to the mobilization of mature cells from the bone marrow (BM) to the bloodstream and their further differentiation in tissues. Atypical chemokine receptors (ACKRs) are regulators of the chemokine system by controlling chemokine bioavailability and chemokine receptor function. Even though ACKRs bind a wide range of chemokines, they appear to have a selective role in the process of neutrophil production and differentiation. The aim of this review is to give an overview of the current evidence regarding the role of chemokines and chemokine receptors in the life of neutrophils with a focus on the regulation exerted by ACKRs.

CCRL2 Expression by Specialized Lung Capillary Endothelial Cells Controls NK-cell Homing in Lung Cancer.

Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C-C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27- CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell-mediated lung immune surveillance.

Reduced G protein signaling despite impaired internalization and ß-arrestin recruitment in patients carrying a CXCR4Leu317fsX3 mutation causing WHIM syndrome.

WHIM syndrome is an inherited immune disorder caused by an autosomal dominant heterozygous mutation in CXCR4. The disease is characterized by neutropenia/leukopenia (secondary to retention of mature neutrophils in bone marrow), recurrent bacterial infections, treatment-refractory warts, and hypogammaglobulinemia. All mutations reported in WHIM patients lead to the truncations in the C-terminal domain of CXCR4, R334X being the most frequent. This defect prevents receptor internalization and enhances both calcium mobilization and ERK phosphorylation, resulting in increased chemotaxis in response to the unique ligand CXCL12. Here, we describe 3 patients presenting neutropenia and myelokathexis, but normal lymphocyte count and immunoglobulin levels, carrying what we believe to be a novel Leu317fsX3 mutation in CXCR4, leading to a complete truncation of its intracellular tail. The analysis of the L317fsX3 mutation in cells derived from patients and in vitro cellular models reveals unique signaling features in comparison with R334X mutation. The L317fsX3 mutation impairs CXCR4 downregulation and ß-arrestin recruitment in response to CXCL12 and reduces other signaling events - including ERK1/2 phosphorylation, calcium mobilization, and chemotaxis - all processes that are typically enhanced in cells carrying the R334X mutation. Our findings suggest that, overall, the L317fsX3 mutation may be causative of a form of WHIM syndrome not associated with an augmented CXCR4 response to CXCL12.

Chemokines as Regulators of Neutrophils: Focus on Tumors, Therapeutic Targeting, and Immunotherapy.

Neutrophils are an important component of the tumor microenvironment, and their infiltration has been associated with a poor prognosis for most human tumors. However, neutrophils have been shown to be endowed with both protumor and antitumor activities, reflecting their heterogeneity and plasticity in cancer. A growing body of studies has demonstrated that chemokines and chemokine receptors, which are fundamental regulators of neutrophils trafficking, can affect neutrophil maturation and effector functions. Here, we review human and mouse data suggesting that targeting chemokines or chemokine receptors can modulate neutrophil activity and improve their antitumor properties and the efficiency of immunotherapy.

Perioperative corticosteroid treatment impairs tumor-infiltrating dendritic cells in patients with newly diagnosed adult-type diffuse gliomas.

Introduction: Adult-type diffuse gliomas are malignant primary brain tumors characterized by very poor prognosis. Dendritic cells (DCs) are key in priming antitumor effector functions in cancer, but their role in gliomas remains poorly understood. Methods: In this study, we characterized tumor-infiltrating DCs (TIDCs) in adult patients with newly diagnosed diffuse gliomas by using multi-parametric flow cytometry and single-cell RNA sequencing. Results: We demonstrated that different subsets of DCs are present in the glioma microenvironment, whereas they are absent in cancer-free brain parenchyma. The largest cluster of TIDCs was characterized by a transcriptomic profile suggestive of severe functional impairment. Patients undergoing perioperative corticosteroid treatment showed a significant reduction of conventional DC1s, the DC subset with key functions in antitumor immunity. They also showed phenotypic and transcriptional evidence of a more severe functional impairment of TIDCs. Discussion: Overall, the results of this study indicate that functionally impaired DCs are recruited in the glioma microenvironment. They are severely affected by dexamethasone administration, suggesting that the detrimental effects of corticosteroids on DCs may represent one of the mechanisms contributing to the already reported negative prognostic impact of steroids on glioma patient survival.

Immunotherapeutic early-phase clinical trials and malignant gliomas: A single-center experience and comprehensive immunophenotyping of circulating leukocytes.

BACKGROUND: Immunotherapeutic early-phase clinical trials (ieCTs) increasingly adopt large expansion cohorts exploring novel agents across different tumor types. High-grade glioma (HGG) patients are usually excluded from these trials. METHODS: Data of patients with recurrent HGGs treated within multicohort ieCTs between February 2014 and August 2019 (experimental group, EG) at our Phase I Unit were retrospectively reviewed and compared to a matched control group (CG) of patients treated with standard therapies. We retrospectively evaluated clinical, laboratory, and molecular parameters through univariate and multivariate analysis. A prospective characterization of circulating leukocyte subpopulations was performed in the latest twenty patients enrolled in the EG, with a statistical significance cutoff of P < .1. RESULTS: Thirty HGG patients were treated into six ieCTs. Fifteen patients received monotherapies (anti-PD-1, anti-CSF-1R, anti-TGFß, anti-cereblon), fifteen patients combination regimens (anti-PD-L1 + anti-CD38, anti-PD-1 + anti-CSF-1R). In the EG, median progression-free survival and overall survival (OS) from treatment initiation were 1.8 and 8.6 months; twelve patients survived more than 12 months, and two of them more than 6 years. Univariate analysis identified O 6-methylguanine DNA methyltransferase (MGMT) promoter methylation and total protein value at six weeks as significantly correlated with a better outcome. Decreased circulating neutrophils and increased conventional dendritic cells levels lead to significantly better OS. CONCLUSIONS: A subgroup of EG patients achieved remarkably durable disease control. MGMT promoter methylation identifies patients who benefit more from immunotherapy. Monitoring dynamic changes of innate immune cell populations may help to predict clinical outcomes.

Control of Cytoskeletal Dynamics by ß-Arrestin1/Myosin Vb Signaling Regulates Endosomal Sorting and Scavenging Activity of the Atypical Chemokine Receptor ACKR2.

The atypical chemokine receptor ACKR2, formerly named D6, is a scavenger chemokine receptor with a non-redundant role in the control of inflammation and immunity. The scavenging activity of ACKR2 depends on its trafficking properties, which require actin cytoskeleton rearrangements downstream of a ß-arrestin1-Rac1-PAK1-LIMK1-cofilin-dependent signaling pathway. We here demonstrate that in basal conditions, ACKR2 trafficking properties require intact actin and microtubules networks. The dynamic turnover of actin filaments is required to sustain ACKR2 constitutive endocytosis, while both actin and microtubule networks are involved in processes regulating ACKR2 constitutive sorting to rapid, Rab4-dependent and slow, Rab11-dependent recycling pathways, respectively. After chemokine engagement, ACKR2 requires myosin Vb activity to promote its trafficking from Rab11-positive recycling endosomes to the plasma membrane, which sustains its scavenging activity. Other than cofilin phosphorylation, induction of the ß-arrestin1-dependent signaling pathway by ACKR2 agonists also leads to the rearrangement of microtubules, which is required to support the myosin Vb-dependent ACKR2 upregulation and its scavenging properties. Disruption of the actin-based cytoskeleton by the apoptosis-inducing agent staurosporine results in impaired ACKR2 internalization and chemokine degradation that is consistent with the emerging scavenging-independent activity of the receptor in apoptotic neutrophils instrumental for promoting efficient efferocytosis during the resolution of inflammation. In conclusion, we provide evidence that ACKR2 activates a ß-arrestin1-dependent signaling pathway, triggering both the actin and the microtubule cytoskeletal networks, which control its trafficking and scavenger properties.

Multiple Roles for Chemokines in Neutrophil Biology.

Chemokines are recognized as the most critical mediators for selective neutrophil recruitment during inflammatory conditions. Furthermore, they are considered fundamental regulators of neutrophil mobilization from the bone marrow (BM) to the bloodstream and for their homing back at the end of their life for apoptosis and clearance. However, chemokines are also important mediators of neutrophil effector functions including oxidative burst, degranulation, neutrophil extracellular trap (NET)osis, and production of inflammatory mediators. Neutrophils have been historically considered as a homogeneous population. In recent years, several maturation stages and subsets with different phenotypic profiles and effector functions were described both in physiological and pathological conditions such as infections, autoimmunity, and cancer. The aim of this review is to give an overview of the current evidence regarding the role of chemokines and chemokine receptors in neutrophil biology, including their possible role in neutrophil maturation, differentiation, and in defining emerging neutrophil subsets.

Neutrophil diversity and plasticity in tumour progression and therapy.

Neutrophils play a key role in defence against infection and in the activation and regulation of innate and adaptive immunity. In cancer, tumour-associated neutrophils (TANs) have emerged as an important component of the tumour microenvironment. Here, they can exert dual functions. TANs can be part of tumour-promoting inflammation by driving angiogenesis, extracellular matrix remodelling, metastasis and immunosuppression. Conversely, neutrophils can also mediate antitumour responses by direct killing of tumour cells and by participating in cellular networks that mediate antitumour resistance. Neutrophil diversity and plasticity underlie the dual potential of TANs in the tumour microenvironment. Myeloid checkpoints as well as the tumour and tissue contexture shape neutrophil function in response to conventional therapies and immunotherapy. We surmise that neutrophils can provide tools to tailor current immunotherapy strategies and pave the way to myeloid cell-centred therapeutic strategies, which would be complementary to current approaches.

Role of myeloid cells in the immunosuppressive microenvironment in gliomas.

Glioma is the most common primary brain cancer, and half of patients present a diagnosis of glioblastoma (GBM), its most aggressive and lethal form. Conventional therapies, including surgery, radiotherapy, and chemotherapy, have not resulted in major ameliorations in GBM survival outcome, which remains extremely poor. Recent immunotherapy improvements for other tumors, coupled with growing knowledge of the complex interactions between malignant glioma cells and the immune system, led to an exponential increase in glioma immunotherapy research. However, immunotherapeutic strategies in GBM have not yet reached their full potential, mainly due to the limited understanding of the strong immunosuppressive microenvironment (TME) characterizing this tumor. Glioma-associated macrophages and microglia (GAMs) are key drivers of the local immunosuppression promoting tumor progression and its resistance to immunomodulating therapeutic strategies. Together with other myeloid cells, such as dendritic cells and neutrophils, GAMs actively shape glioma TME, modulate anti-tumoral immune response and support angiogenesis, tumor cell invasion and proliferation. In this review, we discuss the role of myeloid cells in the complex TME of glioma and the available clinical data on therapeutic strategies focusing on approaches that affect myeloid cells activity in GBM.

One Clever Macrophage Checkpoint.

Macrophages are a key component of the tumor microenvironment (TME). Blocking Clever-1, a molecule expressed in M2-like macrophages, unleashed macrophage and T-cell-mediated antitumor immunity. Myeloid checkpoints including Clever-1 hold promise as alternative or complementary immunotherapy strategies.See related article by Viitala et al., p. 3289.

Chemokines and Chemokine Receptors: New Targets for Cancer Immunotherapy.

Immunotherapy is a clinically validated treatment for many cancers to boost the immune system against tumor growth and dissemination. Several strategies are used to harness immune cells: monoclonal antibodies against tumor antigens, immune checkpoint inhibitors, vaccination, adoptive cell therapies (e.g., CAR-T cells) and cytokine administration. In the last decades, it is emerging that the chemokine system represents a potential target for immunotherapy. Chemokines, a large family of cytokines with chemotactic activity, and their cognate receptors are expressed by both cancer and stromal cells. Their altered expression in malignancies dictates leukocyte recruitment and activation, angiogenesis, cancer cell proliferation, and metastasis in all the stages of the disease. Here, we review first attempts to inhibit the chemokine system in cancer as a monotherapy or in combination with canonical or immuno-mediated therapies. We also provide recent findings about the role in cancer of atypical chemokine receptors that could become future targets for immunotherapy.

Chemokines sound the alarmin: The role of atypical chemokine in inflammation and cancer.

As main drivers of leukocyte recruitment during inflammatory reactions, chemokines act as mediatrs of alarmins in priming host defense responses after tissue exposure to toxic or infectious agents, immunomediated damage, and in inflammation-driven tumors. Chemokines can therefore be considered alarm signals generated by tissues in a broad number of conditions, and mechanisms controlling chemokines biological activities are therefore key to regulate tissue reactions induced by alarmins. By transporting, presenting or scavenging different sets of chemokines, atypical chemokine receptors represent an emerign subfamily of chemokine receptors which operates in tissues as chemokine gatekeepers in order to establish and shape their gradients and coordinate leukocyte recruitment.

Regulation of hematopoiesis by the chemokine system.

Although chemokines are best known for their role in directing cell migration, accumulating evidence indicate their involvement in many other processes. This review focus on the role of chemokines in hematopoiesis with an emphasis on myelopoiesis. Indeed, many chemokine family members are an important component of the cytokine network present in the bone marrow that controls proliferation, retention, and mobilization of hematopoietic progenitors.

ACKR2 in hematopoietic precursors as a checkpoint of neutrophil release and anti-metastatic activity.

Atypical chemokine receptors (ACKRs) are regulators of leukocyte traffic, inflammation, and immunity. ACKR2 is a scavenger for most inflammatory CC chemokines and is a negative regulator of inflammation. Here we report that ACKR2 is expressed in hematopoietic precursors and downregulated during myeloid differentiation. Genetic inactivation of ACKR2 results in increased levels of inflammatory chemokine receptors and release from the bone marrow of neutrophils with increased anti-metastatic activity. In a model of NeuT-driven primary mammary carcinogenesis ACKR2 deficiency is associated with increased primary tumor growth and protection against metastasis. ACKR2 deficiency results in neutrophil-mediated protection against metastasis in mice orthotopically transplanted with 4T1 mammary carcinoma and intravenously injected with B16F10 melanoma cell lines. Thus, ACKR2 is a key regulator (checkpoint) of mouse myeloid differentiation and function and its targeting unleashes the anti-metastatic activity of neutrophils in mice.

Neutrophils in Gliomas.

Neutrophils are the most abundant white blood cells and are the first recruited to inflammatory sites. Neutrophils are an important component of the tumor stroma and can exert both anti-tumoral and pro-tumoral activities, depending on their maturation and activation state. In human gliomas, the number of circulating and infiltrating neutrophils correlates with the severity of the disease, indicating a prognostic and possible pro-tumoral role for these leukocytes. In glioma preclinical models, neutrophils promote tumor growth and orchestrate the resistance to anti-angiogenic therapies. Nevertheless, recent data indicate that neutrophils can be activated to directly kill tumor cells or to orchestrate the anti-tumoral response. Here, we review current knowledge about the role of neutrophils in glioma and their possible involvement in new strategies to improve current cancer therapies.

Corrigendum: ACKR2: An Atypical Chemokine Receptor Regulating Lymphatic Biology.

[This corrects the article on p. 691 in vol. 7, PMID: 28123388.].

CXCL4 and CXCL4L1 Differentially Affect Monocyte Survival and Dendritic Cell Differentiation and Phagocytosis.

Upon inflammation, circulating monocytes leave the bloodstream and migrate into the tissues, where they differentiate after exposure to various growth factors, cytokines or infectious agents. The best defined macrophage polarization types are M1 and M2. However, the platelet-derived CXC chemokine CXCL4 induces the polarization of macrophages into a unique phenotype. In this study, we compared the effect of CXCL4 and its variant CXCL4L1 on the differentiation of monocytes into macrophages and into immature monocyte-derived dendritic cells (iMDDC). Differently to M-CSF and CXCL4, CXCL4L1 is not a survival factor for monocytes. Moreover, the expression of the chemokine receptors CCR2, CCR5 and CXCR3 was significantly higher on CXCL4L1-treated monocytes compared to M-CSF- and CXCL4-stimulated monocytes. IL-1 receptor antagonist (IL-1RN) expression was upregulated by CXCL4 and downregulated by CXCL4L1, respectively, whereas both chemokines reduced the expression of the mannose receptor (MRC). Furthermore, through activation of CXCR3, CXCL4L1-stimulated monocytes released significantly higher amounts of CCL2 and CXCL8 compared to CXCL4-treated monocytes, indicating more pronounced inflammatory traits for CXCL4L1. In contrast, in CXCL4L1-treated monocytes, the production of CCL22 was lower. Compared to iMDDC generated in the presence of CXCL4L1, CXCL4-treated iMDDC showed an enhanced phagocytic capacity and downregulation of expression of certain surface markers (e.g. CD1a) and specific enzymes (e.g. MMP-9 and MMP-12). CXCL4 and CXCL4L1 did not affect the chemokine receptor expression on iMDDC and cytokine production (CCL2, CCL18, CCL22, CXCL8, IL-10) by CXCL4- or CXCL4L1-differentiated iMDDC was similar. We can conclude that both CXCL4 and CXCL4L1 exert a direct effect on monocytes and iMDDC. However, the resulting phenotypes are different, which suggests a unique role for the two CXCL4 variants in physiology and/or pathology.