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

IFN\u3b1 enhances the production of IL-6 by human neutrophils activated via TLR8.

PURPOSE Recently, we reported that human neutrophils produce biologically active IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. Herein, we investigated the effect of IFN\u3b1, a cytokine known to modulate the early innate immune responses toward viral and bacterial infections, on the production of IL-6 by TLR8-activated neutrophils. METHODS Human neutrophils isolated from healthy donors or systemic lupus erythematosus (SLE) patients by negative selection using immunomagnetic beads (99.7 \ub1 0.2 % purity), were incubated for up to 20 h with or without 5 \u3bcM R848 (a TLR8 agonist), in the presence or the absence of 1000 U/ml IFN\u3b1. mRNA expression and cytokine production were then measured by, respectively, RT-qPCR and ELISA, while C/EBP\u3b2 transcription factor recruitment at the IL-6 genomic locus was investigated by chromatin immunoprecipitation (ChIP) assays. RESULTS In this study, we demonstrate that IFN\u3b1 potently enhances the production of IL-6 in neutrophils incubated with R848. Such an effect is not caused by an IFN\u3b1-dependent induction of TLR7, another receptor for R848, but, rather, by an increased release of TNF\u3b1, which in turn amplifies IL-6 expression. Endogenous TNF\u3b1, in fact, was shown to promote an augmented synthesis of the IkB\u3b6 coactivator and an enhanced recruitment of C/EBP\u3b2 to the IL-6 promoter. Moreover, our data uncover that neutrophils from active SLE patients, displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNF\u3b1 in response to R848 as compared to healthy donors. DISCUSSION Altogether, data clarify the molecular bases of the IFN\u3b1-dependent enhancement of IL-6 production in TLR8-activated neutrophils. More in general, we show that TLR8 ligands, IFN\u3b1 and TNF\u3b1, three players often coexisting in many diseases of viral or autoimmune origin, promote a strong production of IL-6 in human neutrophils, placing the same neutrophils among potential targets for immunotherapeutic interventions

IFN\u3b1 enhances the production of IL-6 by human neutrophils activated via TLR8

Purpose: Recently, we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. Herein, is investigated the effect of IFN\u3b1, a potent antiviral cytokine, on IL-6 production. Methods: Human neutrophils isolated from whole blood of healthy donors or systemic lupus erythematosus (SLE) patients by immunomagnetic beads (99.7 \ub1 0.2 % purity), were incubated for up to 20 h with or without R848 (a TLR8 agonists), in the presence or the absence of IFN\u3b1. Cytokine production and mRNA expression were then measured by ELISA and RT-qPCR, respectively, while analysis of C/EBP\u3b2 recruitment at the IL-6 genomic locus was investigated by chromatin immunoprecipitation assays. Results: In this study, data demonstrate that IFN\u3b1 potently enhances the production of IL-6 in neutrophils stimulated with R848. Such an effect is not caused by an IFN\u3b1-dependent induction of TLR7, another receptor for R848, but, rather, it is substantially mediated by an increased release of endogenous TNF\u3b1. The latter cytokine, in an autocrine manner, leads to an augmented synthesis of the I\u3baB\u3b6 co-activator and an enhanced recruitment of the C/EBP\u3b2 transcription factor to the IL-6 promoter. Moreover, data demonstrate that neutrophils from active SLE patients, thus displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNF\u3b1 in response to R848 as compared to healthy donors. Discussion: TLR8 ligands, IFN\u3b1 and TNF\u3b1, three players often coexisting in many diseases of viral or autoimmune origin, promote a strong production of IL-6 in human neutrophils, placing this cell type among potential targets for immunotherapeutic interventions. Conclusions: Altogether, data clarify the molecular bases of the IFN\u3b1-dependent enhancement of IL-6 production in TLR8-activated neutrophil

IFN\u3b1 enhances the production of IL-6 by human neutrophils activated via TLR8.

Recently, we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. In this study, we demonstrate that IFN\u3b1, a cytokine that modulates the early innate immune responses toward viral and bacterial infections, potently enhances the production of IL-6 in neutrophils stimulated with R848, a TLR8 agonist. We also show that such an effect is not caused by an IFN\u3b1-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848, but, rather, it is substantially mediated by an increased production and release of endogenous TNF\u3b1. The latter cytokine, in an autocrine manner, leads to an augmented synthesis of the IkB\u3b6 co-activator and an enhanced recruitment of the C/EBP\u3b2 transcription factor to the IL-6 promoter. Moreover, we show that neutrophils from SLE patients with active disease state, hence displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNF\u3b1 in response to R848 as compared to healthy donors. Altogether, data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils, which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases

Human neutrophils activated by TLR8 agonists, with or without IFNγ, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct α (IL-12A, IL-23A, and IL-27A) with two β (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFNγ for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNFα and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFNγ in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFNγ, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFNγ plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo

CD66b-CD64dimCD115- cells in the human bone marrow represent neutrophil-committed progenitors

Here, we report the identification of human CD66b-CD64dimCD115-neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/- subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA-, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. scRNA-seq analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene (ISG) signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express ISGs in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described eNePs, proNeus and COVID-19 proNeus. Altogether, data shed light on the very early phases of neutrophil ontogeny