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The inflammatory and IFN pathways of innate immunity play a key role in both resistance and pathogenesis of Coronavirus Disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-Associated Molecular Patterns (SAMPs) remain to be completely defined. Here we identify single-stranded RNA (ssRNA) fragments from SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and functions, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identify TLR7/8 as crucial cellular sensors of ssRNAs encoded by SARS-CoV-2 involved in host resistance and disease pathogenesis of COVID-19.

Chemical induced carcinogenesis together with genetically engineered mouse models represent important approaches for the study of the complex mechanisms involving genotype and environmental factors in cancer development, including lung cancer. The induction of lung tumor in mice with urethane (ethyl carbamate) is considered a valuable model of Kras-driven lung cancer. However, inbred mouse strains show variable susceptibility to lung tumor formation, with C57BL/6 background, widely used to study many transgenic and null mutations, highly resistant to lung carcinogenesis. Here is described a protocol of urethane-induced lung cancer effective in lung tumor induction in C57BL/6J strain. Multiple urethane injections are needed to overcome genetic resistance and induce in a reproducible manner lung carcinogenesis in C57BL/6J background mice.

CCRL2 is a seven-transmembrane domain receptor that belongs to the chemokine receptor family. At difference from other members of this family, CCRL2 does not promote chemotaxis and shares structural features with atypical chemokine receptors (ACKRs). However, CCRL2 also differs from ACKRs since it does not bind chemokines and is devoid of scavenging functions. The only commonly recognized CCRL2 ligand is chemerin, a non-chemokine chemotactic protein. CCRL2 is expressed both by leukocytes and non-hematopoietic cells. The genetic ablation of CCRL2 has been instrumental to elucidate the role of this receptor as positive or negative regulator of inflammation. CCRL2 modulates leukocyte migration by two main mechanisms. First, when CCRL2 is expressed by barrier cells, such endothelial, and epithelial cells, it acts as a presenting molecule, contributing to the formation of a non-soluble chemotactic gradient for leukocytes expressing CMKLR1, the functional chemerin receptor. This mechanism was shown to be crucial in the induction of NK cell-dependent immune surveillance in lung cancer progression and metastasis. Second, by forming heterocomplexes with other chemokine receptors. For instance, CCRL2/CXCR2 heterodimers were shown to regulate the activation of β2-integrins in mouse neutrophils. This mini-review summarizes the current understanding of CCRL2 biology, based on experimental evidence obtained by the genetic deletion of this receptor in in vivo experimental models. Further studies are required to highlight the complex functional role of CCRL2 in different organs and pathological conditions.

Dendritic cells (DCs) constitute a complex network of cell subsets with common functions but also with many divergent aspects. All dendritic cell subsets share the ability to prime T cell response and to undergo a complex trafficking program related to their stage of maturation and function. For these reasons, dendritic cells are implicated in a large variety of both protective and detrimental immune responses, including a crucial role in promoting anti-tumor responses. Although cDC1s are the most potent subset in tumor antigen cross-presentation, they are not sufficient to induce full-strength anti-tumor cytotoxic T cell response and need close interaction and cooperativity with the other dendritic cell subsets, namely cDC2s and pDCs. This review will take into consideration different aspects of DC biology, including the functional role of dendritic cell subsets in both fostering and suppressing tumor growth, the mechanisms underlying their recruitment into the tumor microenvironment, as well as the prognostic value and the potentiality of dendritic cell therapeutic targeting. Understanding the specificity of dendritic cell subsets will allow to gain insights on role of these cells in pathological conditions and to design new selective promising therapeutic approaches.

Dendritic cells (DCs) are professional antigen-presenting cells responsible for the activation of specific T-cell responses and for the development of immune tolerance. Immature DCs reside in peripheral tissues and specialize in antigen capture, whereas mature DCs reside mostly in the secondary lymphoid organs where they act as antigen-presenting cells. The correct localization of DCs is strictly regulated by a large variety of chemotactic and nonchemotactic signals that include bacterial products, DAMPs (danger-associated molecular patterns), complement proteins, lipids, and chemokines. These signals function both individually and in concert, generating a complex regulatory network. This network is regulated at multiple levels through different strategies, such as synergistic interactions, proteolytic processing, and the actions of atypical chemokine receptors. Understanding this complex scenario will help to clarify the role of DCs in different pathological conditions, such as autoimmune diseases and cancers and will uncover new molecular targets for therapeutic interventions.

Inflammatory reactions occurring in the central nervous system (CNS), known as neuroinflammation, are key components of the pathogenic mechanisms underlying several neurological diseases. The chemokine system plays a crucial role in the recruitment and activation of immune and non-immune cells in the brain, as well as in the regulation of microglia phenotype and function. Chemokines belong to a heterogeneous family of chemotactic agonists that signal through the interaction with G protein-coupled receptors (GPCRs). Recently, a small subset of chemokine receptors, now identified as “atypical chemokine receptors” (ACKRs), has been described. These receptors lack classic GPCR signaling and chemotactic activity and are believed to limit inflammation through their ability to scavenge chemokines at the inflammatory sites. Recent studies have highlighted a role for ACKRs in neuroinflammation. However, in the CNS, the role of ACKRs seems to be more complex than the simple control of inflammation. For instance, CXCR7/ACKR3 was shown to control T cell trafficking through the regulation of CXCL12 internalization at CNS endothelial barriers. Furthermore, D6/ACKR2 KO mice were protected in a model of experimental autoimmune encephalomyelitis (EAE). D6/ACKR2 KO showed an abnormal accumulation of dendritic cells at the immunization and a subsequent impairment in T cell priming. Finally, CCRL2, an ACKR-related protein, was shown to play a role in the control of the resolution phase of EAE. Indeed, CCRL2 KO mice showed exacerbated, non-resolving disease with protracted inflammation and increased demyelination. This phenotype was associated with increased microglia and macrophage activation markers and imbalanced M1 vs. M2 polarization. This review will summarize the current knowledge on the role of the ACKRs in neuroinflammation with a particular attention to their role in microglial polarization and function.

In spite of their presumed relevance in maintaining interalveolar septal fluid homeostasis, the knowledge of the anatomy of human lung lymphatics is still incomplete. The recent discovery of reliable markers specific for lymphatic endothelium has led to the observation that, contrary to previous assumptions, human lymphatic vessels extend deep inside the pulmonary lobule in association with bronchioles, intralobular arterioles or small pulmonary veins. The aim of this study was to provide a morphometric characterization of lymphatic vessels in the periphery of the human lung. Human lung sections were immunolabelled with the lymphatic marker D2-40, followed by blood vessel staining with von Willebrand Factor. Lymphatic vessels were classified into: intralobular (including those associated with bronchovascular bundles, perivascular, peribronchiolar and interalveolar), pleural (in the connective tissue of the visceral pleura), and interlobular (in interlobular septa). The percentage area occupied by the lymphatic lumen was much greater in the interlobular septa and in the subpleural space than in the lobule. Most of the intralobular lymphatic vessels were in close contact with a blood vessel, either alone or within a bronchovascular bundle, whereas 7% were associated with a bronchiole and < 1% were not connected to blood vessels or bronchioles (interalveolar). Intralobular lymphatic size progressively decreased from bronchovascular through to peribronchiolar, perivascular and interalveolar lymphatics. Lymphatics associated with bronchovascular bundles had similar morphometric characteristics to pleural and interlobular lymphatics. Shape factors were similar across lymphatic populations, except that peribronchiolar lymphatics had a marginally increased roundness and circularity, suggesting a more regular shape due to increased filling, and interlobular lymphatics had greater elongation, due to a greater proportion of conducting lymphatics cut longitudinally. Unsupervised cluster analysis confirmed a marked heterogeneity of lymphatic vessels both within and between groups, with a cluster of smaller vessels specifically represented in perivascular and interalveolar lymphatics within the alveolar interstitium. Our data indicate that intralobular lymphatics are a heterogeneous population, including vessels surrounding the bronchovascular bundle analogous to the conducting vessels present in the pleural and interlobular septa, many small perivascular lymphatics responsible for maintaining fluid balance in the alveolar interstitium, and a minority of intermediate lymphatics draining the peripheral airways. These lymphatic populations could be differentially involved in the pathogenesis of diseases preferentially involving distinct lung compartments.

CCRL2 is a nonsignaling seven-transmembrane domain receptor. CCRL2 binds chemerin, a protein that promotes chemotaxis of leukocytes, including macrophages and natural killer (NK) cells. In addition, CCRL2 controls the inflammatory response in different pathologic settings, such as hypersensitivity, inflammatory arthritis, and experimental autoimmune encephalitis. Here, we investigated the role of CCRL2 in the regulation of lung cancer–related inflammation. The genetic deletion of Ccrl2 promoted tumor progression in urethane-induced and in KrasG12D/+/p53LoxP lung tumor mouse models. Similarly, a Kras-mutant lung tumor displayed enhanced growth in Ccrl2-deficient mice. This phenotype was associated with a reduced inflammatory infiltrate characterized by the impaired recruitment of several leukocyte populations including NK cells. Bone marrow chimeras showed that CCRL2 expression by the nonhematopoietic cell compartment was responsible for the increased tumor formation observed in Kras-mutant Ccrl2-deficient mice. In human and mouse lungs, CCRL2 was expressed by a fraction of CD31+ endothelial cells, where it could control NK infiltration. Elevated CCRL2 expression in biopsies from human lung adenocarcinoma positively correlated with clinical outcome. These results provide evidence for a crucial role of CCRL2 in shaping an anti–lung tumor immune response.

Background & Aims A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. Methods CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14 + with CCA-sphere conditioned medium. Results CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction ( p =0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14 + macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro -educated macrophages. Consistent with invasive features, the largest CD163 + set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect. Conclusion CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease. Lay summary Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.