ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production.

Chemotaxis is an essential physiological process, often harnessed by tumors for metastasis. CXCR4, its ligand CXCL12 and the atypical receptor ACKR3 are overexpressed in many human cancers. Interfering with this axis by ACKR3 deletion impairs lymphoma cell migration towards CXCL12. Here, we propose a model of how ACKR3 controls the migration of the diffused large B-cell lymphoma VAL cells in vitro and in vivo in response to CXCL12. VAL cells expressing full-length ACKR3, but not a truncated version missing the C-terminus, can support the migration of VAL cells lacking ACKR3 (VAL-ko) when allowed to migrate together. This migration of VAL-ko cells is pertussis toxin-sensitive suggesting the involvement of a Gi-protein coupled receptor. RNAseq analysis indicate the expression of chemotaxis-mediating LTB4 receptors in VAL cells. We found that LTB4 acts synergistically with CXCL12 in stimulating the migration of VAL cells. Pharmacologic or genetic inhibition of BLT1R markedly reduces chemotaxis towards CXCL12 suggesting that LTB4 enhances in a contact-independent manner the migration of lymphoma cells. The results unveil a novel mechanism of cell-to-cell-induced migration of lymphoma.

Protection against influenza infection requires early recognition by inflammatory dendritic cells through C-type lectin receptor SIGN-R1.

The early phase of influenza infection occurs in the upper respiratory tract and the trachea, but little is known about the initial events of virus recognition and control of viral dissemination by the immune system. Here, we report that inflammatory dendritic cells (IDCs) are recruited to the trachea shortly after influenza infection through type I interferon-mediated production of the chemokine CCL2. We further show that recruited IDCs express the C-type lectin receptor SIGN-R1, which mediates direct recognition of the virus by interacting with N-linked glycans present in glycoproteins of the virion envelope. Activation of IDCs via SIGN-R1 triggers the production of the chemokines CCL5, CXCL9 and CXCL10, which initiate the recruitment of protective natural killer (NK) cells in the infected trachea. In the absence of SIGN-R1, the recruitment and activation of NK cells is impaired, leading to uncontrolled viral proliferation. In sum, our results provide insight into the orchestration of the early cellular and molecular events involved in immune protection against influenza.

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal. Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.