Neutrophil-macrophage interplay is a fine-tuning mechanism that regulates the innate immune response during infection and inflammation. Cell surface receptors play an essential role in neutrophil and macrophage functions. The same receptor can provide different outcomes within diverse leukocyte subsets in different inflammatory conditions. Understanding the variety of responses mediated by one receptor is critical for the development of anti-inflammatory treatments. In this study, we evaluated the role of a leukocyte adhesive receptor, integrin αDβ2, in the development of acute inflammation. αDβ2 is mostly expressed on macrophages and contributes to the development of chronic inflammation. In contrast, we found that αD-knockout dramatically increases mortality in the cecal ligation and puncture sepsis model and LPS-induced endotoxemia. This pathologic outcome of αD-deficient mice is associated with a reduced number of monocyte-derived macrophages and an increased number of neutrophils in their lungs. However, the tracking of adoptively transferred fluorescently labeled wild-type (WT) and αD−/− monocytes in WT mice during endotoxemia demonstrated only a moderate difference between the recruitment of these two subsets. Moreover, the rescue experiment, using i.v. injection of WT monocytes to αD-deficient mice followed by LPS challenge, showed only slightly reduced mortality. Surprisingly, the injection of WT neutrophils to the bloodstream of αD−/− mice markedly increased migration of monocyte-derived macrophage to lungs and dramatically improves survival. αD-deficient neutrophils demonstrate increased necrosis/pyroptosis. αDβ2-mediated macrophage accumulation in the lungs promotes efferocytosis that reduced mortality. Hence, integrin αDβ2 implements a complex defense mechanism during endotoxemia, which is mediated by macrophages via a neutrophil-dependent pathway.

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDß2 (CD11d/CD18) and αMß2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDß2-transfected HEK293 cells, WT, and αD-/- mouse M1- polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDß2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDß2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDß2- recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

La cellule NK fait partie du système immunitaire inné et participe à l’immunosurveillance anti-tumorale. La compréhension des facteurs affectant leur biologie, telle que la génétique, est donc cruciale. Dans un premier temps, nous avons évalué le rôle de la sous-unité d’intégrine CD11d, sur le phénotype et l'expression d’autres sous-unités (CD11a, CD11b, CD11c, CD18) de diverses cellules immunitaires entre un modèle murin CD11d-KO et une souris C57BL/6 (B6). Nous avons remarqué que l'effet de la délétion de CD11d sur l'expression des autres sous-unités d'intégrine est spécifique à chaque type de cellule immunitaire. La différence la plus notable dans l'expression de CD11b et CD11c a été observée dans les cellules NK. La perte de CD11d dans les cellules NK a entraîné une diminution de l'expression de CD107a après leur activation, ce qui suggère une altération de la dégranulation des cellules NK. Ensuite, malgré une croissance de mélanome similaire, une plus grande proportion de cellules NK issues de CD11d-KO se sont accumulées dans les lits tumoraux par rapport à leur homologue B6. Dans un deuxième temps, nous avons exploité le modèle murin NOD, connu d’avoir des défauts immunitaires importants. L’acquisition des fonctions cytotoxiques des cellules NK se fait par un processus appelé maturation fonctionnelle où une cellule NK est d’abord CD27-CD11b-, suivi du stade CD27+CD11b-, puis CD27+CD11b+ et finalement CD27-CD11b+, soit le stade le plus mature et cytotoxique. Nous avons démontré que les cellules NK de la souris NOD produisent nettement moins d’IFN-γ, de TNFα et de Granzyme B et échouent à réguler l’expression du récepteur d’activation NKG2D pour chaque stade de maturation fonctionnelle. Finalement, nous avons traité des souris immunodéficientes porteuses de tumeurs avec des cellules NK de NOD et B6. Nous avons démontré que, tout comme pour les cellules NK de B6, ce sont surtout des cellules NK CD27+ de NOD qui s’accumulent dans les tumeurs. Par contre, les souris injectées avec des cellules de NOD montraient une croissance tumorale significativement plus importante. De manière générale, ces études sont les premières à élucider les impacts de l’absence de CD11d sur le phénotype et la fonction des cellules NK ainsi que leurs défauts fonctionnels dans la souris NOD au courant de leur maturation fonctionnelle. / NK cells are part of the innate immune system and participate in anti-tumor immunosurveillance. Understanding the factors affecting their biology, such as genetics, is therefore crucial. First, we evaluated the role of the integrin subunit CD11d on the phenotype and expression of other subunits (CD11a, CD11b, CD11c, CD18) of various immune cells between a CD11d-KO mouse model and a C57BL/6 (B6) mouse. We noted that the effect of CD11d deletion on the expression of other integrin subunits is specific to each immune cell type. The most notable difference in CD11b and CD11c expression was observed in NK cells. Loss of CD11d in NK cells resulted in decreased CD107a expression after their activation, suggesting impaired NK cell degranulation. Second, despite similar melanoma growth, a greater proportion of CD11d-KO-derived NK cells accumulated in tumor beds compared to their B6 counterpart. We then exploited the NOD mouse model, known to have significant immune defects. The acquisition of cytotoxic functions of NK cells occurs through a process called functional maturation where an NK cell is first CD27-CD11b-, followed by the CD27+CD11b- stage, then CD27+CD11b+ and finally CD27-CD11b+, the most mature and cytotoxic stage. We demonstrated that NK cells from NOD mice produce significantly less IFN-γ, TNFα, and Granzyme B and fail to regulate the expression of the activation receptor NKG2D for each stage of functional maturation. Finally, we treated immunodeficient tumor-bearing mice with NOD and B6 NK cells. We demonstrated that, as with B6 NK cells, NOD CD27+ NK cells predominantly accumulated in tumors. However, mice injected with NOD NK cells showed significantly greater tumor growth. Overall, these studies are the first to elucidate the impact of the absence of CD11d on the phenotype and function of NK cells as well as their functional defects in NOD mice during their functional maturation.

Chronic inflammation is essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration/accumulation of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages in damaged tissue. The mechanism of macrophage migration and subsequent accumulation is still not fully understood. Currently, the amoeboid adhesion-independent motility is considered essential for leukocyte migration in the three-dimensional environment. We challenge this hypothesis by studying the contribution of leukocyte adhesive receptors, integrins αMβ2, and αDβ2, to three-dimensional migration of M1-polarized, M2-polarized, and resident macrophages. Both integrins have a moderate expression on M2 macrophages, while αDβ2 is upregulated on M1 and αMβ2 demonstrates high expression on resident macrophages. The level of integrin expression determines its contribution to macrophage migration. Namely, intermediate expression supports macrophage migration, while a high integrin density inhibits it. Using in vitro three-dimensional migration and in vivo tracking of adoptively-transferred fluorescently-labeled macrophages during the resolution of inflammation, we found that strong adhesion of M1-activated macrophages translates to weak 3D migration, while moderate adhesion of M2-activated macrophages generates dynamic motility. Reduced migration of M1 macrophages depends on the high expression of αDβ2, since αD-deficiency decreased M1 macrophage adhesion and improved migration in fibrin matrix and peritoneal tissue. Similarly, the high expression of αMβ2 on resident macrophages prevents their amoeboid migration, which is markedly increased in αM-deficient macrophages. In contrast, αD- and αM-knockouts decrease the migration of M2 macrophages, demonstrating that moderate integrin expression supports cell motility. The results were confirmed in a diet-induced diabetes model. αD deficiency prevents the retention of inflammatory macrophages in adipose tissue and improves metabolic parameters, while αM deficiency does not affect macrophage accumulation. Summarizing, β2 integrin-mediated adhesion may inhibit amoeboid and mesenchymal macrophage migration or support mesenchymal migration in tissue, and, therefore, represents an important target to control inflammation.