Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection

Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation

The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell-APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser(5) phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons