The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.

Protease-activated receptor 2 (PAR2) signaling controls skin barrier function and inflammation, but the roles of immune cells and PAR2-activating proteases in cutaneous diseases are poorly understood. To dissect PAR2 signaling contributions to skin inflammation with new genetic and pharmacological tools. We found markedly increased numbers of PAR2⁺ infiltrating myeloid cells in skin lesions of allergic contact dermatitis (ACD) patients and in the skin of contact hypersensitivity (CHS) in mice, a murine ACD model for T cell-mediated allergic skin inflammation. Cell type-specific deletion of PAR2 in myeloid immune cells as well as mutation-induced complete PAR2 cleavage insensitivity significantly reduced skin inflammation and hapten-specific Tc1/Th1 cell response. Pharmacological approaches identified individual proteases involved in PAR2 cleavage and demonstrated a pivotal role of tissue factor (TF) and coagulation factor Xa (FXa) as upstream activators of PAR2 in both the induction and effector phase of CHS. PAR2 mutant mouse strains with differential cleavage sensitivity for FXa versus skin epithelial cell-expressed proteases furthermore uncovered a time-dependent regulation of CHS development with an important function of FXa-induced PAR2 activation during the late phase of skin inflammation. Myeloid cells and the TF-FXa-PAR2 axis are key mediators and potential therapeutic targets in inflammatory skin diseases.

Hepatocytes comprise the majority of the liver and largely exert metabolic functions, whereas non-parenchymal cells (NPCs)—comprising Kupffer cells, dendritic cells and liver sinusoidal endothelial cells—control the immunological state within this organ. Here, we compared the suitability of two isolation methods for murine liver NPCs. Liver perfusion (LP) with collagenase/DNase I applied via the portal vein leads to efficient liver digestion, whereas the modified liver dissociation (LD) method combines mechanical dissociation of the retrieved organ with enzymatic degradation of the extracellular matrix. In cases of both LP and LD, NPCs were enriched by subsequent gradient density centrifugation. Our results indicate that LP and LD are largely comparable with regards to the yield, purity, and composition of liver NPCs. However, LD-enriched liver NPCs displayed a higher degree of activation after overnight cultivation, and accordingly were less responsive towards stimulation with toll-like receptor ligands that are frequently used as adjuvants, e.g., in nano-vaccines. We conclude that LP is more suitable for obtaining liver NPCs for subsequent in vitro studies, whereas LD as the less laborious method, is more convenient for parallel isolation of larger numbers of samples for ex vivo analysis.

Heterodimeric β2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common β2 (CD18) subunit, which hampers the analysis of the cell type-specific role of β2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of β2 integrins, specifically in dendritic cells (DCs). Stimulated β2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2–6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific β2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of β2 integrins in vivo.

Background: Stimulated dendritic cells (DCs), which constitute the most potent population of antigen-presenting cells (APCs), express the actin-bundling protein Fascin-1 (Fscn1). In tumor cells, de novo expression of Fscn1 correlates with their invasive and metastatic properties. Therefore, Fscn1 inhibitors have been developed to serve as antitumor agents. In this study, we were interested in better understanding the impact of Fscn1 inhibitors on DCs. Methods: In parallel settings, murine spleen cells and bone-marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide in the presence of Fscn1 inhibitors (NP-G2-044 and BDP-13176). An analysis of surface expression of costimulatory and coinhibitory receptors, as well as cytokine production, was performed by flow cytometry. Cytoskeletal alterations were assessed by confocal microscopy. The effects on the interactions of BMDCs with antigen-specific T cells were monitored by time lapse microscopy. The T-cell stimulatory and polarizing capacity of BMDCs were measured in proliferation assays and cytokine studies. Results: Administration of Fscn1 inhibitors diminished Fscn1 expression and the formation of dendritic processes by stimulated BMDCs and elevated CD273 (PD-L2) expression. Fscn1 inhibition attenuated the interaction of DCs with antigen-specific T cells and concomitant T-cell proliferation. Conclusions: Systemic administration of Fscn1 inhibitors for tumor therapy may also modulate DC-induced antitumor immune responses.