ß2 Integrins on Dendritic Cells Modulate Cytokine Signaling and Inflammation-Associated Gene Expression, and Are Required for Induction of Autoimmune Encephalomyelitis.

Heterodimeric ß2 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 ß2 (CD18) subunit, which hampers the analysis of the cell type-specific role of ß2 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 ß2 integrins, specifically in dendritic cells (DCs). Stimulated ß2-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 ß2 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 ß2 integrins in vivo.

Inhibitors of the Actin-Bundling Protein Fascin-1 Developed for Tumor Therapy Attenuate the T-Cell Stimulatory Properties of Dendritic Cells.

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.

Density of Conjugated Antibody Determines the Extent of Fc Receptor Dependent Capture of Nanoparticles by Liver Sinusoidal Endothelial Cells.

Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB-antibody conjugates by in vivo imaging and flow cytometry. We observed that PB-antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB-antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab')2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab')2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells.

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.

Friend virus limits adaptive cellular immune responses by imprinting a maturation-resistant and T helper type 2-biased immunophenotype in dendritic cells.

The murine Friend virus (FV) retrovirus model has been widely used to study anti-viral immune responses, and virus-induced cancer. Here we analyzed FV immune evasion mechanisms on the level of dendritic cells (DC) essential for the induction of primary adaptive immune responses. Comparative quantitative proteome analysis of FV-infected DC (FV-DC) of different genotypes (BALB/c, C57BL/6) and non-infected DC revealed numerous genotype-independently regulated proteins rergulating metabolic activity, cytoskeletal rearrangements, and antigen processing/presentation. These alterations may promote virion production in FV-DC. Stimulation of FV-DC with LPS resulted in strongly enhanced IL-10 production which was partially responsible for their attenuated T cell (CD4+, CD8+) stimulatory capacity. Stimulated FV-DC induced less IFN-γ production in T cells required for cellular anti-viral responses, but more T helper cell type 2 (Th2)-associated cytokines (IL-4, IL-5, IL-13). We conclude that FV reprograms DC to promote viral spreading and immune deviation by imprinting a largely maturation-resistant, Th2-biased immunophenotype.

Vaccination with trifunctional nanoparticles that address CD8+ dendritic cells inhibits growth of established melanoma.

AIM: We wanted to assess the potency of a trifunctional nanoparticle (NP) that targeted and activated CD8+ dendritic cells (DC) and delivered an antigen to induce antitumor responses. MATERIALS & METHODS: The DC targeting and activating properties of ferrous NPs conjugated with immunostimulatory CpG-oligonucleotides, anti-DEC205 antibody and ovalbumin (OVA) as a model antigen to induce antigen-specific T-cell responses and antitumor responses were analyzed. RESULTS: OVA-loaded NP conjugated with immunostimulatory CpG-oligonucleotides and anti-DEC205 antibody efficiently targeted and activated CD8+ DC in vivo, and induced strong OVA-specific T-cell activation. Vaccination of B16/OVA tumor-burdened mice with this NP formulation resulted in tumor growth arrest. CONCLUSION: CD8+ DC-targeting trifunctional nanocarriers bear significant potential for antitumor immunotherapy.

Selective uptake of cylindrical poly(2-oxazoline) brush-antiDEC205 antibody-OVA antigen conjugates into DEC-positive dendritic cells and subsequent T-cell activation.

To achieve specific cell targeting by various receptors for oligosaccharides or antibodies, a carrier must not be taken up by any of the very many different cells and needs functional groups prone to clean conjugation chemistry to derive well-defined structures with a high biological specificity. A polymeric nanocarrier is presented that consists of a cylindrical brush polymer with poly-2-oxazoline side chains carrying an azide functional group on each of the many side chain ends. After click conjugation of dye and an anti-DEC205 antibody to the periphery of the cylindrical brush polymer, antibody-mediated specific binding and uptake into DEC205(+) -positive mouse bone marrow-derived dendritic cells (BMDC) was observed, whereas binding and uptake by DEC205(-) negative BMDC and non-DC was essentially absent. Additional conjugation of an antigen peptide yielded a multifunctional polymer structure with a much stronger antigen-specific T-cell stimulatory capacity of pretreated BMDC than application of antigen or polymer-antigen conjugate.

A trifunctional dextran-based nanovaccine targets and activates murine dendritic cells, and induces potent cellular and humoral immune responses in vivo.

Dendritic cells (DCs) constitute an attractive target for specific delivery of nanovaccines for immunotherapeutic applications. Here we tested nano-sized dextran (DEX) particles to serve as a DC-addressing nanocarrier platform. Non-functionalized DEX particles had no immunomodulatory effect on bone marrow (BM)-derived murine DCs in vitro. However, when adsorbed with ovalbumine (OVA), DEX particles were efficiently engulfed by BM-DCs in a mannose receptor-dependent manner. A DEX-based nanovaccine containing OVA and lipopolysaccharide (LPS) as a DC stimulus induced strong OVA peptide-specific CD4(+) and CD8(+) T cell proliferation both in vitro and upon systemic application in mice, as well as a robust OVA-specific humoral immune response (IgG1>IgG2a) in vivo. Accordingly, this nanovaccine also raised both a more pronounced delayed-type hypersensitivity response and a stronger induction of cytotoxic CD8(+) T cells than obtained upon administration of OVA and LPS in soluble form. Therefore, DEX-based nanoparticles constitute a potent, versatile and easy to prepare nanovaccine platform for immunotherapeutic approaches.

Perforin deficiency attenuates inflammation and tumor growth in colitis-associated cancer.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have a markedly increased risk to develop colon cancer, but there are only limited data about the host antitumor response in such colitis-associated cancer. In the present study we aimed at assessing the role of perforin-dependent effector mechanisms in the immune response in a murine model of colitis-associated colon cancer. METHODS: Wildtype and perforin-deficient mice were analyzed in a mouse model of colitis-associated colon cancer using azoxymethane (AOM) and dextran sodium sulfate (DSS). RESULTS: Tumors of wildtype mice showed infiltration of CD4+, CD8+ T cells, natural killer (NK) cells, high numbers of apoptotic cells, and expression of the transcription factor eomesodermin and cytotoxic effector proteins, suggesting a potential role of the antitumor immune response in AOM/DSS tumorigenesis. Furthermore, perforin deficiency resulted in reduced apoptosis of epithelial cells as compared to wildtype mice, whereas tumor infiltration by NK cells, CD8+, and CD4+ T cells was unchanged. However, perforin-deficient mice surprisingly developed significantly fewer tumors than wildtype mice. Subsequent studies identified an important role of perforin in regulating colitis activity, as perforin deficiency caused a significant reduction of DSS colitis activity and proinflammatory cytokine production as compared to wildtype controls. CONCLUSIONS: Perforin is involved in both the antitumor immune response and the regulation of activity of mucosal inflammation in colitis-associated cancer. Our data emphasize the possible consequences for therapeutic strategies targeting colitis-associated colon cancer.

Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue.

Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis. Finally, the isolated cells can be used for further investigations like comparative studies of mRNA expression, cell-proliferation assay or protein analysis. This protocol can be completed within 6-7 h.

Protection from lethal septic peritonitis by neutralizing the biological function of interleukin 27.

The immune response to bacterial infections must be tightly controlled to guarantee pathogen elimination while preventing tissue damage by uncontrolled inflammation. Here, we demonstrate a key role of interleukin (IL)-27 in regulating this critical balance. IL-27 was rapidly induced during murine experimental peritonitis induced by cecal ligation and puncture (CLP). Furthermore, mice deficient for the EBI3 subunit of IL-27 were resistant to CLP-induced septic peritonitis as compared with wild-type controls, and this effect could be suppressed by injection of recombinant single-chain IL-27. EBI3-/- mice displayed significantly enhanced neutrophil migration and oxidative burst capacity during CLP, resulting in enhanced bacterial clearance and local control of infection. Subsequent studies demonstrated that IL-27 directly suppresses endotoxin-induced production of reactive oxygen intermediates by isolated primary granulocytes and macrophages. Finally, in vivo blockade of IL-27 function using a newly designed soluble IL-27 receptor fusion protein led to significantly increased survival after CLP as compared with control-treated mice. Collectively, these data identify IL-27 as a key negative regulator of innate immune cell function in septic peritonitis. Furthermore, in vivo blockade of IL-27 is a novel potential therapeutic target for treatment of sepsis.

EBV-induced gene 3 transcription is induced by TLR signaling in primary dendritic cells via NF-kappa B activation.

The EBV-induced gene 3 (EBI3) is expressed in dendritic cells (DCs) and part of the cytokine IL-27 that controls Th cell development. However, its regulated expression in DCs is poorly understood. In the present study we demonstrate that EBI3 is expressed in splenic CD8(-), CD8(+), and plasmacytoid DC subsets and is induced upon TLR signaling. Cloning and functional analysis of the EBI3 promoter using in vivo footprinting and mutagenesis showed that stimulation via TLR2, TLR4, and TLR9 transactivated the promoter in primary DCs via NF-kappaB and Ets binding sites at -90 and -73 bp upstream of the transcriptional start site, respectively. Furthermore, we observed that NF-kappaB p50/p65 and PU.1 were sufficient to transactivate the EBI3 promoter in EBI3-deficient 293 cells. Finally, induced EBI3 gene expression in DCs was reduced or abrogated in TLR-2/TLR4, TLR9, and MyD88 knockout mice, whereas both basal and inducible EBI3 mRNA levels in DCs were strongly suppressed in NF-kappaB p50-deficient mice. In summary, these data suggest that EBI3 expression in DCs is transcriptionally regulated by TLR signaling via MyD88 and NF-kappaB. Thus, EBI3 gene transcription in DCs is induced rapidly by TLR signaling during innate immune responses preceding cytokine driven Th cell development.