Lipocalin 2 modulates dendritic cell activity and shapes immunity to influenza in a microbiome dependent manner.

Lipocalin 2 (LCN2) is a secreted glycoprotein with roles in multiple biological processes. It contributes to host defense by interference with bacterial iron uptake and exerts immunomodulatory functions in various diseases. Here, we aimed to characterize the function of LCN2 in lung macrophages and dendritic cells (DCs) using Lcn2-/- mice. Transcriptome analysis revealed strong LCN2-related effects in CD103+ DCs during homeostasis, with differential regulation of antigen processing and presentation and antiviral immunity pathways. We next validated the relevance of LCN2 in a mouse model of influenza infection, wherein LCN2 protected from excessive weight loss and improved survival. LCN2-deficiency was associated with enlarged mediastinal lymph nodes and increased lung T cell numbers, indicating a dysregulated immune response to influenza infection. Depletion of CD8+ T cells equalized weight loss between WT and Lcn2-/- mice, proving that LCN2 protects from excessive disease morbidity by dampening CD8+ T cell responses. In vivo T cell chimerism and in vitro T cell proliferation assays indicated that improved antigen processing by CD103+ DCs, rather than T cell intrinsic effects of LCN2, contribute to the exacerbated T cell response. Considering the antibacterial potential of LCN2 and that commensal microbes can modulate antiviral immune responses, we speculated that LCN2 might cause the observed influenza phenotype via the microbiome. Comparing the lung and gut microbiome of WT and Lcn2-/- mice by 16S rRNA gene sequencing, we observed profound effects of LCN2 on gut microbial composition. Interestingly, antibiotic treatment or co-housing of WT and Lcn2-/- mice prior to influenza infection equalized lung CD8+ T cell counts, suggesting that the LCN2-related effects are mediated by the microbiome. In summary, our results highlight a novel regulatory function of LCN2 in the modulation of antiviral immunity.

Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.

Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs.

Skin inflammation is not sufficient to break tolerance induced against a novel antigen.

Depending on the cellular and molecular microenvironment, immune responses generated by skin-associated lymphoid tissues can lead to protective immunity against pathogens or to tolerance. In this study, we investigated immune responses to an Ag expressed de novo in adult skin under homeostatic conditions by generating transgenic mice producing the Ag Ova in a Cre-inducible manner in keratinocytes. Expression of Ova was induced in adult mice with a tamoxifen-inducible K5-CreER transgenic line. Although Ova was efficiently expressed by keratinocytes and presented by Langerhans cells after Cre-mediated transgene recombination, adult transgenic mice did not develop any obvious autoimmune disease symptoms like hair or weight loss. Ag-specific T cells were activated after Ova expression as indicated by up-regulation of CD44 and CD69. After in vitro restimulation Ova-specific T cells showed reduced IFN-gamma production suggesting induction of tolerance after Ova expression in the skin. After transfer into Ova-expressing mice, naive OT-1 T cells transiently proliferated in skin-draining lymph nodes, infiltrated the skin but did not cause disease. Topical application of danger signals at the time of Ova induction did also not induce autoimmune disease. The unresponsiveness of Ag-specific T cells after induction of Ova expression could only be circumvented by simultaneous priming with CpG-matured, bone marrow-derived dendritic cells. Our data suggest that low amount of Ag expressed in the induction phase of the immune response results in tolerance even in the presence of danger signals and thereby helps to preserve homeostasis in the skin under normal and pathologic conditions.

The AP-1 transcription factors c-Jun and JunB are essential for CD8α conventional dendritic cell identity.

Dendritic cell (DC) development is orchestrated by lineage-determining transcription factors (TFs). Although, members of the activator-protein-1 (AP-1) family, including Batf3, have been implicated in conventional (c)DC specification, the role of Jun proteins is poorly understood. Here, we identified c-Jun and JunB as essential for cDC1 fate specification and function. In mice, Jun proteins regulate extrinsic and intrinsic pathways, which control CD8α cDC1 diversification, whereas CD103 cDC1 development is unaffected. The loss of c-Jun and JunB in DC progenitors diminishes the CD8α cDC1 pool and thus confers resistance to Listeria monocytogenes infection. Their absence in CD8α cDC1 results in impaired TLR triggering and antigen cross-presentation. Both TFs are required for the maintenance of the CD8α cDC1 subset and suppression of cDC2 identity on a transcriptional and phenotypic level. Taken together, these results demonstrate the essential role of c-Jun and JunB in CD8α cDC1 diversification, function, and maintenance of their identity.

Psoriatic skin inflammation is promoted by c-Jun/AP-1-dependent CCL2 and IL-23 expression in dendritic cells.

Toll-like receptor (TLR) stimulation induces innate immune responses involved in many inflammatory disorders including psoriasis. Although activation of the AP-1 transcription factor complex is common in TLR signaling, the specific involvement and induced targets remain poorly understood. Here, we investigated the role of c-Jun/AP-1 protein in skin inflammation following TLR7 activation using human psoriatic skin, dendritic cells (DC), and genetically engineered mouse models. We show that c-Jun regulates CCL2 production in DCs leading to impaired recruitment of plasmacytoid DCs to inflamed skin after treatment with the TLR7/8 agonist Imiquimod. Furthermore, deletion of c-Jun in DCs or chemical blockade of JNK/c-Jun signaling ameliorates psoriasis-like skin inflammation by reducing IL-23 production in DCs. Importantly, the control of IL-23 and CCL2 by c-Jun is most pronounced in murine type-2 DCs. CCL2 and IL-23 expression co-localize with c-Jun in type-2/inflammatory DCs in human psoriatic skin and JNK-AP-1 inhibition reduces the expression of these targets in TLR7/8-stimulated human DCs. Therefore, c-Jun/AP-1 is a central driver of TLR7-induced immune responses by DCs and JNK/c-Jun a potential therapeutic target in psoriasis.

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.

Specific roles for dendritic cell subsets during initiation and progression of psoriasis.

Several subtypes of APCs are found in psoriasis patients, but their involvement in disease pathogenesis is poorly understood. Here, we investigated the contribution of Langerhans cells (LCs) and plasmacytoid DCs (pDCs) in psoriasis. In human psoriatic lesions and in a psoriasis mouse model (DKO* mice), LCs are severely reduced, whereas pDCs are increased. Depletion of pDCs in DKO* mice prior to psoriasis induction resulted in a milder phenotype, whereas depletion during active disease had no effect. In contrast, while depletion of Langerin-expressing APCs before disease onset had no effect, depletion from diseased mice aggravated psoriasis symptoms. Disease aggravation was due to the absence of LCs, but not other Langerin-expressing APCs. LCs derived from DKO* mice produced increased IL-10 levels, suggesting an immunosuppressive function. Moreover, IL-23 production was high in psoriatic mice and further increased in the absence of LCs. Conversely, pDC depletion resulted in reduced IL-23 production, and therapeutic inhibition of IL-23R signaling ameliorated disease symptoms. Therefore, LCs have an anti-inflammatory role during active psoriatic disease, while pDCs exert an instigatory function during disease initiation.

How imiquimod licenses plasmacytoid dendritic cells to kill tumors.

We have provided evidence for a multifaceted antitumor-function of the Toll-like receptor 7 (TLR7) agonist imiquimod, which rapidly recruits plasmacytoid dendritic cells and possibly other immune cells into tumors by inducing the secretion of CCL2 by dermal cells. Imiquimod induces pDC maturation and their conversion into cytolytic killer cells, which are capable of eliminating tumors independently from the adaptive immune system.

Imiquimod clears tumors in mice independent of adaptive immunity by converting pDCs into tumor-killing effector cells.

Imiquimod is a synthetic compound with antitumor properties; a 5% cream formulation is successfully used to treat skin tumors. The antitumor effect of imiquimod is multifactorial, although its ability to modulate immune responses by triggering TLR7/8 is thought to be key. Among the immune cells suggested to be involved are plasmacytoid DCs (pDCs). However, a direct contribution of pDCs to tumor killing in vivo and the mechanism of their recruitment to imiquimod-treated sites have never been demonstrated. Using a mouse model of melanoma, we have now demonstrated that pDCs can directly clear tumors without the need for the adaptive immune system. Topical imiquimod treatment led to TLR7-dependent and IFN-α/ß receptor 1-dependent (IFNAR1-dependent) upregulation of expression of the chemokine CCL2 in mast cells. This was essential to induce skin inflammation and for the recruitment of pDCs to the skin. The recruited pDCs were CD8α+ and induced tumor regression in a TLR7/MyD88- and IFNAR1-dependent manner. Lack of TLR7 and IFNAR1 or depletion of pDCs or CD8α+ cells from tumor-bearing mice completely abolished the effect of imiquimod. TLR7 was essential for imiquimod-stimulated pDCs to produce IFN-α/ß, which led to TRAIL and granzyme B secretion by pDCs via IFNAR1 signaling. Blocking these cytolytic molecules impaired pDC-mediated tumor killing. Our results demonstrate that imiquimod treatment leads to CCL2-dependent recruitment of pDCs and their transformation into a subset of killer DCs able to directly eliminate tumor cells.