Loredana Frasca

Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.

In systemic lupus erythematosus, neutrophils release peptide/self-DNA complexes that trigger plasmacytoid dendritic cell activation and autoantibody formation. Lupus Neutrophils Cast a Wide NET Systemic lupus erythematosus, also known as SLE or lupus, is a systemic, chronic autoimmune disease that can affect the skin, joints, kidneys, and other organs. In lupus, the body’s immune system turns against antigens in the body’s own nuclei, with activated B cells producing antibodies against self-DNA and associated proteins. The resulting immune complexes accumulate in the body, causing inflammation and tissue damage. Now, two new studies, by Lande et al. and Garcia-Romo et al., demonstrate a role for neutrophils and the “neutrophil extracellular traps,” a specialized structure they release when activated, in the pathogenesis of the disease. A key characteristic of lupus is the presence of chronically activated plasmacytoid dendritic cells, which secrete type I interferons. Lupus patients also display increased numbers of immature neutrophils in the blood, but the exact role of neutrophils in the disease had been unclear. Lande et al. began with the observation that patient serum contains immunogenic complexes that include the antimicrobial peptide LL37, human neutrophil peptide (HNP), and self-DNA. These complexes are taken up by and activate dendritic cells, and patients carry antibodies directed against LL37, HNP, and self-DNA. What is the origin of these complexes? Activated neutrophils can undergo NETosis, a particular type of cell death in which their nuclear DNA is released in long chromatin filaments that form web-like structures, neutrophil extracellular traps (NETs). NETs contain antimicrobial peptides, and can entrap bacteria, enabling them to be killed. Lande et al. now show that the anti-LL37 and anti-HNP antibodies present in lupus patient serum can activate neutrophils and induce them to release NETs. Patient-derived neutrophils release more NETs upon exposure to antibody than control neutrophils. In a parallel study, Garcia-Romo et al. look in detail at neutrophils in lupus, and show that lupus patient neutrophils undergo accelerated cell death in culture. Anti-ribonucleoprotein antibodies present in patient serum induce NETosis, and the released NETs contain LL37 and another neutrophil protein, HMGB1. Induction of NETosis requires FcRIIa, signaling through the pattern recognition receptor Toll-like receptor 7, and formation of reactive oxygen species. Garcia-Romo et al. also show that these NETs potently activate dendritic cells, leading to secretion of high levels of interferon-α. Together, these findings portray an important role for neutrophils in lupus pathogenesis, whereby neutrophils activated by anti-self antibodies release NETs. These NETs, which contain antimicrobial peptides complexed with self-DNA, activate plasmacytoid dendritic cells, leading to interferon release and furtherment and aggravation of inflammation and disease. Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.

The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis

Psoriasis is a common T-cell-mediated skin disease with 2-3% prevalence worldwide. Psoriasis is considered to be an autoimmune disease, but the precise nature of the autoantigens triggering T-cell activation remains poorly understood. Here we find that two-thirds of patients with moderate-to-severe plaque psoriasis harbour CD4(+) and/or CD8(+) T cells specific for LL37, an antimicrobial peptide (AMP) overexpressed in psoriatic skin and reported to trigger activation of innate immune cells. LL37-specific T cells produce IFN-γ, and CD4(+) T cells also produce Th17 cytokines. LL37-specific T cells can infiltrate lesional skin and may be tracked in patients blood by tetramers staining. Presence of circulating LL37-specific T cells correlates significantly with disease activity, suggesting a contribution to disease pathogenesis. Thus, we uncover a role of LL37 as a T-cell autoantigen in psoriasis and provide evidence for a role of AMPs in both innate and adaptive immune cell activation.

Lipopolysaccharides from Bordetella pertussis and Bordetella parapertussis Differently Modulate Human Dendritic Cell Functions Resulting in Divergent Prevalence of Th17-Polarized Responses

Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B. parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1β production in human monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B. pertussis LOS, with the notable exception of IL-1β, which was equally produced. Only B. parapertussis LPS was able to induce IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14, B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and B. pertussis LOS equally induced IκBα and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88-independent gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute to the severity of pertussis in humans.

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca2+ release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter‐receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte‐derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down‐modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN‐γ > CD40 cross‐linking). Although weak, IFN‐γ consistently induces DC maturation. De novo‐synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF‐κB activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti‐CD38 mAb induces up‐regulation of CD83 expression and IL‐12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL‐12 secretion and MDDC‐induced allogeneic T cell proliferation.

IFN-γ Arms Human Dendritic Cells to Perform Multiple Effector Functions

Dendritic cells (DCs) are central players in immunity and are used in immune-adoptive vaccine protocols in humans. IFN-γ, mandatory in Th-1 polarization and endowed with regulatory properties, is currently used to condition monocyte-derived DCs (MDDC) in cancer therapy and in clinical trials to treat chronic infectious diseases. We therefore performed a wide analysis of IFN-γ signaling consequences on MDDC multiple effector functions. IFN-γ itself induced IL-27p28 expression and survival but did not promote relevant CCR7-driven migration or activated Th-1 cell recruitment capacity in MDDC. Administered in association with classical maturation stimuli such as CD40 or TLR-4 stimulation, IFN-γ up-regulated IL-27 and IL-12 production, CCR7-driven migration, and activated Th-1 cell recruitment, whereas it decreased IL-10 production and STAT3 phosphorylation. CD38 signaling, which orchestrates migration, survival, and Th-1 polarizing ability of mature MDDC, was involved in IFN-γ-mediated effects. Thus, IFN-γ is a modulator of multiple DC effector functions that can be helpful in MDDC-based vaccination protocols. These data also help understand the dual role exerted by this cytokine as both an inducer and a regulator of inflammation and immune response.

Cationic antimicrobial peptides in psoriatic skin cooperate to break innate tolerance to self-DNA

Psoriasis is a T‐cell‐mediated skin autoimmune disease characterized by the aberrant activation of dermal dendritic cells (DCs) and the sustained epidermal expression of antimicrobial peptides. We have previously identified a link between these two events by showing that the cathelicidin antimicrobial peptide LL37 has the ability to trigger self‐nucleic acid mediated activation of plasmacytoid DCs (pDCs) in psoriatic skin. Whether other cationic antimicrobial peptides exert similar activities is unknown. By analyzing heparin‐binding HPLC fractions of psoriatic scales, we found that human beta‐defensin (hBD)2, hBD3, and lysozyme are additional triggers of pDC activation in psoriatic skin lesions. Like LL37, hBD2, hBD3, and lysozyme are able to condense self‐DNA into particles that are endocytosed by pDCs, leading to activation of TLR9. In contrast, other antimicrobial peptides expressed in psoriatic skin including elafin, hBD1, and psoriasin (S100A7) did not show similar activities. hBD2, hBD3, and lysozyme were detected in psoriatic skin lesions in the vicinity of pDCs and found to cooperate with LL37 to induce high levels of IFN production by pDCs, suggesting their concerted role in the pathogenesis of psoriasis.

Use of Whole-Blood Samples in In-House Bulk and Single-Cell Antigen-Specific Gamma Interferon Assays for Surveillance of Mycobacterium tuberculosis Infections

ABSTRACT Tests based on the gamma interferon (IFN-γ) assay (IGA) are used as adjunctive tools for the diagnosis of Mycobacterium tuberculosis infection. Here we compared in-house and commercial whole-blood IGAs to identify a suitable assay for the surveillance of tuberculosis in population studies. The IGAs were selected on the basis of the ease with which they are performed and because they require a small amount of a biological sample and do not require cell purification. Since a “gold standard” for latently M. tuberculosis-infected individuals is not available, the sensitivities and the specificities of the IGAs were determined with samples from patients with clinically diagnosed active tuberculosis and in Mycobacterium bovis BCG-unvaccinated healthy controls. The in-house tests consisted of a bulk assay based on diluted whole blood and a single-cell assay based on IFN-γ intracellular staining. The commercial assays used were the QuantiFERON-TB-Gold (Q-TB) and the Q-TB in-tube tests. When the purified protein derivative was used as the antigen, in-house whole-blood intracellular staining was found to be highly discriminatory between active tuberculosis patients and BCG-vaccinated healthy controls, whereas the other IGAs did not discriminate between the two categories of patients. When M. tuberculosis-specific antigens were used, a very strong agreement between the results of the Q-TB in-tube assay and the clinical diagnosis was observed, while the Q-TB assay, performed according to the manufacturers instructions, showed a significantly lower performance. Intriguingly, when the test was performed with RD1 proteins instead of peptides, its sensitivity was significantly increased. The in-house assay with diluted whole blood showed an elevated sensitivity and an elevated specificity, and the results agreed with the clinical diagnosis. Considering that the in-house assay uses 1/20 of the sample compared with the amount of sample used in the commercial IGA, it appears to be particularly promising for use in pediatric studies. Overall, the different assays showed different performance characteristics that need to be considered for surveillance of tuberculosis in population studies.

Liquid-crystalline ordering of antimicrobial peptide–DNA complexes controls TLR9 activation

Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs 1-5). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases.

Defensins and cathelicidins are anti-microbial peptides (AMPs) that act as natural antibiotics and are part of the innate immune defence in many species. We consider human defensins and LL37, the only human member of the cathelicidin family. In particular, we refer to the human alpha-defensins called human neutrophil peptides (HNP1 through 4), which are produced by neutrophils, HD5 and HD6, mainly expressed in Paneth cells of intestine, the human beta-defensins HBD1, HBD2 and HBD3, synthesized by epithelial cells and LL37, which is located in granulocytes, but is also produced by epithelial cells of the skin, lungs, and gut. In the last years, the study of AMPs activity and regulation has allowed to understand the important role of these peptides not only in the innate defence mechanisms against bacteria, viruses, fungi, but also in the regulation of immune cell activation and migration. Complementary studies have disclosed a role for AMPs in modulating many physiological processes that involve non-immune cells, such as activation of wound healing, angiogenesis, cartilage remodeling. Due to the pleiotropic tasks of these peptides, many of them are now being discovered to contribute to immune pathology of chronic diseases that affect skin, gut, joints; this is supported by many examples of immune-mediated pathologies in which their expression is disregulated. In this article we review the current literature that suggests a role for human defensins and LL37 in pathogenic mechanisms of several chronic diseases that are considered of auto-immune or auto-inflammatory origin.

Differential mechanisms of memory CD8 T cell maintenance by individual myeloid cell types

This study tested the hypothesis that individual myeloid subsets have a differential ability to maintain memory CD8 T cells via IL‐15. Although DCs support IL‐15‐mediated homeostasis of memory CD8 T cells in vivo, whether various DC subsets and other myeloid cells similarly mediate homeostasis is unknown. Therefore, we studied the ability of different myeloid cells to maintain memory CD8 T cells in vitro. Using an in vitro cocoulture system that recapitulated known roles of DCs and IL‐15 on memory CD8 T cells, all in vitro‐derived or ex vivo‐isolated DCs maintained CD8 T cells better than rIL‐15 alone, and FLT‐3L‐DCs are the most efficient compared with GM‐DCs, BM‐derived macrophages, or freshly isolated DCs. Although FLT‐3L‐DCs were the least effective at inducing CD8 T cell proliferation, FLT‐3L‐DCs promoted better CD8 T cell survival and increased Bcl‐2 and MCL‐2 expression in CD8 T cells. T cell maintenance correlated only partially with DC expression of IL‐15Rα and IL‐15, suggesting that DCs provided additional support signals. Indeed, in the absence of IL‐15 signals, CD70/CD27 further supported CD8 T cell maintenance. IFN‐α enhanced CD70 expression by DCs, resulting in increased proliferation of CD8 T cells. Overall, this study supports our hypothesis by demonstrating that specific DC subtypes had a greater capacity to support memory CD8 T cell maintenance and did so through different mechanisms. Furthermore, this study shows that IL‐15 trans‐presentation can work in conjunction with other signals, such as CD70/CD27 interactions, to mediate CD8 T cell homeostasis efficiently.