Roberto Lande

Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide

Plasmacytoid dendritic cells (pDCs) sense viral and microbial DNA through endosomal Toll-like receptors to produce type 1 interferons. pDCs do not normally respond to self-DNA, but this restriction seems to break down in human autoimmune disease by an as yet poorly understood mechanism. Here we identify the antimicrobial peptide LL37 (also known as CAMP) as the key factor that mediates pDC activation in psoriasis, a common autoimmune disease of the skin. LL37 converts inert self-DNA into a potent trigger of interferon production by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger Toll-like receptor 9. Thus, our data uncover a fundamental role of an endogenous antimicrobial peptide in breaking innate tolerance to self-DNA and suggest that this pathway may drive autoimmunity in psoriasis.

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.

Plasmacytoid dendritic cells prime IL-10–producing T regulatory cells by inducible costimulator ligand

Although there is evidence for distinct roles of myeloid dendritic cells (DCs [mDCs]) and plasmacytoid pre-DCs (pDCs) in regulating T cell–mediated adaptive immunity, the concept of functional DC subsets has been questioned because of the lack of a molecular mechanism to explain these differences. In this study, we provide direct evidence that maturing mDCs and pDCs express different sets of molecules for T cell priming. Although both maturing mDCs and pDCs upregulate the expression of CD80 and CD86, only pDCs upregulate the expression of inducible costimulator ligand (ICOS-L) and maintain high expression levels upon differentiation into mature DCs. High ICOS-L expression endows maturing pDCs with the ability to induce the differentiation of naive CD4 T cells to produce interleukin-10 (IL-10) but not the T helper (Th)2 cytokines IL-4, -5, and -13. These IL-10–producing T cells are T regulatory cells, and their generation by ICOS-L is independent of pDC-driven Th1 and Th2 differentiation, although, in the later condition, some contribution from endogenous IL-4 cannot be completely ruled out. Thus, in contrast to mDCs, pDCs are poised to express ICOS-L upon maturation, which leads to the generation of IL-10–producing T regulatory cells. Our findings demonstrate that mDC and pDCs are intrinsically different in the expression of costimulatory molecules that drive distinct types of T cell responses.

Viral infection and Toll-like receptor agonists induce a differential expression of type I and λ interferons in human plasmacytoid and monocyte-derived dendritic cells

In humans, the type I interferon (IFN) family consists of 13 IFN‐α subtypes, IFN‐β and IFN‐o the newly discovered IFN‐like family consists of IFN‐λ1, ‐λ2 and ‐λ3. We have investigated the expression of type I and λ IFN genes following virus infections or Toll‐like receptor (TLR) triggering in monocyte‐derived DC (MDDC) and plasmacytoid DC (pDC). We found thatall IFN‐α, ‐β, ‐o and ‐λ subtypes are expressed in influenza‐virus‐infected MDDC or pDC. Conversely, differential type I IFN gene transcription was induced in MDDC and pDC stimulated by specific TLR agonists. TLR‐9 stimulation by CpG DNA induced the expression of all IFN‐α, ‐β, ‐o and ‐λ subtypes in pDC, whereas TLR‐4 stimulation by LPS, or TLR‐3 stimulation bypoly I:C, induced only IFN‐β and IFN‐λ gene expression in MDDC. The expression pattern of IFN regulatory factor (IRF)‐5 and IRF‐7 in MDDC and pDC was also determined. IRF‐5 was constitutively expressed in the two DC subsets whereas IRF‐7 was constitutive in pDC but its expression was induced along MDDC maturation. Overall, our data indicate that the coordinated expression of IFN‐λ with IFN‐β would be of crucial importance for the maturation of DC.

Self-RNA–antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8

Dendritic cell (DC) responses to extracellular self-DNA and self-RNA are prevented by the endosomal seclusion of nucleic acid–recognizing Toll-like receptors (TLRs). In psoriasis, however, plasmacytoid DCs (pDCs) sense self-DNA that is transported to endosomal TLR9 upon forming a complex with the antimicrobial peptide LL37. Whether LL37 also interacts with extracellular self-RNA and how this may contribute to DC activation in psoriasis is not known. Here, we report that LL37 can bind self-RNA released by dying cells, protect it from extracellular degradation, and transport it into endosomal compartments of DCs. In pDC, self-RNA–LL37 complexes activate TLR7 and, like self-DNA–LL37 complexes, trigger the secretion of IFN-α without inducing maturation or the production of IL-6 and TNF-α. In contrast to self-DNA–LL37 complexes, self-RNA–LL37 complexes also trigger the activation of classical myeloid DCs (mDCs). This occurs through TLR8 and leads to the production of TNF-α and IL-6, and the differentiation of mDCs into mature DCs. We also found that self-RNA–LL37 complexes are present in psoriatic skin lesions and are associated with mature mDCs in vivo. Our results demonstrate that the cationic antimicrobial peptide LL37 converts self-RNA into a trigger of TLR7 and TLR8 in human DCs, and provide new insights into the mechanism that drives the auto-inflammatory responses in psoriasis.

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.

Plasmacytoid dendritic cells: key players in the initiation and regulation of immune responses

Plasmacytoid dendritic cells (pDCs) are a rare population of circulating cells that selectively express Toll‐like receptors (TLR)7 and TLR9 and have the capacity to produce large amounts of type I interferons (IFNs) in response to viruses or host‐derived nucleic acid‐containing complexes. Through the production of type I IFNs, pDCs initiate protective immunity by activating classical DCs, T cells, natural killer cells, and B cells. Upon activation, pDCs also differentiate into mature DCs and may contribute to the contraction of T‐cell responses. In this review, we describe how pDCs are key players in the initiation and regulation of immune responses and discuss their emerging role in the pathogenesis of human diseases.

Antimicrobial peptides and self-DNA in autoimmune skin inflammation.

Toll-like receptor (TLR)-mediated detection of viral nucleic acids and production of type I interferons (IFNs) by plasmacytoid dendritic cells (pDCs) are key elements of antiviral defense. By contrast, inappropriate recognition of self-nucleic acids with induction of IFN responses in pDCs can lead to autoimmunity. In this review we describe how pDC responses to self-DNA are normally avoided and focus on our recent finding that in psoriasis, a common autoimmune disease of the skin, these barriers can be breached by the cationic antimicrobial peptide LL37. LL37 binds extracellular self-DNA fragments into aggregated particles that enter pDCs and trigger robust IFN responses by activating endosomal TLR9 as if they were viruses. We also describe the mechanisms that normally control production and activity of LL37 in human skin and propose that the persistent overexpression of LL37 in psoriasis leads to uncontrolled IFN responses that drive autoimmune skin inflammation.

IFN-αβ Released by Mycobacterium tuberculosis-Infected Human Dendritic Cells Induces the Expression of CXCL10: Selective Recruitment of NK and Activated T Cells

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-γ-inducing cytokines, IFN-αβ and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-αβ. Indeed, IFN-αβ neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4+, and CD8+ cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-αβ-induced expression of CXCL10, neutralization of IFN-αβ significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-αβ may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.

Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions.

As a result of their close association with the blood-brain barrier, astrocytes play an important role in regulating the homing of different leukocyte subsets to the inflamed central nervous system (CNS). In this study, we investigated whether human astrocytes produce chemokines that promote the migration of myeloid dendritic cells (DCs). By reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, we show that cultured human astrocytes stimulated with interleukin-1β and tumor necrosis factor produce CCL2, CCL3, CCL4, CCL5, CCL20, and CXCL12 that act on immature DCs, but not CCL19 and CCL21, 2 chemokines specific for mature DCs. Compared with controls, supernatants of cytokine-stimulated astrocytes are more effective in promoting the migration of immature monocyte-derived DCs (iMDDCs). Desensitization of CXCR4 (receptor for CXCL12), CCR1-3-5 (shared receptors for CCL3-4-5), and CCR6 (receptor for CCL20) on iMDDC reduces cell migration toward astrocyte supernatants, indicating that astrocytes release biologically relevant amounts of iMDDC-attracting chemokines. By immunohistochemistry, we show that CXCL12 and, to a lesser extent, CCL20 are expressed by reactive astrocytes in multiple sclerosis lesions. These data lend support to the idea that astrocyte-derived chemokines may contribute to immature DC recruitment to the inflamed CNS.