Valeria Facchinetti

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.

SIN1/MIP1 Maintains rictor-mTOR Complex Integrity and Regulates Akt Phosphorylation and Substrate Specificity

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function.

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.

A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB−/− cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation‐ and stress‐induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome‐to‐nucleus signalling mechanism that involves TFEB and mTOR.

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 mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C

The target of rapamycin (TOR), as part of the rapamycin‐sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl‐terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination‐mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide

The mechanisms that couple translation and protein processing are poorly understood in higher eukaryotes. Although mammalian target of rapamycin (mTOR) complex 1 (mTORC1) controls translation initiation, the function of mTORC2 in protein synthesis remains to be defined. In this study, we find that mTORC2 can colocalize with actively translating ribosomes and can stably interact with rpL23a, a large ribosomal subunit protein present at the tunnel exit. Exclusively during translation of Akt, mTORC2 mediates phosphorylation of the nascent polypeptide at the turn motif (TM) site, Thr450, to avoid cotranslational Akt ubiquitination. Constitutive TM phosphorylation occurs because the TM site is accessible, whereas the hydrophobic motif (Ser473) site is concealed in the ribosomal tunnel. Thus, mTORC2 can function cotranslationally by phosphorylating residues in nascent chains that are critical to attain proper conformation. Our findings reveal that mTOR links protein production with quality control.

Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells

Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I IFN responses in plasmacytoid dendritic cells (pDCs). Previous studies suggest the presence of myeloid differentiation primary response gene 88 (MyD88)-dependent DNA sensors other than TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)-binding proteins from human pDCs, pDC-cell lines, and interferon regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively bound the aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36), whereas CpG-B selectively bound DExD/H-box helicase 9 (DHX9). Although the aspartate-glutamate-alanine-histidine box motif (DEAH) domain of DHX36 was essential for CpG-A binding, the domain of unknown function 1605 (DUF1605 domain) of DHX9 was required for CpG-B binding. DHX36 is associated with IFN-α production and IRF7 nuclear translocation in response to CpG-A, but DHX9 is important for TNF-α and IL-6 production and NF-κB activation in response to CpG-B. Knocking down DHX9 or DHX36 significantly reduced the cytokine responses of pDCs to a DNA virus but had no effect on the cytokine responses to an RNA virus. We further showed that both DHX9 and DHX36 are localized within the cytosol and are directly bound to the Toll-interleukin receptor domain of MyD88 via their helicase-associated domain 2 and DUF domains. This study demonstrates that DHX9/DHX36 represent the MyD88-dependent DNA sensors in the cytosol of pDCs and suggests a much broader role for DHX helicases in viral sensing.

DHX9 Pairs with IPS-1 To Sense Double-Stranded RNA in Myeloid Dendritic Cells

The innate immune system is equipped with many molecular sensors for microbial DNA/RNA to quickly mount antimicrobial host immune responses. In this paper, we identified DHX9, a DExDc helicase family member, as an important viral dsRNA sensor in myeloid dendritic cells (mDCs). Knockdown of DHX9 expression by small heteroduplex RNA dramatically blocked the ability of mDCs to produce IFN-α/β and proinflammatory cytokines in response to polyinosine-polycytidylic acid, influenza A, and reovirus. DHX9 could specifically bind polyinosine-polycytidylic acid via its double-strand RNA binding motifs. DHX9 interacted with IPS-1 via the HelicC-HA2-DUF and CARD domains of DHX9 and IPS-1, respectively. Knockdown of DHX9 expression in mDCs blocked the activation of NF-κB and IFN regulatory factor 3 by dsRNA. Collectively, these results suggest that DHX9 is an important RNA sensor that is dependent on IPS-1 to sense pathogenic RNA.

Sin1-mTORC2 suppresses rag and il7r gene expression through Akt2 in B cells.

Mammalian target of rapamycin (mTOR) is an important mediator of phosphoinositol-3-kinase (PI3K) signaling. PI3K signaling regulates B cell development, homeostasis, and immune responses. However, the function and molecular mechanism of mTOR-mediated PI3K signaling in B cells has not been fully elucidated. Here we show that Sin1, an essential component of mTOR complex 2 (mTORC2), regulates B cell development. Sin1 deficiency results in increased IL-7 receptor (il7r) and RAG recombinase (rag1 and rag2) gene expression, leading to enhanced pro-B cell survival and augmented V(D)J recombinase activity. We further show that Akt2 specifically mediates the Sin1-mTORC2 dependent suppression of il7r and rag gene expression in B cells by regulating FoxO1 phosphorylation. Finally, we demonstrate that the mTOR inhibitor rapamycin induces rag expression and promotes V(D)J recombination in B cells. Our study reveals that the Sin1/mTORC2-Akt2 signaling axis is a key regulator of FoxO1 transcriptional activity in B cells.