The TLR‐independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature

Abstract

Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of IFN-b. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the endosomal TLR response. To address these questions, we compared the two most commonly used types of DNA ligands (IFN-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure -activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-b induction, while phosphorylation at the 5 0 -end is dispensable. Altogether, our data suggest that, in murine macrophages, only one major cytosolic DNA recognition pathway is operational. IntroductionInnate immunity represents the first line of defense against invading pathogens. PRR recognize molecular features that are conserved among many pathogens, so called PAMP. PRR are localized on the plasma membrane, the endosome or the cytosol and belong to distinct classes, most notably the TLR, the NOD-like receptors and the RIG-I-like helicases (RLH) [1,2]. Engagement of PRR by specific ligands triggers intracellular signaling cascades that culminate in the production and secretion of type-I IFN, cytokines and chemokines.An invading virus can either be sensed by endosomal TLR (TLR3, 7/8 or 9) or by cytosolic RLH [3], whereas, TLR3 and TLR7/8 recognize viral RNA, TLR9 senses the presence of viral DNA containing CpG motifs [4]. The RLH detect viral RNA in the cytosol [5]. This raises the question of how the host cell Eur. J. Immunol. 2009. 39: 1929-1936 DOI 10.1002 Innate immunity 1929distinguishes between viral and cellular RNA that are simultaneously present in the same subcellular compartment. It has recently been shown that many viral RNA, unlike cellular RNA, contain a triphosphate moiety at the 5 0 -end, which is recognized by RIG-I, the founding member of the RLH family [6,7]. Despite ''thousands of man-years worth of DNA transfections'', it has only recently been noticed that introduction of dsDNA into the cytosol triggers a potent innate immune response, leading to the production of IL-1b, IFN-b and other cytokines [8][9][10]. IL-1b secretion is governed by the DNA sensor AIM2 that has recently been identified [11][12][13][14]. IFN-b production is regulated by the DNA-dependent activator of IFN-regulatory factors (DAI, formerly known as DLM-1/ZBP1) [15] and as-yet unknown DNA sensors [16,17]. With regard to the IFN pathway, it has been suggested that the B-conformation of DNA is stimulatory, whereas Z-DNA is not [9]. Another report suggests that the exchange of the phosphate backbone for a phosphorothioate backbone renders the DNA inert, implying that the DNA backbone is the site of recog...

Structure of Human cGAS Reveals a Conserved Family of Second-Messenger Enzymes in Innate Immunity

“…S4C). This finding is consistent with previous results demonstrating that at least 20–30 base pairs of dsDNA are required for efficient stimulation of innate immunity (Ablasser et al, 2009; Karayel et al, 2009; Stetson and Medzhitov, 2006). …”

DNase Sda1 Allows Invasive M1T1 Group A Streptococcus to Prevent TLR9-Dependent Recognition

“…In contrast to our ex vivo data and tissue culture experiments carried out by others [25], IFN-α and TNF-α secretion in our in vivo experiments was strictly TLR9-dependent. This finding suggests that TLR9 recognition of GAS is of true physiological relevance, and the described TLR9-independent pathways elicited ex vivo may be of diminished importance in the in vivo setting.…”

“…2C ). The stimulation of IFN-α secretion from TLR9-deficient BMDMs, albeit at a reduced level, is most likely explained by a ubiquitous interferon response to immunostimmulatory nucleic acids, mediated by cytosolic DNA sensors amongst others [25]. Similarly, recent work shows that IFN-β is secreted after challenge of TLR9-deficient macrophages with live GAS or GAS DNA complexed with RNA [16].…”

Cytosolic DNA-Activated Human Dendritic Cells Are Potent Activators of the Adaptive Immune Response

“…Much of the efforts focus on identifying DNA receptors and pathways leading to cell activation, especially in cells with antigen presenting capacity. Recent studies, in cell lines and mouse knock out systems, have elegantly demonstrated that cytosolic dsDNA activates APCs by interacting with unknown intracellular nucleic acid sensor(s) (11, 27, 34, 36, 43). In humans, myeloid DC activation by cytosolic DNA has been observed but only partly characterized (35, 44).…”