Pathogen Recognition and Innate Immunity

The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.

Toll-like receptors.

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses

https://www.cell.com/cell/pdf/S0092-8674(05)00816-0.pdf

Identification and Characterization of MAVS, a Mitochondrial Antiviral Signaling Protein that Activates NF-κB and IRF3

Summary: The innate immune system constitutes the first line of defense against invading microbial pathogens and relies on a large family of pattern recognition receptors (PRRs), which detect distinct evolutionarily conserved structures on pathogens, termed pathogen-associated molecular patterns (PAMPs). Among the PRRs, the Toll-like receptors have been studied most extensively. Upon PAMP engagement, PRRs trigger intracellular signaling cascades ultimately culminating in the expression of a variety of proinflammatory molecules, which together orchestrate the early host response to infection, and also is a prerequisite for the subsequent activation and shaping of adaptive immunity. In order to avoid immunopathology, this system is tightly regulated by a number of endogenous molecules that limit the magnitude and duration of the inflammatory response. Moreover, pathogenic microbes have developed sophisticated molecular strategies to subvert host defenses by interfering with molecules involved in inflammatory signaling. This review presents current knowledge on pathogen recognition through different families of PRRs and the increasingly complex signaling pathways responsible for activation of an inflammatory and antimicrobial response. Moreover, medical implications are discussed, including the role of PRRs in primary immunodeficiencies and in the pathogenesis of infectious and autoimmune diseases, as well as the possibilities for translation into clinical and therapeutic applications.

Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses

The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction. Using mice deficient in the Irf7 gene (Irf7-/- mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7-/- fibroblasts. Consistently, Irf7-/- mice are more vulnerable than Myd88-/- mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8+ T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.

IRF-7 is the master regulator of type-I interferon-dependent immune responses

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-α/β) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal “input” can be processed through MyD88 to “output” the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.

https://www.pnas.org/content/pnas/101/43/15416.full.pdf

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

Heterologous production of a useful carotenoid astaxanthin was achieved in a cyanobacterium Synechocystis sp. PCC 6803 with the aid of marine bacterial genes. Astaxanthin and its intermediates emerged at high levels, whereas β-carotene and zeaxanthin disappeared in the strain. Total carotenoid accumulation was nearly two fold compared with wild type. The astaxanthin-producing strain was capable of only growing heterotrophically, which was likely due to the absence of β-carotene. Further enhanced accumulation was pursued by gene overexpression for possible rate-limiting steps in the biosynthesis pathway.

/pdf/astaxanthin-production-in-a-model-cyanobacterium-2k6nxm1kpp.pdf

Astaxanthin production in a model cyanobacterium Synechocystis sp. PCC 6803

There is growing demand for a novel tool that measures mechanical properties of cellular proteins such as cytoskeletal protein. Conventionally, for such studies, optical tweezers using micro-beads has widely utilized. This method, however, has two significant problems: unchanged contact-area, and irradiation of strong laser close to the proteins. First, this method cannot change contact-area between micro-beads and target proteins because the moved micro-beads are only spherical shape. Secondly, the irradiation of strong laser thermally damages protein because the laser approaches near target. To solve these problems, herein we developed “optically driven nano-beam”. It is specialized to perform quasi-static mechanical analysis about cellular proteins. The nano-beam had a movable plate and a fixed wall which are same sizes. The plate has cylindrical object named “trap-point” for moved by optical tweezers. The movable plate was contacted to a fixed block by a thin beam. The beam was 20μm long, 647nm height, and 147nm wide in average. When target proteins were placed between the palate and wall, the nano-beam could mechanically analyze elastic modulus of target proteins by slowly moved the plate using optical tweezers. The nano-beam had two advantages; variable contact-area and strong laser far from the target proteins. Therefore, our device can reveal cellular life phenomena which conventional method cannot perform.

Mechanical properties analyzer for nano-size protein with optically driven nano-beam

We have been developing optically driven micro-robots for biosensing such as cellular mechanical properties. In this report, we have created a novel optically driven micro-robot to analyze mechanical properties of cellular proteins. The micro-robot has two parallel beams with rectangular shaped walls on the end, extending from a base block. One of the beams can be manipulated to bend to and fro. When proteins are placed between the two walls, the movement of the manipulated beam will be mechanically transferred by proteins to the other wall causing it to move. By measuring both the input and output, mechanical properties of proteins can be identified. This device becomes unique tool for cellular biology.

Naoya Shimada

Papers

IRF-7 is the master regulator of type-I interferon-dependent immune responses

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

Astaxanthin production in a model cyanobacterium Synechocystis sp. PCC 6803

Mechanical properties analyzer for nano-size protein with optically driven nano-beam

Nanoscale dynamic viscoelasticity analyzer by using optically driven nanobeams for study of biomolecules