Pattern Recognition Receptors and the Innate Immune Response to Viral Infection

The innate immune response to viral pathogens is critical in order to mobilize protective immunity. Cells of the innate immune system detect viral infection largely through germline-encoded pattern recognition receptors (PRRs) present either on the cell surface or within distinct intracellular compartments. These include the Toll-like receptors (TLRs), the retinoic acid-inducble gene I-like receptors (RLRs), the nucleotide oligomerization domain-like receptors (NLRs, also called NACHT, LRR and PYD domain proteins) and cytosolic DNA sensors. While in certain cases viral proteins are the trigger of these receptors, the predominant viral activators are nucleic acids. The presence of viral sensing PRRs in multiple cellular compartments allows innate cells to recognize and quickly respond to a broad range of viruses, which replicate in different cellular compartments. Here, we review the role of PRRs and associated signaling pathways in detecting viral pathogens in order to evoke production of interferons and cytokines. By highlighting recent progress in these areas, we hope to convey a greater understanding of how viruses activate PRR signaling and how this interaction shapes the anti-viral immune response

Role of toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils

In vitro studies as well as clinical trials indicate that the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) enhance the ability of neutrophils (polymorphonuclear leukocytes) to eliminate microbial organisms. Toll-like receptor (TLR) proteins, homologs of the Drosophila protein Toll, have been found on the surface of mammalian cells and are important in the responses of macrophages to bacterial, viral, and fungal antigens. TLR4 is critical for the response to lipopolysaccharide (LPS) of gram-negative bacteria, while TLR2 is important for response to gram-positive bacteria, bacterial peptides, and yeast zymosan. We demonstrate that TLR2, but very little TLR4, is present on the surface of human neutrophils. In addition we demonstrate that GM-CSF and G-CSF dramatically up-regulate TLR2 and CD14 surface expression. GM-CSF treatment also up-regulates TLR2 and CD14 mRNA levels in neutrophils. In addition to increasing receptor expression, GM-CSF treatment enhanced the interleukin 8 (IL-8) secretion and superoxide priming responses of neutrophils to stimulation with TLR2 ligands, including zymosan, peptidoglycan, and lipoarabinomannan. The human monocyte response to crude bacterial LPS is composed of a TLR4-specific response to the pure LPS component and a TLR2-dependent response to associated lipopeptides. The removal of TLR2 lipopeptide components from LPS by phenol re-extraction substantially reduced both the IL-8 and superoxide response of the stimulated neutrophils, indicating that, unlike monocytes, the neutrophil response is preferentially directed to TLR2 ligands. Thus, our studies demonstrate that GM-CSF dramatically enhances the functional response of neutrophils to TLR2 ligands, including LPS-associated lipopeptides

STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis. Deficiency of STING prevented IRF3 phosphorylation by ethanol or ER stress, and absence of IRF3 prevented hepatocyte apoptosis. The pathogenic role of IRF3 in ALD was independent of inflammation or Type-I interferons. Thus, STING and IRF3 are key determinants of ALD, linking ER stress signaling with the mitochondrial pathway of hepatocyte apoptosis

miR-718 represses proinflammatory cytokine production through targeting phosphatase and tensin homolog (PTEN)

Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS. LPS binds Toll-like receptor (TLR) 4, which leads to the release of proinflammatory cytokines that are essential for a potent innate immune response against pathogens. The innate immune system is tightly regulated, as excessive inflammation can lead to organ failure and death. MicroRNAs have recently emerged as important regulators of the innate immune system. Here we determined the function of miR-718, which is conserved across mammals and overlaps with the 5\u27 UTR of the interleukin 1 receptor-associated kinase (IRAK1) gene. As IRAK1 is a key component of innate immune signaling pathways that are downstream of most TLRs, we hypothesized that miR-718 helps regulate the innate immune response. Activation of TLR4, but not TLR3, induced the expression of miR-718 in macrophages. miR-718 expression was also induced in the spleens of mice upon LPS injection. miR-718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production. Phosphorylated Akt induces let-7e expression, which, in turn, down-regulates TLR4 and further diminishes TLR4-mediated proinflammatory signals. Decreased miR-718 expression is associated with bacterial burden during Neisseria gonorrhoeae infection and alters the infection dynamics of N. gonorrhoeae in vitro Furthermore, miR-718 regulates the induction of LPS tolerance in macrophages. We propose a role for miR-718 in controlling TLR4 signaling and inflammatory cytokine signaling through a negative feedback regulation loop involving down-regulation of TLR4, IRAK1, and NF-kappaB

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction

IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice

Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1beta. IL-1beta, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1beta maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra-treated mice as well as 3 mouse models deficient in regulators of IL-1beta activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1beta signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1beta was due to upregulation of Casp-1 activity and inflammasome activation. The pathogenic role of IL-1 signaling in ALD was attributable to the activation of the inflammasome in BM-derived Kupffer cells. Importantly, in vivo intervention with a recombinant IL-1Ra blocked IL-1 signaling and markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. Furthermore, physiological doses of IL-1beta induced steatosis, increased the inflammatory and prosteatotic chemokine MCP-1 in hepatocytes, and augmented TLR4-dependent upregulation of inflammatory signaling in macrophages. In conclusion, we demonstrated that Casp-1-dependent upregulation of IL-1beta and signaling mediated by IL-1R1 are crucial in ALD pathogenesis. Our findings suggest a potential role of IL-1R1 inhibition in the treatment of ALD

Endoplasmic reticulum stress-induced hepatocellular death pathways mediate liver injury and fibrosis via Stimulator of Interferon Genes.

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon Regulatory Factor 3 (IRF3) regulates hepatocyte apoptosis and production of Type-I interferons (IFNs). In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the ER adapter, Stimulator of Interferon Genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically-induced liver fibrogenesis. To test this, we performed acute or chronic carbontetrachloride (CCl4) administration to WT, IRF3-, TRAM-, TRIF-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3 and Type-I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4. Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4. In contrast, mice deficient in Type-I IFN receptors or in TLR4-signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis, and indicate that innate immune signaling modulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver

Micro RNAs from DNA Viruses are Found Widely in Plasma in a Large Observational Human Population

Viral infections associate with disease risk and select families of viruses encode miRNAs that control an efficient viral cycle. The association of viral miRNA expression with disease in a large human population has not been previously explored. We sequenced plasma RNA from 40 participants of the Framingham Heart Study (FHS, Offspring Cohort, Visit 8) and identified 3 viral miRNAs from 3 different human Herpesviridae. These miRNAs were mostly related to viral latency and have not been previously detected in human plasma. Viral miRNA expression was then screened in the plasma of 2763 participants of the remaining cohort utilizing high-throughput RT-qPCR. All 3 viral miRNAs associated with combinations of inflammatory or prothrombotic circulating biomarkers (sTNFRII, IL-6, sICAM1, OPG, P-selectin) but did not associate with hypertension, coronary heart disease or cancer. Using a large observational population, we demonstrate that the presence of select viral miRNAs in the human circulation associate with inflammatory biomarkers and possibly immune response, but fail to associate with overt disease. This study greatly extends smaller singular observations of viral miRNAs in the human circulation and suggests that select viral miRNAs, such as those for latency, may not impact disease manifestation

UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

UNC93B1 associates with Toll-Like Receptor (TLR) 3, TLR7 and TLR9, mediating their translocation from the endoplasmic reticulum to the endolysosome, hence allowing proper activation by nucleic acid ligands. We found that the triple deficient ‘3d’ mice, which lack functional UNC93B1, are hyper-susceptible to infection with Toxoplasma gondii. We established that while mounting a normal systemic pro-inflammatory response, i.e. producing abundant MCP-1, IL-6, TNFα and IFNγ, the 3d mice were unable to control parasite replication. Nevertheless, infection of reciprocal bone marrow chimeras between wild-type and 3d mice with T. gondii demonstrated a primary role of hemopoietic cell lineages in the enhanced susceptibility of UNC93B1 mutant mice. The protective role mediated by UNC93B1 to T. gondii infection was associated with impaired IL-12 responses and delayed IFNγ by spleen cells. Notably, in macrophages infected with T. gondii, UNC93B1 accumulates on the parasitophorous vacuole. Furthermore, upon in vitro infection the rate of tachyzoite replication was enhanced in non-activated macrophages carrying mutant UNC93B1 as compared to wild type gene. Strikingly, the role of UNC93B1 on intracellular parasite growth appears to be independent of TLR function. Altogether, our results reveal a critical role for UNC93B1 on induction of IL-12/IFNγ production as well as autonomous control of Toxoplasma replication by macrophages

CD200R1 Supports HSV-1 Viral Replication and Licenses Pro-Inflammatory Signaling Functions of TLR2

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1−/− mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1−/− peritoneal macrophages demonstrated a 70–75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam2CSK4 and to HSV-1. CD200R1−/− macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1−/− mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1−/− mice and CD200R1−/− fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in “licensing” pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence