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The relationship between sovereignty, order, and conflict and the challenges to this relationship are important topics in international life. Although sovereign states are the primary actors in world politics, they do not always possess sovereign reach or maintain political order across their territories. Zones of conflict, informal control, and uncertain rule exist both within states and across borders. Terrorist networks, rebel insurgencies, and crime syndicates are all examples of forms of order that begin where sovereign control ends. The purpose behind this multidisciplinary seminar series is to explore the latest research on these themes that cut across the social sciences. The working group will not only involve faculty and doctoral students from various departments at Syracuse University, but will also take a multi-method approach to these challenges.
September 9. Megan Stewart (Assistant Professor, American University)
Title: Governing for Revolution
October 28. Gladys McCormick (Associate Professor, Syracuse University)
Title: Mexico's Open Secret: From the Dirty War to the Drug War
November 18. Hein Goemans (Associate Professor, Rochester University)
Title: War From the Ground Up
January 27. Corri Zoli (Associate Professor, Syracuse University)
Title: The Return of Sovereignty in International Law
February 10. Mona Bhan (Associate Professor, Syracuse University)
Title: Infrastructures of Occupation: Dams, Development, and the Politics of Integration in Kashmir
February 24. David Skarbek (Associate Professor, Brown University)
Title: The Puzzle of Prison Order: Why Life Behind Bars Varies Around the World
September 14. Stuart Brown (Professor, Syracuse University) and Margaret Hermann (Professor, Syracuse University)
Title: Transnational Crime and Black Spots: Rethinking Sovereignty and the Global Economy
September 25. Alexander Cooley (Professor, Barnard College) and Daniel Nexon (Professor, Georgetown University)
Title: Exit from Hegemony: The Unraveling of the American Global Order
October 8. Jacob Mundy (Associate Professor, Colgate University)
Title: Making Hegemony in the Middle East
October 19. Anna Stilz (Professor, Princeton University)
Title: Redistributing Territory?
November 6. Hendrik Spruyt (Professor, Northwestern University)
Title: The World Imagined: Collective Beliefs and Political Order in the Sinocentric, Islamic and Southeast Asian International Societies
Contact: Ryan Griffiths


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text: 23 0 3Start case one and watch the first part of the game. Achievement unlocked.Showing all 3 comments. Leave a comment. lol you're kidding! Looks like "The Simpsons Game" has some competition for easiest achievement ;)Posted by Rapjackal on 30 Oct 09 at 18:55 How the bloody hell could someone give this a negative vote? What an idiot.Posted by OMGWTH00111111 on 11 Jun 12 at 18:13 Thank you so much, i missed this and it was really hard to get it.Posted by segatasanshiro2 on 01 Sep 17 at 17:08 Leave a comment


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text: American By Nino Marchetti / July 6, 2020 One Eight Distilling, noted for being Washington D.C.'s second oldest modern distillery, has been producing over the last number of years the Untitled Whiskey series. Each of these are noted for being a way for the distillery team there to flex their "creativity muscles and create uncommon spirits for the discerning drinker who believes they've tasted everything." We reviewed the first 7 back in 2016, and now word is out of the new 18th release.The new One Eight Distilling Untitled Whiskey No. 18, according to those behind it, marks a special moment for the distillery. What's so special about it is that, according to the brand, "for the first time, the distillery blended its own grain-to-glass District Made Rye Whiskey with a barrel of the finest 14-year-old high-rye bourbon selected from its rickhouse and bottled it at barrel strength to preserve its complexity of flavor."One Eight Distilling Untitled Whiskey No. 18 (image via One Eight Distilling)The launch of this whiskey comes alongside continued production of hand sanitizer for the D.C. City Government and other organizations serving the frontlines of the battle against the ongoing COVID-19 pandemic.."We had to stop producing spirits and focus all of our efforts on making hand sanitizer," said Sandy Wood, One Eight Co-Founder and CEO, in a prepared statement. It's been quite a big change for the business, but we're grateful we can find a revenue stream from that, and also that we're able to stay busy.""We hope you enjoy this special release, a nod to our name, and an exciting exploration into new territories for Untitled," added Alex Laufer, One Eight Co-Founder and Head Distiller.As it stands now plans call for No. 18 to be a distillery only release, bottled at 61% ABV and pricing around $80. You'll find some official tasting notes below."The nose on this whiskey leads to notes of rock candy and grapefruit peel. Upon sipping, the complex, earthy notes of leather and walnuts are readily apparent, followed by fruity notes of cherry cola and molasses. The finish is long and warming with hints of rose, cedar and black licorice."


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The Q Continuum Future Proves Past – Lock Down Look Up
A Dutch model on the cover of a magazine. COVFEFE for 5G. Atlas Mission. JFK Junior is alive. Rumors, chatter, Q Codes and different realities. While the world is in a corona daze a plot is unfolding behind the scenes of the silent war. First Lady of the United States, Melania Trump. "When 'non-dogmatic' information becomes FREE & TRANSPARENT it becomes a threat to those who attempt to control the narrative and/or the stable. When you are awake, you stand on the outside of the stable ('group-think' collective), and have 'free thought'. "Free thought" is a philosophical viewpoint which holds that positions regarding truth shouldRead More →
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Autocar Pro News Desk ,
17 Apr 2019
The Suzuki Jimny has won the 2019 World Urban Car award. This makes a Suzuki car in the 'Top three in the World' finalist in the World Urban Car category for three consecutive years. This year, the Jimny was also a top three finalist in the World Car Design of the Year category.
Toshihiro Suzuki, representative director and president of Suzuki Motor Corporation said, "We at Suzuki are greatly honoured to receive this award. The Jimny is the one-and-only, compact, authentic off-roader, and we have kept true to this concept for almost 50 years. It now has many fans around the world, who enjoy its excellent performance and one-of-a-kind functional design, both on and off-road. We accept this award on behalf of our fans, as well as with pride and confidence in our passion to build exciting cars to be the foot of our customers everyday lives."
Hiroyuki Yonezawa collecting the Urban Car of the Year trophy for the Suzuki Jimny at the 2019 World Car Awards at the New York Motor Show.
The 2019 World Urban Car winner was chosen from an initial entry list of five cars from all over the world, then a short list of three finalists as announced in Geneva on March 5th. The 2019 Top Three in the World finalists are: the Hyundai AH2/Santro, the Kia Soul and the Suzuki Jimny.
The World Urban Car award was introduced in 2017 because it's an award whose time has come. World Car Awards Program co-chair, Mike Rutherford, commented, "Everyday cars in many -- perhaps most -- parts of the world will have to become smaller if road and parking space is to be found for them in increasingly packed towns and cities whose populations are swelling annually."
Also read: Jaguar I-Pace the big winner at the 2019 World Car Awards
Audi A7 wins 2019 World Luxury Car title
Glanza sells over 25,000 units in first year, brand Toyota deciding fa...
Autocar India
02 Jul 2020
Suzuki's new Across is Toyota RAV4-based SUV
Felix Page, Autocar UK
02 Jul 2020
Porsche India director Pavan Shetty steps down
Rivan RS, Autocar India
01 Jul 2020


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One of the more intriguing passages of compassion in the Bible is Mark 6:30-34 (which appears in Matthew 14, Luke 9, and John 6 as well), which reads: "Then the apostles gathered to Jesus and told Him all things, both what they had done and what they had taught. And He said to them, "Come aside by yourselves to a deserted place and rest a while." For there were many coming and going, and they did not even have time to eat. So they departed to a deserted place in the boat by themselves. But the multitudes saw them departing, and many knew Him and ran there on foot from all the cities. They arrived before them and came together to Him. And Jesus, when He game out, saw a great multitude and was moved with compassion for them, because they were like sheep not having a shepherd. So He began to teach them many things."
Why was Jesus moved with compassion that they were like sheep without a shepherd? It is the job of leadership in an institution or society to provide direction for most people. Any successful work requires strong leadership at the top, with a broad and deep enough development of leadership to continue the institution or movement after the original leader dies. This is not an easy matter, as passing on a righteous legacy [1] is among the most difficult tasks a leader has to accomplish to be successful for the long term. As we are involved in projects that go on far beyond our own lifetimes, extending into the past for many generations and extending into the future as long as we remain on this earth. Even as much as we might enjoy rest and relaxation, we may be moved by our compassion for those who struggle in darkness to teach and instruct those who hunger and thirst for knowledge, even if we are tired ourselves.
There is another passage where this sort of compassion plays a major role in a biblical circumstances, a passage that is much more obscure, 2 Chronicles 28:8-11: "And the children of Israel carried away captive of their brethren two hundred thousand women, sons, and daughters, and they also took away much spoil from them, and brought the spoil to Samaria. But a prophet of the Eternal was there, whose name was Oded; and he went out before the army that came to Samaria, and said to them: "Look, because the Eternal God of your fathers was angry with Judah, He has delivered them into your hand; but you have killed them in a rage that reaches up to heaven. And now you propose to force the children of Judah and Jerusalem to be your male and female slaves, but are you not also guilty before the Eternal your God? Now here me, therefore, and return the captives, whom you have taken captive from your brethren, for the fierce wrath of the Eternal is upon you."
Here we see a different sort of compassion, although it too was shown to people who were leaderless, whose leaders had been slaughtered in battle for their unbelief. Yet those who had won the victory were also under God's wrath, as their nation would soon go into captivity for its own unbelief and its own rebellion against God's ways. Not coincidentally, the people of Judah were not long for their own freedom, as within 40 years of the crucifixion of Jesus Christ, the autonomy of the Jews as well as the existence of the Temple and the priestly aristocracy would be ended after the revolt of 67. A corrupt leadership that had failed to provide for the well-being of their people would soon be removed from that leadership position because of God's wrath. Just as God provides leaders with offices and commands respect of those offices, so too God commands leaders to enforce His ways and to provide for the well-being of those that they serve.
Why is it so hard to find good shepherds, or to be a good shepherd? To be a godly leader is a very difficult task, made more difficult by the fact that many of us do not trust others easily and tend to be critical and skeptical of those who lead. As leaders, all of us will be imperfect because of our own limitations or flaws and because the overall plan that we lead with will not be easy to see or understand by others. Yet though we may inevitably be judged harshly by some others, especially by those who are not experienced at governing themselves, so long as we dedicate ourselves to service based on the talents and opportunities that we have to serve, we can help prevent a situation in our own institutions and societies where there are many sheep without a shepherd, upon whom God looks with compassion even as their societies face judgment.
[1] See, for example:


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The positive COVID-19 test percentage dipped Tuesday, after four days of positive tests results of more than 5% in Wisconsin.
The Wisconsin Department of Health Services reported 4.7% of all COVID-19 tests performed over the last 24 hours were positive.
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Type III IFNs (IFN-λs) are secreted factors that are well-known for their antiviral activities. However, their regulation and functions during bacterial infections are unclear. In this article, we report that the regulation of IFN-λ genes did not track with mechanisms that control type I IFN expression in response to TLRs. Whereas type I IFNs were only expressed from TLRs present on endosomes, type III IFNs could be induced by TLRs that reside at the plasma membrane and that detect various bacterial products. The mechanisms that regulate type III IFN gene expression tracked with those that promote inflammatory cytokine and chemokine expression. Importantly, rIFN-λs enhanced epithelial barriers in vitro, preventing transcellular bacteria dissemination. We therefore propose that in addition to their functions in cell-intrinsic antiviral immunity, type III IFNs protect epithelial barrier integrity, an activity that would benefit the host during any infectious encounter.
Type III IFNs are factors that are secreted by immune cells during microbial infections (1, 2). In contrast with their more extensively studied counterparts, the type I IFNs, we know little of how these genes are regulated or the functions of their products in host defense. Much of the study of type III IFNs (also known as IFN-λs) has focused on their roles in viral infections. Early studies demonstrated that these genes are coregulated with type I IFNs during many infectious encounters (3, 4). However, our recent work established that during viral infections, type I and III IFNs can be induced via distinct pathways (5). Functionally, type III IFNs are most distinct from type I IFNs in that they are particularly important at barrier surfaces. These locations are rich in epithelial cells that express high levels of the IFN-λ receptor (6), and produce various factors that protect the host from microbial encounters. Type III IFNs also have roles in protecting endothelial cells at the blood–brain barrier from viral infection (7). Thus, this family of proteins has increasingly important functions in host defense.
Recent work has suggested a role for the type III IFN family in bacteria–host interactions. For example, infection with a number of bacterial pathogens and stimulation with LPSs from the outer membrane of Gram-negative bacteria induce the expression of type III IFN genes (8–10). In addition, Listeria monocytogenes encodes virulence determinants that modulate the expression of type III IFN–responsive genes (11). These findings suggest an important role of type III IFNs in antibacterial defense, but the spectrum of bacteria that induce expression of these genes, and the function of their products, have yet to be revealed.
TLRs of the innate immune system are common sensors of bacterial infection. These receptors are expressed on various cells, in particular phagocytes and barrier epithelial cells. Detection of cognate microbial ligands, collectively known as pathogen-associated molecular patterns (PAMPs), by TLRs leads to inflammatory and adaptive immune responses that restrict bacterial spread and provide long-term immunity (12). The spectrum of activities induced by TLRs is an active area of research. Although all TLRs induce the expression of inflammatory chemokines and cytokines, a limited set of receptors induce type I IFN gene expression (13).
TLRs are one of several families of pattern recognition receptors (PRRs) that sense and respond to PAMPs. PRRs can be found in various subcellular locations, including the plasma membrane, endosomes, cytosol, endoplasmic reticulum, and the nucleus (14). Several of these families induce type I and III IFN expression, including the cytosolic DNA sensor cyclic GMP-AMP synthase and cytosolic RNA sensors of the RIG-I–like receptor (RLR) family (1, 2, 15). Our cell biological analysis of the RLR pathway revealed that these receptors can induce type III IFN or type I IFN expression from distinct subcellular locations (5). The mechanisms underlying these differential sites of signaling, and the generality of these observations as they relate to other receptor families, remain to be established. Like the RLRs, TLRs signal from diverse organelles. These receptors can signal from the plasma membrane or endosomes, and some receptors signal from both locations (16, 17). Mechanistically, TLR signaling pathways depend on the ability of these receptors to assemble large supramolecular organizing centers in the cytosol after microbial detection (18). These organizing centers serve as the principal sites of signals that promote inflammatory and immunoregulatory gene expression. The most commonly used supramolecular organizing center in the TLR pathway is the myddosome. The myddosome consists minimally of the adaptors TIRAP and MyD88, and several members of the IRAK family of serine/threonine kinases (19–22). The myddosome is best understood to promote NF-κB– and AP-1–dependent upregulation of genes encoding inflammatory cytokines and chemokines (23). A subset of TLRs also activates a myddosome-independent response that relies on the adaptor TRIF (24). TRIF signaling leads to the expression of type I IFNs. This TRIF-dependent pathway is thought to be only activated by TLR4 and TLR3, although recent studies have expanded the possible roles for TRIF to other TLRs (25).
Several TLRs induce the expression of type III IFN genes, most notably those that detect viral nucleic acids (4, 8, 15). However, the full spectrum of TLRs that induce type III IFN gene expression, and the signaling pathways that promote these putative responses, are unknown. In this study, we sought to identify potential functions of type III IFNs during host–bacteria encounters, and define the means by which the genes encoding these factors are induced by bacteria. Analogous to their ability to reinforce the endothelial blood–brain barrier to prevent viral dissemination (26), we found that recombinant type III IFNs increase the barrier functions of a polarized human intestinal epithelial cell line. This barrier reinforcement prevents the transcellular passage of infectious Gram-negative bacteria. Furthermore, we found that a variety of bacterial PAMPs activate individual TLRs to promote type III IFN expression, and that myddosome components are required for TLR-dependent type III IFN gene expression. Finally, we identify type III IFNs as, to our knowledge, the first example of IFN genes whose expression can be induced by plasma membrane localized receptors. Overall, these results highlight type III IFNs as factors that are commonly upregulated by bacteria-sensing TLRs, and suggest their general importance in barrier immunity.
Materials and Methods
Cell culture
Wild-type (WT), MyD88, TRIF, or TLR5 knockout (KO) mice were purchased from JAX. Bone marrow–derived dendritic cells (BMDCs) were differentiated from mouse bone marrow in IMDM (Life Technologies), 10% GM-CSF, and 10% FBS. 293T cells were cultured in complemented DMEM supplemented with 10% FBS. WT THP1 cells, along with IRAK2 or IRAK4 CRISPR KO cells, were a kind gift from Iain Fraser (National Institutes of Health). MyD88 KO THP1 cells were constructed in WT/Dual THP1 background (InvivoGen). THP1 cells were cultured in complete RPMI 1640 supplemented with 10% FBS and were differentiated into macrophages using 50 ng/ml PMA for 48 h. T84 epithelial cells were obtained from the Harvard Digestive Disease Center. T84 cells were cultured and polarized as previously described (27). In brief, cells were plated on collagen-coated 3-μm polyester filters and grown for 7 d until transepithelial electrical resistance (TEER) reached 1650–2970 Ω/cm2. TEER was measured with an epithelial Voltohmmeter (WPI). Human PBMCs were isolated from blood of healthy individuals using Lympholyte and SepMate columns, according to the manufacturer's instructions. Cells were then plated on tissue culture plates in RPMI 1640 (Life Technologies) + 10% FBS and allowed to adhere for 24 h followed by washing, to enrich the monocyte fraction.
ELISA and Abs
Infections and stimulations in vitro
Where needed, cells were treated with 1 μg/ml cytochalasin D (Sigma) or 5 μM 34-2 Dynole (Applied Biochemicals). Dynole was applied in serum-free medium. Where indicated, 50 μg/ml 4 kDa FITC-dextran (Sigma) was applied on the apical side of polarized T84 cells for 90 min. Cells were then treated with 0.1 μg/ml recombinant human IFN-λ1 or IFN-β (PeproTech) for 2 h before infection as described later. At the time points indicated, TEER and fluorescence in the basal chamber were measured, or bacteria in the basal compartment were quantified by CFU plating and enumeration.
Salmonella SL1344 and fliC mutant strains were obtained from D. Monack (Stanford), Shigella M90T strain was obtained from N. Alto (UT Southwestern), whereas a Shigella sonnei strain was obtained from A. Clements (Imperial College). Shigella flexneri was used for the majority of the experiments including the functional assays in Fig. 1. Shigella sonnei was used in Fig. 2C, 2D, 2H, and 2I, to respect regulations on work with Shigella flexneri in the U.K. Salmonella was grown in Luria Bertani broth, whereas Shigella was grown in tryptic soy broth. Unless otherwise specified, to minimize expression of type III secretion systems, we used overnight cultures directly for infection. When virulent bacteria were required (Fig. 1), Salmonella were subcultured 1:33 for 3 h OD600 nm = 2, and Shigella were subcultured 1:60 in tryptic soy broth until OD600 nm = 0.6. LPS was supplied by Enzo (ALX-581-012-L002). Phosphorothioate-linked CpG DNA (5′-TCCATGACGTTCCTGACGTT-3′) was synthesized by IDT. All other synthetic ligands were supplied by InvivoGen: Pam3CSK4 (tlrl-pms), Pam2CSK4 (tlrl-pm2s-1), Zymosan (tlrl-zyn), and VacciGrade flagellin (vac-fla). Where indicated, ligands were treated with polymyxin B (Sigma) for 30 min at room temperature.
When indicated, cell culture media from BMDCs stimulated with TLR ligands were collected and incubated onto fresh 293T cells for 1 h. Cells were then lysed and processed for western immunoblotting using standard techniques.
Quantitative real-time PCR
Cells were lysed in RNA Lysis buffer (Ambion) supplemented with 2-MΕ and passed through QIAshredder columns (Qiagen). RNA was then isolated using PureLink RNA Mini Kit columns (Ambion) following the manufacturer's recommendations. RNA samples were treated with DNase I (Thermo Scientific) for 30 min at 37°C followed by 10 min EDTA deactivation at 65°C. Quantitative RT-PCR (qRT-PCR) was carried out with a Bio-Rad CFX384 real-time cycler with TaqMan probes as directed by the manufacturer. Data were normalized against the expression of a control gene (GAPDH) followed by the uninduced or uninfected (control) sample (set to 1). When no counts were detected in a sample, an arbitrary number of 45 cycles was selected.
Type III IFNs protect epithelial barrier integrity during bacterial infections
Although much work has been focused on the role of type III IFNs in antiviral immunity at epithelial surfaces, the functions of these factors during bacterial infections are less clear. The IFN-λ receptor protects the endothelial blood–brain barrier to prevent viral dissemination (26). In an analogous sequence of events, several enteric bacterial pathogens disrupt epithelial barriers to enable their spread to the underlying tissues of the body. We therefore determined whether type III IFNs protect the intestinal barrier from damage inflicted by bacteria. As a control and complementary experiment, we studied the role of type I IFNs in the same assays.
For these studies, we examined polarized colonic human T84 cells because these cells are routinely used as an in vitro model to study the interactions between enteric bacteria and intestinal epithelial cells (27, 28). Cells were treated with rIFN-λ and rIFN-β, and TEER, a marker of epithelial barrier integrity, was monitored over time. We found that treatment with IFN-λ led to a significant increase in TEER. IFN-β treatment also significantly increased TEER, but the effect was less sustained (Fig. 1A). Because increases in TEER correspond to an increase in barrier integrity, we examined the role of type III IFNs in bacteria-induced epithelial disruptions. We first used Salmonella enterica serovar Typhimurium (Salmonella), a human pathogen that naturally disrupts intestinal epithelia, to promote systemic dissemination (29, 30). Polarized T84 cells were treated with IFN-β or IFN-λ for 2 h, followed by apical infection with Salmonella. These bacteria were grown to induce the Salmonella pathogenicity island 1 type III secretion system, which is required for bacterial invasion (31–33) and epithelial damage (30, 34). We found that Salmonella infection disrupted TEER (Fig. 1B, black line). In cells pretreated with IFN-β, TEER was disrupted to an extent similar to control conditions (Fig. 1B, blue line). By contrast, pretreatment with IFN-λ protected T84 monolayers from Salmonella-induced TEER disruption, with the cells recovering by 2 h postinfection (Fig. 1B, red line). As an additional readout of epithelial barrier integrity, we measured the transcellular passage of fluorescent dextrans across the monolayer. After apical IFN and dextran application, cells were infected apically, and fluorescence in the basal compartment was measured. Salmonella infection led to an increase in dextran passage into the basal compartment (Fig. 1C, black line), indicating a disruption of the epithelial barrier. Treatment with both IFNs reduced dextran passage across the monolayer, but the effect of IFN-λ was more pronounced (Fig. 1C, red line). To determine whether the ability of IFN-λ to protect against dextran passage through the epithelial barrier correlated with bacterial passage, we quantified the number of bacteria in the basal chamber by plating and enumerating CFUs. Two hours after apical infection, we found that 60% less bacteria were able to cross a monolayer treated with IFN-λ, as compared with control conditions (Fig. 1D). Again, the effect of IFN-β treatment was less pronounced. To determine whether the ability of IFN-λ to protect epithelial barriers during Salmonella infections extended to other bacteria, we examined Shigella flexneri. Like Salmonella, Shigella is an enteric pathogen whose virulence relies on damage of the epithelial barrier (35). IFN-λ efficiently prevented the transmigration of bacteria across the monolayer, as we measured 70% less CFUs in the basal chamber of monolayers treated with IFN-λ, with IFN-β having a smaller effect (Fig. 1E). Taken together, these data establish that IFN-λs protect epithelial barrier integrity from bacteria-induced damage and efficiently block the ability of bacterial pathogens to cross the epithelium.
Extracellular bacteria induce type III IFN expression
We reasoned that if type III IFNs are important for protecting against bacteria-induced epithelial damage, then the genes encoding these factors may be expressed during bacterial encounters. We therefore analyzed the transcriptional response of cells infected with Salmonella and Shigella. Specifically, quantitative RT-PCR was used to examine the expression of the type III IFN IFN-λ1 in human cells, because it is the most prominent type III IFN produced (36). However, because IFN-λ1 (IL-29) is a pseudogene in mice (37), we measured IFN-λ2 (IL-28A) in mouse cells. IFN-β was used to monitor type I IFN expression. Basal expression of each IFN gene was first established. We found that basal type I and III IFN expression differed from one another in different cell types, with mouse BMDCs and PMA-differentiated human THP1 monocytes containing higher levels of basal IFN-β transcripts, and polarized T84 cells containing higher levels of IFN-λ1 (Supplemental Fig. 1A–C). Based on this differential basal expression, we monitored relative fold induction over control, rather than transcript abundance, in all subsequent experiments. Relative fold induction was calculated as the ratio of mRNA present in resting cells as compared with infected or stimulated cells. Infection of BMDCs (Fig. 2A), polarized T84 cells (Fig. 2B), and PMA-differentiated human THP1 cells (Fig. 2C) demonstrated that IFN-λ fold induction was at least as strong as IFN-β during Salmonella and Shigella infection. In addition, we observed secreted IFN-λ1 protein by ELISA in Salmonella- and Shigella-infected THP1 cells (Fig. 2D). To determine whether phagocytosis was required for bacteria-induced IFN-λ expression, we blocked bacterial entry using the actin polymerization inhibitor cytochalasin D. Cytochalasin D treatment did not affect baseline levels of IFN-β or IFN-λ1/2 in BMDCs (Supplemental Fig. 1D) or T84 cells (Supplemental Fig. 1E). Cytochalasin D treatment of BMDCs (Fig. 2E) or T84 cells (Fig. 2F) prevented IFN-β expression, as compared with vehicle treatment. Interestingly, cytochalasin D did not inhibit the upregulation of IFN-λ in response to Salmonella in BMDCs (Fig. 2E), or Salmonella or Shigella in T84 cells (Fig. 2F). These results suggest that bacterial entry is not required for IFN-λ expression.
Because bacteria can activate multiple innate immune receptor families, we sought to determine the receptor(s) responsible for IFN expression during infection. TLR-dependent pathways can be identified by the examination of cells lacking the signaling adaptor proteins MyD88 and TRIF. We therefore infected BMDCs lacking MyD88 or TRIF (Fig. 2G) and THP1 cells that were rendered MyD88-deficient using CRISPR/Cas (Fig. 2H). Baseline levels of IFN transcripts in WT, MyD88 KO, and TRIF KO cells were comparable in BMDCs and THP1 cells (Supplemental Fig. 1F, 1G). IFN-β fold induction during infection was largely TRIF dependent, whereas IFN-λ expression was strongly dependent on MyD88 and TRIF in BMDCs (Fig. 2G). Similarly, in THP1 cells, IFN-λ expression upon Salmonella and Shigella infection was MyD88 dependent (Fig. 2H). IFN-β expression in response to Salmonella and Shigella was not mediated by MyD88. Finally, we monitored IFN-λ secretion by ELISA postinfection of THP1 cells that are deficient for IRAK4, a component of the MyD88 signaling pathway (38). IRAK4 KO cells secreted minimal amounts of IFN-λ after bacterial infection (Fig. 2I). These data indicate that bacteria-induced type I and III IFN expression are TLR dependent, and further reveal that these IFN genes are subject to differential regulation. IFN-λ expression activity depends on MyD88 signaling and does not depend on phagocytosis.
Endosomal and cell surface resident TLRs activate type III IFN gene expression
To better understand the means by which bacteria induce IFNs, we took the reductionist approach of identifying individual PAMPs that promote type I and III IFN expression. PMA-differentiated THP1 monocytes (Fig. 3A), polarized T84 cells (Fig. 3B), and BMDCs (Fig. 3C) were treated with cognate ligands for all well-defined TLRs. Endosome-localized TLR3, TLR7/8, and TLR9 were respectively activated with poly(I:C), R848, and CpG DNA. These ligands induced IFN-β and IFN-λ similarly (Fig. 3A–C). As expected, these ligands also upregulated the inflammatory cytokines IL-6 and IL-8 (Supplemental Fig. 2A). TLR4 activation by bacterial LPS also induced expression of both IFN species. TLR2/1 heterodimers were activated with the bacterial lipoprotein mimic Pam3Cys and TLR2/6 heterodimers with Pam2Cys. Interestingly, TLR2 promoted a greater increase in the expression of IFN-λ than IFN-β in these different cell types. We obtained comparable results with the yeast cell wall component zymosan, an additional TLR2 agonist (Fig. 3A–C). TLR5 was activated by Salmonella flagellin. Much like TLR2, TLR5 induced IFN-λ and IFN-β expression, although IFN-λ fold induction was an order of magnitude higher than that of IFN-β, especially in THP1 cells and dendritic cells (Fig. 3A, 3C). These findings with TLR2 and TLR5 were somewhat unexpected, because cell surface localized receptors are not typically considered to be capable of inducing IFN expression.
To further study IFN expression by cell surface TLRs, we stimulated different cell types with varying concentrations of LPS, Pam3Cys, and flagellin for 2 or 5 h. In THP1 cells (Fig. 3D), polarized T84 cells (Fig. 3E), and BMDCs (Fig. 3F), IFN-λ expression in response to TLR2 or TLR5 stimulation was higher than IFN-β, especially at the later time point (Fig. 3D, 3F). IL-6 or IL-8 expression was also measured under the same conditions. As expected, these cytokines were induced by all ligands (Supplemental Fig. 2B–D). To rule out LPS contamination in our flagellin preparations, we compared IFN induction in TLR5 KO BMDCs. Baseline expression levels of all genes examined were comparable in WT and TLR5 KO cells (Supplemental Fig. 1H). Flagellin- but not LPS-mediated induction of IFNs and cytokines was completely dependent on TLR5 (Supplemental Fig. 2E). In addition, treatment with the LPS inhibitor polymyxin B affected only LPS-mediated responses (Supplemental Fig. 2F). These results indicate that all TLRs are capable of inducing type III IFNs, and that TLR signaling from the plasma membrane favors the induction of type III IFN genes.
To determine whether the amount of IFN-λ expressed by cell surface TLRs was functional, we took three approaches. We first examined the release of IFN-λ protein from THP1 cells stimulated with various TLR ligands (Fig. 3G). We found that all TLRs ligands, including cell surface TLRs, caused the secretion of IFN-λ, as detected by ELISA (Fig. 3G). In addition, we monitored IFN-λ protein secretion from primary human peripheral blood–derived monocytes stimulated with the same TLR ligands (Fig. 3H, 3I). Again, treatment with ligands against all TLRs caused the production of IFN-λ (Fig. 3H, 3I). In fact, type III IFN production was stronger upon stimulation of TLR4 and TLR2 than of all endosome-localized TLRs. Second, we analyzed the phosphorylation of STAT1 in T84 cells treated with various TLR ligands (Supplemental Fig. 2G). We found that STAT1 was phosphorylated after Pam3Cys or flagellin treatment to levels at least comparable with LPS or poly(I:C) (Supplemental Fig. 2G). We also found that the IFN-stimulated gene viperin was induced by treatment with all TLRs (Supplemental Fig. 2G). These data demonstrate that functional IFN proteins were produced upon TLR activation. To specifically investigate type III IFN signaling, we analyzed JAK2 phosphorylation, because we and others had previously demonstrated that JAK2 regulates the type III IFN signaling pathway (5, 39). Interestingly, JAK2 phosphorylation was induced by Pam3Cys and flagellin to levels comparable with LPS (Supplemental Fig. 2G). Third, we took advantage of our finding that mouse IFN-λ2 (but not IFN-β, IFN-α, or IFN-γ) can signal in human cells (5) (Supplemental Fig. 2H). Thus, we treated human 293T cells with cell culture supernatants from mouse BMDCs that had been challenged with TLR ligands. Bioactivity of mouse IFNs was measured by western analysis of STAT1 phosphorylation (Fig. 3J). Densitometric analyses were performed using ImageJ (Supplemental Fig. 2I). Interestingly, we found that stimulation with bacterial PAMPs that activate cell surface TLRs led to the release of bioactive type III IFNs that induced STAT1 phosphorylation to levels comparable with that obtained with endosomal TLRs (Fig. 3J). Collectively, these data establish that a range of plasma membrane and endosomal TLRs induce IFN-λ gene expression and induce the release of bioactive IFN-λ proteins.
TLR5 preferentially induces type III IFNs
Our results have thus far indicated that flagellin was able to upregulate IFN-λ (Fig. 3). To analyze this observation further, we used flagellin mutant Salmonella (fliC KO). We infected polarized T84 cells (Fig. 4A) and BMDCs (Fig. 4B) with WT and fliC KO Salmonella. Interestingly, despite the fact that Salmonella encodes many PAMPs, IFN-λ induction during infection with this bacterium was dependent on TLR5, because fliC KO Salmonella induced marginal amounts of type III IFNs (Fig. 4A, 4B). Similar data were obtained in THP1 cells, by monitoring IFN-λ1 secretion (Fig. 4C). As a complementary experiment, we compared the ability of WT and TLR5 KO BMDCs to respond to WT Salmonella and Shigella. IL-6 induction was independent of TLR5, whereas some IFN-β was induced at slightly lower levels in TLR5 KO cells (Fig. 4D). Notably, IFN-λ induction was almost completely TLR5 dependent (Fig. 4D). These data further demonstrate the ability of TLR5 to induce type III IFNs and suggest that TLR5 is an important contributor of IFN-λ expression in infections with Gram-negative pathogens.
Type III IFN induction by TLRs depends on the myddosome components MyD88 and IRAK4
We next sought to determine the signaling pathways that differentially drive type I and III IFN production in response to TLR activation. We first focused on TLR4, because we found it induces IFNs of both families (Fig. 3). In the TLR4 pathway, genes encoding inflammatory cytokines are induced by myddosome component MyD88 (40). However, TLR4 also activates a myddosome-independent pathway that is required for TLR4-induced type I IFN expression (41). We sought to determine the role of the myddosome in TLR4-dependent IFN-λ expression. We therefore examined murine BMDCs that were genetically deficient for MyD88 (Fig. 5A). We found that LPS-induced IFN-β expression was MyD88 independent, whereas IL-6 expression was MyD88 dependent. These findings are consistent with the work of Akira and colleagues (42), who generated MyD88-deficient mice. Interestingly, we observed a requirement of MyD88 for LPS-induced IFN-λ expression (Fig. 5A). To corroborate these findings, we performed similar stimulations in THP1 monocytes lacking MyD88. MyD88-deficient THP1 cells retained the ability to express IFN-β. In contrast, IFN-λ expression in response to LPS was reduced in the absence of MyD88 (Fig. 5B). In addition, the myddosome components IRAK2 and IRAK4 were not required for IFN-β expression in THP1 cells, whereas expression of the inflammatory chemokine IL-8 was dependent on these kinases (Fig. 5C). Importantly, IFN-λ expression in these cells was IRAK2 and IRAK4 dependent (Fig. 5C). Baseline levels of IFN and IL-8 transcripts are shown in Supplemental Fig. 1I. The small differences we observed in IFN-λ baseline levels did not affect the effects we observed upon cell stimulation. Finally, IRAK4 was required for LPS-induced IFN-λ secretion, as assessed by ELISA in THP1 cells (Fig. 5D). Collectively, these studies in human and mouse cells reveal a fundamental difference between the signaling pathways used by TLR4 to promote type I and III IFN expression, with IFN-λ (but not IFN-β) expression depending on the myddosome.
To determine whether other TLRs also require the myddosome components to promote type III IFN expression, we examined the requirement of MyD88 on the ability of TLR2 and TLR5 to induce IFN-λ expression. We found that MyD88 was required for the induction of IFN-λ in response to Pam3Cys and flagellin in BMDCs and THP1 cells (Fig. 5E, 5F, 5I, 5J). As expected, IL-6/IL-8 induction in response to both ligands was MyD88 dependent. Interestingly, the low induction of IFN-β observed in response to these ligands also required MyD88. Similar findings were obtained when we examined IRAK2- and IRAK4-deficient THP1 monocytes (Fig. 5G, 5K), and IFN-λ secretion upon Pam3Cys or flagellin treatment was also IRAK dependent (Fig. 5H, 5L). Overall, these findings likely explain how a broad range of TLRs can induce type III IFN expression, because IFN-λ is induced by the signaling hub most commonly used by TLRs—the myddosome.
TLR4 induces type III IFNs from the cell surface
Although TLR4 detects LPS at the plasma membrane, and induces myddosome signaling from this location, type I IFN induction does not occur until TLR4 reaches endosomes (43). In fact, no receptor is known to induce the expression of any IFN gene from the plasma membrane. Our finding that TLR4-induced IFN-λ expression depends on the myddosome therefore raised the possibility that the signals inducing this gene emanate from the cell surface. This possibility was examined by preventing endocytosis with Dynole, a chemical inhibitor of dynamin family GTPases (44). Baseline levels of all genes in BMDCs and T84 epithelial cells were not affected by Dynole treatment (Supplemental Fig. 1J, 1K). As expected, Dynole treatment blocked the LPS-induced expression of IFN-β in BMDCs (Fig. 6A) and polarized T84 cells (Fig. 6B). Also, as expected, the LPS-induced expression of the myddosome-dependent genes IL-6 and IL-8 was insensitive to endocytosis inhibition (Fig. 6A, 6B). Interestingly, IFN-λ expression was not blocked by endocytosis inhibition (Fig. 6A, 6B). These data suggest that pharmacological inhibition of TLR4 endocytosis differentially affects type I and III IFN expression in response to LPS. To complement this analysis, we used a genetic approach by taking advantage of the fact that TLR4 endocytosis is dependent on CD14 (45). We therefore examined the requirement of CD14 for LPS-induced gene expression. CD14 KO cells had baseline levels of all genes comparable with WT cells (Supplemental Fig. 1L). CD14 was required for IFN-β, but not IL-6, expression (Fig. 6C). Most notably, IFN-λ expression was independent of CD14 (Fig. 6C). These collective data establish a unique aspect of type III IFNs, in that they can be induced by TLRs present at the plasma membrane (Fig. 6D).
In this study, we describe an unexpected aspect of the TLR signaling pathways, in that these pathways are general inducers of type III IFNs during bacterial encounters. Much of the work on type III IFNs has focused on the regulation of their expression and functions during viral infections. However, several recent reports suggest a role for this IFN family in bacteria–host interactions as well. These reports include observations that some bacterial infections or LPS challenges induce type III IFN expression (8–10, 46). In addition, the bacterium L. monocytogenes encodes virulence factors that fine-tune the expression of IFN-λ–responsive genes (11). The generality of these observations, and the means by which type III IFN gene expression is regulated by TLRs, has been unclear.
TLRs have been examined extensively for their abilities to induce cytokine, chemokine, and type I IFN expression. The collective studies from several laboratories have led to a model whereby all TLRs are potent inducers of cytokine/chemokine expression, and that the signals that induce these genes can emanate from the plasma membrane or endosomes (14, 47). In contrast, a subset of TLRs are strong inducers of type I IFNs, and the expression of these genes can only be initiated by TLRs that are present on endosomes. This statement applies to TLRs that detect viral nucleic acids, which sense their ligands within endosomes, but also applies to TLR4. Indeed, TLR4 can detect bacterial LPS at the plasma membrane, but cannot induce the expression of type I IFN genes until after CD14-dependent endocytosis (43, 45, 48). Studies in recent years have extended this principle to TLR2, which induces the weak (but detectable) expression of type I IFN genes (25, 49–51). Like TLR4, TLR2 must be internalized into endosomes before type I IFN can be induced (25, 49–51). In considering this paradigm, our finding that type III IFN genes can be induced by TLR4 at the plasma membrane is notable, because it provides an intriguing exception to the apparent cell biological rule that IFNs cannot be induced from the plasma membrane.
Several lines of evidence support this conclusion. First, LPS-induced IFN-λ expression is dependent on signaling proteins that TLR4 engages at the plasma membrane, such as the myddosome components MyD88, IRAK2, and IRAK4 (Fig. 6D). In contrast, LPS-induced IFN-β expression is mainly myddosome independent. Second, LPS-induced IFN-λ expression is not sensitive to the endocytosis inhibitor Dynole, whereas IFN-β expression is. Third, IFN-λ expression induced by LPS proceeds normally in the absence of CD14, which is an essential regulator of TLR4 endocytosis. IFN-β expression, in contrast, is lost in the absence of CD14. Thus, at least in the case of TLR4, this receptor induces type I and III IFNs from different organelles. Our recent studies on virus-induced type III IFN expression led to a similar conclusion, but with different receptors and organelles (5). During viral infections, RLRs induce the expression of type I and III IFN genes. These receptors can signal from peroxisomes and mitochondria (52, 53). Signaling from mitochondria induces both sets of IFNs, whereas signaling from peroxisomes induces the expression of type III IFNs specifically. Thus, distinct receptor families in the innate immune system can separate the subcellular sites of type I and III IFN expression.
The aspects of IFN-λ gene regulation described in this article are reminiscent of those governing the expression of gene-encoding inflammatory cytokines and chemokines, such as IL-8 and IL-6 (Fig. 6D). These aspects include the ability to be transcriptionally upregulated by all TLRs, the dependence on myddosome components, and the ability to be induced from the plasma membrane. Unlike type I IFNs, which are most important for antiviral defense, inflammatory cytokines and chemokines are important to defend against any microbial infection. It therefore stands to reason that the common regulatory mechanisms governing the expression of cytokines, chemokines, and type III IFNs reflect a function of IFN-λs that extends beyond antiviral defense.
What could be the function of type III IFNs in general antimicrobial defense? Although this answer is likely to be context dependent, our observation that rIFN-λ can reinforce the barrier functions of polarized T84 epithelial cells provides some clues. IFN-λ treatment was sufficient to increase the intrinsic TEER of T84 cells and prevent the barrier damage induced by pathogenic Salmonella and Shigella. This protection from epithelial damage resulted in a diminished number of bacteria that can pass into the basal chamber of our in vitro culture system. These findings complement recent findings that the IFN-λ receptor is required to prevent virus-induced damage to the blood–brain barrier (26), and that type III IFN signaling appears to have a protective role in colitis models (54). Thus, using in vitro and in vivo systems with viruses and bacteria, type III IFNs appear to play an important role of reinforcing epithelial and endothelial barriers. Based on the diverse functions of other immunomodulatory cytokines, it is likely that additional activities of type III IFNs exist. This study should provide a mandate to explore these possibilities in various infectious settings.
The authors have no financial conflicts of interest.
We thank members of the Kagan and Zanoni laboratories for helpful discussions, and in particular Dr. Charles Rosadini for instrumental help in purifying mouse dendritic cells.
This work was supported by National Institutes of Health Grants AI093589, AI072955, and P30 DK34854 (to J.C.K.), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund (to J.C.K.), a King's College London Prize Fellowship (to C.O.), and a Sir Henry Dale Fellowship from the Royal Society and the Wellcome Trust (Grant 206200/Z/17/Z to C.O.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- bone marrow–derived dendritic cell
- knockout
- pathogen-associated molecular pattern
- pattern recognition receptor
- qRT-PCR
- quantitative RT-PCR
- RIG-I–like receptor
- transepithelial electrical resistance
- wild-type.
- Received February 27, 2017.
- Accepted August 28, 2017.
- Copyright © 2017 by The American Association of Immunologists, Inc.


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Yesterday it rained most of the day and I completed a few boat projects. Today the sun came out again and I took the dinghy to the Cochrane Islands early in the morning. There I hiked along a part of the Sunshine Coast Trail, which runs for 180 km along Malaspina Peninsula. Currently, the trail is only open for day hikes. I saw some bear scat, but no bears, nor did I meet any people.
In the afternoon I took the boat to Squirrel Cove on Cortes Island, a favourite anchorage. Now it is time to get dinner going, it will be an early night tonight, as I am rather tired.


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Obituary: Jack LeRoy Merritt, 1938 - 2020
Jack "Jackie" LeRoy Merritt, 81, of Cottonwood, Arizona passed away at home on Wednesday, March 11, 2020.
He was born in Grand Junction, Colorado on May 13, 1938 to the late William Guy Merritt Jr. and Mary (Durr) Merritt.
Jack was raised in the Los Angeles area, and joined the United States Army. His work experience included delivering newspapers, working for Fuller Brush Co., and finally finding his place as a semi-truck driver covering the Los Angeles area for 30 years.
Retirement brought Jack and his wife, Loleta to Cottonwood in 1998 where he continued his love of flying and was an active member of EAA volunteering to fly "Young Eagles".
He was also an avid bowler.
Jack served faithfully as a member of The Church of Jesus Christ of Latter-Day Saints in various callings, including as an ordinance worker for the Mesa and Phoenix Temples.
He served in the community as a Boy Scout leader, a Juvenile Justice Committee volunteer, Cottonwood Airport president, and delivered Meals on Wheels.
Jack is survived by his wife, Loleta; their children, Cliff Cordy, Joel Merritt, Charles (April) Cordy, Dennis (Janice) Merritt; and their grandchildren: Benjamin (Jasmine) Cordy, Julianna Cordy, Matthew (Shandra) Cordy, Kristina (Blake) Bowen, Molly (Kenneth) Lathen, Andrew Merritt, and Jason Going; and eight great-grandchildren. He was preceded in death by his parents, his brother, William, and his son, Brian.
The family wishes to have a Celebration of Life at a future date.
You can leave your condolences for the family at www.buelerfuneralhome.com.
Information provided by survivors.
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- Rimrock woman charged with manslaughter in wreck that killed daughter
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- Arizona sets new high mark for one-day gain in COVID-19 cases: 1,127
- New single-day high for Arizona COVID-19 cases: 1,654
- Mask mandate for Cottonwood
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