Many solutions Purposefully create a closed system, which can be tedious to maneuver away from as soon as you begin. But with a platform named Ecwid, you no longer have to worry about it. In fact, the whole goal of the company, which began in 2009, would be to quickly and quickly make an online store system on your existing websites.
This increased my Attention, because I understand the struggles that come along with trying to migrate sites, especially in regards to ecommerce. So, I wanted to put together a comprehensive review of the Ecwid system to find out if it is worth the time.
It is quite Remarkable what Ecwid has achieved since 2009, with more than 1 million merchants getting signed up. What's even more cool is that the Ecwid retailers span over 175 different countries, showing that this is not only a platform for most people in the US or Europe. Even companies in developing countries have a chance to get an internet store going, and you do not even have to squander your previous website if you have one.
The usership Continues to grow, particularly considering Ecwid is currently PCI DSS Level 1 accredited. If you are not sure what this is, it is pretty much the gold standard for safety in the ecommerce world. You can rest easy knowing that you and your customers' information is safe and secure.
In addition to The total merchant count, over 20,000 active users are utilizing the Ecwid Shopping Cart for WordPress. The difference between that 20,000 along with also the 1 million is the fact that you don't require a WordPress site to implement Ecwid. So, the 20,000 simply identifies WordPress users with the plugin.
Ecwid is a hosted Alternative for building an online shop. Unlike competing products such as Shopify, Bigcommerce and Volusion, which permit you to construct a new website with e-commerce functionality, Ecwid is geared more towards people who currently have a site and simply want to add an online store for this.
Much like other E-commerce platforms, Ecwid lets you install'catalogs' of goods, and add photos, pricing, weight etc. for each. You can define shipping prices, accept credit card payments etc — all of the key things that you would expect to be able to do with an e-commerce alternative.
However it differs Apart from competing products such as Shopify, Volusion, Squarespace and Bigcommerce since it is not really designed to let you produce a fully-featured, standalone e-commerce website; rather, it's designed to enable you add an internet store to an present online existence.
The Product works by giving you a widget that gets placed on other websites — hence the title Ecwid: it's short for'E-commerce Widget'. You get a few lines of HTML code (the widget) to add to an existing site or social media profile, and your store is displayed wherever you've added this code.
Ecwid makes Strides in the market by catering to companies that don't need to start a brand-new site. The main feature is that you can take your old website and quickly integrate the Ecwid system inside a couple of minutes.
You can also Mirror the machine onto various different sites if you plan on selling elsewhere online. Manage all of these sites from one location instead of switching between multiple dashboards.
Another pleasant Feature is the syncing between multiple devices. Sell on-the-go with your smartphone. This usually means you could go to a tradeshow to generate a number of sales and each one these earnings are represented onto the back part of your website.
The system is Also made to integrate well with social networks, so that you can sell your things on Facebook if needed. 1 part of Ecwid that we especially enjoy is the shipping facet. With real-time transport integrations you constantly know what is happening with your requests and at which they are being shipped to.
Thanks Into Ecwid's point-of-sale (POS) performance, you can use your Ecwid shop to market not only online but in physical locations too — in shops, market stalls, in theatres and so on.
Together with Ecwid's POS Performance, irrespective of whether a customer buys a product in store, online, in their phone or through Facebook, everything remains in sync – i.e., the retailer's catalog, inventory and client / trade info.
There is a few methods that you can take advantage of this attribute: the easiest is likely to use'Paypal Here.' This entails downloading the Ecwid iOS app from the Apple's App shop, connecting it to the Paypal Here service, then taking payments utilizing a cell card reader (pictured above).
You have two options when it comes to POS Hardware and Paypal Here: you can buy a processor and swipe reader (for $24.99) or a processor and also tap reader (for $79.99). Both may be ordered straight from Ecwid.
If you're based outside That the US / UK, or need to work with another mobile device type, the fantastic news is that Ecwid also functions with all the Vend, Clover, NCR Silver and Square POS systems. These all facilitate using a wider range of hardware at a physical retail location (full-sized card readers, receipt printers, cash drawers pill racks etc.) and are usable in more countries.
Although you could avail Of this Paypal Here POS choice on all paid-for plans, you will want to be on the most expensive'Unlimited' plan if you would like to avail of a Vend, Clover, NCR Silver or Square admin.
Granted, it's a pretty Basic plan, where features such as discount coupons and service aren't available, and it merely lets you sell up to 10 products in two classes — but this may actually be sufficient for some customers, and using it's a good way to try the system out.
However, A vital point to note about the free program is that it isn't great from an SEO standpoint — you have to be on a paid program to make certain your product pages speak to hunt engines accurately (more on SEO below).
Ecwid is forward of the Pack in that its storefronts can be automatically translated into nearly 50 different languages — customers can view your store in their own language, without you needing to worry about creating alternate versions of it. Ecwid provides this functionality by detecting visitors' language mechanically based on their browser settings / IP address.
Competing products like Shopify And Bigcommerce are yet to provide this sort of functionality — that they force you to rely on third party programs, the production of multiple stores or Google Translate to supply different language versions of your store, which can be less than perfect. Therefore a definite win for Ecwid here.
1 area where Ecwid does not perform quite so Well on the search engine optimization front entails AMP — accelerated mobile pages. As its name implies, AMP pages load faster on cellular devices. This motivates people to remain longer on your page, thus increasing'dwell time' –something that's believed by many SEO experts consider to be rewarded by Google with preferential treatment in search results. Furthermore, Google sometimes highlights AMP webpages in carousels in search results, giving AMP content an extra little boost.
The Export option allows you to export product data, orders and customers (in CSV format) and ensures that if you ever feel the need to migrate your shop to another e-commerce platform, you should not have any significant problems doing so.
WordPress doesn't Provided any e-commerce performance from the box, so anyone wanting to sell on the platform will require a third-party solution like Ecwid. There is an Ecwid plugin accessible, therefore adding an Ecwid shop to your WordPress site is quite straightforward — you can get up and running with a couple clicks.
Squarespace includes Increasingly good e-commerce features, but the amount of payment gateway options is quite limited — you need to use Stripe or Paypal. (And using Stripe only functions if you're selling products from particular countries). The other main problem with the built-in Squarespace selling software is that they don't facilitate dropshipping, and full export of merchandise isn't currently possible.
Integrating Ecwid with Squarespace permits you to bypass these limitations, and adding it into some Squarespace site is quite easy: it is a simple matter of including a code block to a webpage and pasting some HTML to it.
As With Squarespace, there is currently no dropshipping option for Wix users, so again Ecwid can offer a good workaround. A dedicated app is provided by Ecwid to allow you integrate the item onto a Wix store easily.
Ecwid provides a pretty Restricted number integrations with other apps that are well-known. Integrations do exist with services Mailchimp, Xero and Freshbooks; but to be fair the assortment of integrations could be extensive — competing products such as Shopify offer a much wider range of programs. Notable omissions in the Ecwid store include Quickbooks and Zendesk, for example (that said, a Quickbooks integration is coming shortly ).
Ecwid's Interface is pretty user friendly. Like most similar online shop builders, you receive a vertical menu on the left that allows you to access key functionality, and the place on the right can be used to edit or display associated products, site content and reports.
As with any e-commerce tool, you'll need to Spend a fair amount of time getting your head around creating catalogues and product variants, establishing shipping rates and so on…but there is nothing here that should represent also a lot of a learning curve.
If You're stuck nonetheless, you can always get in touch with Ecwid's support team — more on this later — or, if you're on a'Business' or'Unlimited', you are able to take advantage of the included customization service (you get two hours and 12 hours personalization time from Ecwidon these programs ).
I'm not a Lawyer, so please note that the below observations shouldn't be interpreted as legal information, but I'm planning to do my very best to spell out a number of the key GDPR issues facing Ecwid consumers below.
With the introduction of That the EU's new GDPR laws, there are several legal steps that site owners now should take to ensure they are adequately protecting EU customers' and visitors' privacy. There are severe financial penalties for not doing this (to the point where it's usually a good idea to consult an attorney on what to do); and even when your business isn't based in the EU, then you still will need to comply with the regulations where any website visits in the EU are worried.
There doesn't seem to be Anything that stops you fulfilling the first few requirements easily enough with Ecwid, although you will have to devote time and potentially money generating adequate notices and crafting information capture forms so they are GDPR compliant.
Now, out of the box at There is not a means to deal with the cookie consent issue with Ecwid. Nor is there an adequate program available from the Ecwid app store (the only option on front I can locate, the'EU cookie banner', doesn't appear to provide the essential functionality — despite costing $1.99 a month to use).
It Seems to me that the only alternative available to Ecwid consumers is to use a specialist product such as CookiePro, which does supply all the essential functionality to generate cookie approval fully GDPR compliant. Despite being a strong product nonetheless, Cookiepro does require quite a great deal of configuration and you'll want to set quite a lot of time apart to set this up.
I really enjoy The simple fact which you can run a website without paying for anything. This makes the installation process that much easier, because you simply need to punch in your email and make a password to go straight to the backend port.
The system has Wizards for each platform you want to place your store on. If you have a WordPress site, just walk through three steps to get up and running. You can also click on options like Facebook and Joomla, depending on the place you want to market.
The backend Design is perfect, because it walks you through every site creation and integration measure with a few fine text and photos. Once you obtain your store implemented on a website or societal page it looks fairly slick.
The design Depends upon the theme you have on your website, however as you can see below, it shows photos well and looks clean and easy to navigate. I particularly enjoy the Shopping Bag area, which is prominent and easy to locate for customers when they wish to market.
Though many Sites are going to be mobile friendly, its fine knowing that Ecwid includes a native shopping application for both iOS and Android. Merchants now get a native shopping program for iOS and Android.
The final Design thing that makes sense to mention is your vocabulary instrument. Since conducting an online store generally requires one to attain out to individuals from all over the planet, you are bound to stumble upon a few clients who'd love to read product pages and other content in their native language. Ecwid supports 45 languages, so if a user would like to translate from English to Spanish, that is no problem at all.
A Few of the programs Will help you out with rating and reviews, rewards programs, automated advertising tools, analytics, custom design themes and much more.
Ecwid is a Third party remedy to your present site, so you would typically have to implement additional SEO and marketing tools to truly get out the word. That said, Ecwid supplies quality SEO features to help merchants increase their visibility in Google searches.
Moreover, Google now indexes AJAX sites, so all Ecwid stores are indexed by Google without any extra effort. It gets even better with WordPress sites, as you acquire access to additional search engine optimization plugins as well as also the default search engine functionality around the dashboard.
Each step is Clear and concise, so that you may just go in there and type in a product name, price and image then save the merchandise for afterwards. Obviously you can always open up the Powerful Options, but they separate these for when you are all set to start ramping up your store.
Ecwid offers A Chat With Earnings feature which is one of my favorite ways to converse with companies. Only send them a query and you can work on something else until they respond.
It's not necessarily Simple to find an ecommerce platform which doesn't charge transaction fees, but that is just what you get with Ecwid. They never charge any kind of transaction fee with any of the plans they provide, which makes it desirable for any shop that is trying to keep costs down and to improve margins.
Remember that Ecwid is not a payment processor, so you've got to setup something like PayPal or Stripe to process all the trades that come through your site. It is pretty easy to employ one of these systems, because Ecwid integrates with over 40 global payment options. However, each of these processors have their particular trade fees so you need to prepare for them.
· The'starter site' option is a fantastic stopgap measure for merchants who want a shop quickly, but have yet to develop a complete website in order to bring the Ecwid widget to.
Ecwid comes With four strategies, among which is totally free. They market the free plan like crazy, and mention that a number of different platforms drag you in free trial, even where you will finally need to pay.
The Free plan Lets you sell 10 products, while the Venture program ups that count to 100 products and you get items like an iPhone management program. The company program goes up to 2500 merchandise for a wonderful price and you also get live chat support. Priority support comes along with the Unlimited plan and you might also market an unlimited number of products.
Ecwid is one of The cheapest solutions on the marketplace. You can see that it's actually cheaper than a lot of the other programs we've reviewed previously, so if you're looking for value, this could be the right plugin for you.


text:
Righteous Wrath
A place for you to stop, join in, and have some fun with
like minded, all around good hearted, and
fun lovin people.
Discuss with Us
We have lots of great discussions and would love for you to join us!
We're retro, we use good old posting forums!
RW Forums
If you want a sample of what we are disucssing, check out our latest forum posts below. See what the community is saying, and maybe join in the conversation?
Online Community with Long Roots!
The Righteous Wrath is a group of like minded people that come together in our Online Community to share our thoughts, ideas, and impressions of the world around us. We started out as a guild/community of gamers out of Edmonton, Alberta, Canada in the late 1990s and have since grew into so much more. We welcome anyone whom may be interested in joining our little corner of the internet, just drop us a line on our forums to get started!
Discuss with Us!
Nothing is better than the good old forum postings that have survived the ages of the internet. With our long roots online, The Righteous Wrath has always had some method of discussing a host of various topics in some form. Come join us, post something or respond to something, and expand our lives with your ideas, thoughts, stories and anything else you can think of. Your imagination can be your only limit, confined only by the technology of today.
Join the Discussion: RW Forums
Happy New Year! Here's to 2023!
Hope you all had a great 2022 and can't wait for 2023
Happy New Year everyone 🥳
Holiday cheers to all of you!
Biden pardons Americans convicted of pot possession
Well, I never thought I'd see the day...
https://www.cbc.ca/news/world/biden-marijuana-simple-possession-pardons-1.6608858
She-Hulk: Attorney-at-Law
So I consume pretty much everything Marvel these days, including the Marvel TV shows. I've been watching She-Hulk and enjoying it; it's kinda whimsical and tries to show a female super's life, with occasional fourth-wall breaking just like Hulk and the Agents of S.M.A.S.H.
Last episode saw Charlie CoyleCox enter as Daredevil and Matt Murdock (same guy that played Daredevil in that awesome tv series a few years ago), this episode saw a lot more of him. I'm excited to see how that's all going to pan out!
I don't wanna say more cuz spoilers. But I'm wondering, is anyone else watching it?
Sent from my SM-A520W using Tapatalk
© 2019 The Righteous Wrath Online Community

redpajama_set_name: RedPajamaCommonCrawl


Inside this new magazine you'll find advice on how to manage relationships and expectations, keep everyone happy and still make time for you.
Mindful Christmas Magazine is packed with ideas for how to spend less and enjoy more this festive season, there are lots of creative ideas for a stylish home, tasty vegan recipes everyone will love and plastic-free gift ideas for all the family.
Pre-order your copy today for only £7.99 with FREE UK delivery, EUR price £9.99, ROW price £11.99.


text:
You are here: Home Excavations The Big Dig
The Big Dig
After 15 days of hard graft, great camaraderie, fun and palpable daily excitement, the Big Dig at Carwynnen Quoit came to a fitting finale on 3 October: the excavation had revealed the full extent of the original footprint of this megalithic monument.
Massive socketholes for the three principal granite uprights were discovered. Two were investigated in full and the third was identified but was not fully dug (it lay under one of the main excavation baulks). Our major discovery was a largely intact and well-preserved artificial chamber "floor". That this survived at all was partly to do with the fact that when the three granite uprights and the massive mushroom-shaped capstone collapsed in the 1960s earth tremor, they had fallen right on top of the area where they had once stood, and that this heap of stones had therefore protected the ground beneath.
The "floor" of the monument, an intact stone pavement, is made up of two elements. One is a narrow strip of compacted small stones which formed a hard-standing surface arranged in a doughnut-like circuit. At the front end of the monument, a fine narrow strip of the pavement extended well beyond the shelter of the capstone.
This is the first time that the original footprint of a monument of this type and great antiquity has been revealed by excavation in Cornwall. It shows that wider Megalithic architectural styles in far south-west Britain accommodated tremendous variety.
We plotted all the finds which were found across the entire area of the main excavation trench. As this plan shows some interesting trends are apparent. By looking at the numbers, distribution and character of early finds that were left behind on site – that is the prehistoric pottery, the flints and worked stone – the patterns about where were they left can reveal something about how the monument was used as well as the level of preservation site which can be confidently dated to signs of prehistoric activities.
This preliminary study was carried out once all the finds were assessed and catalogued and the patterns show a marked quantity of prehistoric finds moreorless in and around the immediate edges of the monument. Some of these bits of pots and flints may have moved slightly from their original positions – but there is a notable density of objects on and close to the stone pavement and particularly at the back or "rear" end of the monument where uprights stones 3 and 4 once stood.
When we combine measurements taken from some of the 19th century measured surveys – such as J T Blight's c 1850s as shown here – and superimpose it onto the plan recorded during our excavation in September we can see that the capstone does not cover the central part of the stone pavement, but that the outer pavement skirt, particularly on the north-western edge of the monument is not protected by the shelter of the capstone. This makes it all the more remarkable that this pavement has survived intact! This would suggest that the front end of the monument is facing north and it is from this side that the visitor to the site in the Neolithic period would have approached the site.
The mysteries of this site as uncovered to date are tantalising, but have yet to be fully revealed. This one-time Neolithic community project built over 5-6,000 years ago can be reawakened as a new twenty-first century community challenge to restore the monument to its majestic glory!
...Gallery Loading...
The Investigative Dig


text:
William Thomas "Bill" Medley (19 de septiembre de 1940; Los Ángeles, Estados Unidos) es un cantante y compositor estadounidense que ha compuesto varios éxitos a lo largo de su carrera. Fue miembro del grupo musical The Righteous Brothers en los 60.

Biografía

Nació en Los Ángeles y se crio en el Condado de Orange, Santa Ana, en el seno de una familia de músicos, su padre dirigió una big band y tocaba saxofón y su madre tocaba el piano y cantaba. Medley, que desde niño sintió pasión por la música, se presentó a varios concursos de canto aficionado durante su juventud, pero no fue hasta que escuchó la música de Ray Charles cuando decidió ganarse la vida con ella. "Cuando escuché a Ray Charles, no fue tanto que quería hacerlo," reflexionó. "Era que necesitaba hacer eso".

Medley aprendió solo a tocar el piano, lo que le condujo a escribir canciones. Conoció a su compañero Bobby Hatfield cuando ambos fueron emparejados por el saxofonista John Wimber , que más tarde pasó a fundar el movimiento Vineyard Church, en una banda llamada The Paramours y comenzaron a cantar a dúo en 1962 ya como The Righteous Brothers. Juntos grabarían singles de enorme éxito como " You've Lost That Lovin' Feelin'" o "Unchained Melody".

Su asociación duró cuatro décadas, aunque Medley desarrolló una carrera en solitario al margen del dúo. The Righteous Brothers se separaron a finales de los años 60 durante seis años para volver de nuevo en 1974. Sin embargo en 1976, tras la muerte de su esposa, Bill Medley se retiró de la música para criar a su hijo, haciendo apariciones eventualmente como solista en los primeros años ochenta. Demostró su versatilidad al unir fuerzas con, Jennifer Warnes, cantando el tema principal de la película "Dirty Dancing" (1987), "(I've Had) The Time of My Life" con el que ganó un Grammy a la mejor interpretación vocal a dúo, un Oscar a la mejor canción original y un Globo de oro.

Medley también ha realizado duetos públicamente de vez en cuando con su hija, McKenna, que tiene su propio espectáculo en Las Vegas. Pero, tras la muerte de Hatfield en 2003, Medley no tiene ningún plan para encontrar a otro compañero de canto permanente. En su lugar, Medley espera emprender una vez más por su cuenta, con la etapa de servir como red de seguridad de la que es todavía capaz de realizar una conexión-con los aficionados que siguen para adorar Righteous Brothers tanto bajo potente de Medley, sino también con su propia pasión por la música. No es que su Don raro lo hace diferente de las personas que habitan la audiencia. "Mis amigos que veo todos los días son chicos con que me fui a la secundaria y preparatoria", señala. "Sólo ha mantenido me muy, muy sensato. Por lo que cuando salgo en el escenario para realizar, me siento realmente como yo soy uno de los fanáticos. Entiendo cómo se sienten"

Discografía

Solista

Sencillos

Referencias

Enlaces externos

Nacidos en Los Ángeles
Cantantes de California
Actores de California
Ganadores del Premio Grammy
Ganadores del premio Globo de Oro

redpajama_set_name: RedPajamaWikipedia


text:
» Time after time
» Blu-ray
(Blu-ray)
Meyer, Nicholas, 1945- film director,
Jaffe, Herb, 1921-1991, film producer.
McDowell, Malcolm, 1943- actor.
Warner, David, 1941-2022, actor.
Steenburgen, Mary, actor.
Cioffi, Charles, 1935- actor.
Williams, Kent, 1950- actor.
Rózsa, Miklós, 1907-1995, composer (expression)
Cambern, Donn, editor of moving image work.
Lohmann, Paul (Cinematographer), director of photography.
Warner Bros. Pictures (1969- ) production company,
Warner Home Video (Firm), publisher.
Burbank, CA : Warner Home Video, [2016].
MPAA rating: PG.
Adult Blu-Ray (1st floor Audio/Video)
BLU TIM
Fugitives from justice -- Drama.
Jack, -- the Ripper -- Drama.
Science fiction films.
Steampunk films.
Thrillers (Motion pictures)
Time travel -- Drama.
Video recordings.
Wells, H. G. -- (Herbert George), -- 1866-1946 -- Drama.
Adult Blu-Ray (1st floor Audio/Video) BLU TIM On Shelf
Title from screen.
Widescreen (2.40:1).
Special features: commentary soundtrack by malcom McDowell and Nicholas Miller; theatrical trailer.
Director of photography, Paul Lohmann ; editor, Donn Cambern ; music, Miklos Rozsa.
Malcolm McDowell, David Warner, Mary Steenburgen, Charles Cioffi, Kent Williams.
Blu-ray; 16:9 presentation; DTS-HD master audio 2.0; requires Blu-ray player.
English subtitles for the deaf and hard of hearing (SDH).
Meyer, N., Jaffe, H., McDowell, M., Warner, D., Steenburgen, M., Cioffi, C., Williams, K., Rózsa, M., Cambern, D., & Lohmann, P. (. (2016). Time after time . Warner Home Video.
Nicholas Meyer et al.. 2016. Time After Time. Burbank, CA: Warner Home Video.
Nicholas Meyer et al.. Time After Time Burbank, CA: Warner Home Video, 2016.
Meyer, Nicholas, et al. Time After Time Warner Home Video, 2016.


text:
Research ArticleImmunologyInfectious disease Open Access | 10.1172/jci.insight.150060
Early IL-10 promotes vasculature-associated CD4+ T cells unable to control Mycobacterium tuberculosis infection
Catarina M. Ferreira, Ana Margarida Barbosa, Palmira Barreira-Silva, Ricardo Silvestre, Cristina Cunha, Agostinho Carvalho, Fernando Rodrigues, Margarida Correia-Neves, António G. Castro, and Egídio Torrado
Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal. ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
Address correspondence to: Egídio Torrado, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4810-057 Braga, Portugal. Phone: 351.604.907; Email: egidiotorrado@med.uminho.pt.
Find articles by Ferreira, C. in: JCI | PubMed | Google Scholar |
Find articles by Barbosa, A. in: JCI | PubMed | Google Scholar
Find articles by Barreira-Silva, P. in: JCI | PubMed | Google Scholar |
Find articles by Silvestre, R. in: JCI | PubMed | Google Scholar |
Find articles by Cunha, C. in: JCI | PubMed | Google Scholar
Find articles by Carvalho, A. in: JCI | PubMed | Google Scholar |
Find articles by Rodrigues, F. in: JCI | PubMed | Google Scholar
Find articles by Correia-Neves, M. in: JCI | PubMed | Google Scholar |
Find articles by Castro, A. in: JCI | PubMed | Google Scholar
Find articles by Torrado, E. in: JCI | PubMed | Google Scholar |
Published September 23, 2021 - More info
Published in Volume 6, Issue 21 on November 8, 2021
© 2021 Ferreira et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Published September 23, 2021 - Version history
Received: March 29, 2021; Accepted: September 22, 2021
Cytokine-producing CD4+ T cells play a crucial role in the control of Mycobacterium tuberculosis infection; however, there is a delayed appearance of effector T cells in the lungs following aerosol infection. The immunomodulatory cytokine IL-10 antagonizes control of M. tuberculosis infection through mechanisms associated with reduced CD4+ T cell responses. Here, we show that IL-10 overexpression only before the onset of the T cell response impaired control of M. tuberculosis growth; during chronic infection, IL-10 overexpression reduced the CD4+ T cell response without affecting the outcome of infection. IL-10 overexpression early during infection did not, we found, significantly impair the kinetics of CD4+ T cell priming and effector differentiation. However, CD4+ T cells primed and differentiated in an IL-10–enriched environment displayed reduced expression of CXCR3 and, because they did not migrate into the lung parenchyma, their ability to control infection was limited. Importantly, these CD4+ T cells maintained their vasculature phenotype and were unable to control infection, even after adoptive transfer into low IL-10 settings. Together our data support a model wherein, during M. tuberculosis infection, IL-10 acts intrinsically on T cells, impairing their parenchymal migratory capacity and ability to engage with infected phagocytic cells, thereby impeding control of infection.
Tuberculosis (TB) remains the worldwide leading cause of death from a single infectious agent. While active intervention is lowering the global incidence of this disease, new tools are required to reach the WHO's ambitious goal of ending TB epidemics by 2035. These tools include improved diagnostics of clinical and subclinical disease and more effective drugs and vaccines (1). Specifically, a more effective vaccination regimen would make a crucial impact to interrupt TB transmission and control TB epidemics (1). Indeed, the current TB vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG), while effective in preventing disseminated forms of pediatric TB (2), is not efficient against adult pulmonary disease (3, 4). Therefore, a novel vaccine regimen to protect against pulmonary TB is among the highest global health priorities.
The rational design of more effective vaccines requires a clear understanding of the immunological mechanisms of protection against pulmonary TB. In this regard, a substantial number of data support IFN-γ–producing CD4+ T cells (Th1 cells) as appropriate targets for novel vaccines because these cells are critical to TB control (5–9). Therefore, over the last decade significant efforts have gone into developing new vaccine regimens that elicit strong IFN-γ–producing CD4+ T cell responses or that boost BCG-induced IFN-γ–producing CD4+ T cells. However, the IFN-γ response is not a reliable correlate of protection against TB in humans (10–12). Accordingly, in a recent efficacy trial of modified vaccinia virus Ankara expressing Ag85A from M. tuberculosis, the Th1-boosting TB vaccine candidate failed to improve protection in BCG-vaccinated infants (13). While novel concepts for TB vaccination are emerging (14), these data suggest that the rational design of novel TB vaccines relying on cytokine-producing CD4+ T cells requires a better understanding of the crucial components of an effective CD4+ T cell response against M. tuberculosis infection.
Recent data show that different populations of CD4+ T cells develop in the lungs following M. tuberculosis infection (15). CD4+ T cells expressing programmed cell death 1 protein (PD-1) are a self-renewing population that produce low levels of IFN-γ whereas CD4+ T cells expressing the killer cell lectin-like receptor G1 (KLRG1) are short-lived but produce high levels of IFN-γ (15). Crucially, adoptive transfer experiments have unraveled a critical role of PD-1–expressing CD4+ T cells, but not KLRG1-expressing CD4+ T cells, in the control of M. tuberculosis growth (16). Moreover, while PD-1 cells express CXCR3 and locate within the lung parenchyma, KLRG1-expressing cells coexpress CX3CR1 and locate in the lung vasculature (16–18). Indeed, KLRG1-expressing cells cuff around blood vessels and do not engage with infected macrophages in the parenchyma, which is critical for the protective function of CD4+ T cells (19). Taken together, these data show that the parenchymal migratory capacity and the ability to engage with infected phagocytes within the lung lesions are critical features of protective T cells. Therefore, it is critical to identify the factors impeding the rapid migration of CD4+ T cells into the lung parenchyma. This will lead to the rational design of vaccines that generate T cells capable of entering the lung lesion and thereby mediate long-lasting protection against TB.
IL-10 has been implicated in increased susceptibility to TB, both in humans and in animal models, an event highly associated with decreased CD4+ T cell responses (20–26). In humans, IL-10 is elevated in the pleural fluid (27), bronchoalveolar lavage (28), sputum (29), and serum (30, 31) of patients with active TB compared with healthy controls. Mechanistically, IL-10 impairs the proliferation of IFN-γ–producing T cells stimulated with M. tuberculosis (22, 23). These observations are supported by mouse data wherein the absence of IL-10 in the genetically resistant strains C57BL/6 and BALB/c enhanced the influx of CD4+ T cells into the lungs, resulting in elevated production of T cell–recruiting chemokines and protective cytokines, including IFN-γ, TNF, and GM-CSF (32). Furthermore, the genetically susceptible CBA/J mouse line, and transgenic lines overexpressing IL-10, display enhanced bacterial burdens associated with reduced Th1 responses and impaired macrophage bactericidal functions (24, 25, 33). These data taken together demonstrate that IL-10 has an antagonistic role in the control of M. tuberculosis infection; however, the immunological consequences of IL-10 production during TB are currently unknown. Crucially, recent data show that IL-10 blockade during BCG vaccination and following M. tuberculosis infection enhances Ag-specific responses and provides significantly greater protection against aerogenic M. tuberculosis challenge (34). Therefore, in this work we used a novel mouse model of controlled IL-10 overexpression recently described (pMT-10) (35) to define the temporal role and the mechanisms whereby this cytokine modulates protective immunity to TB. Using this model, IL-10 overexpression was induced in pMT-10 mice by feeding the mice a 2% sucrose solution with 50 mM of zinc sulfate (35).
Our data show that IL-10 overexpression impairs the protective response, prompting uncontrolled bacterial proliferation and severe immunopathological consequences to the host, during the early stages of M. tuberculosis infection but not during chronic infection. During the early stages of infection, IL-10 overexpression altered neither populations of antigen-presenting cells (APCs) in the mediastinal lymph nodes (MLNs) nor the kinetics of CD4+ T cell activation. Instead, CD4+ T cells primed in the IL-10–enriched environment were recruited to the lungs but accumulated in the lung vasculature and did not translocate into the parenchyma. This migratory deficit prevented antigen sensing, which limited proliferation and effector cytokine production. Crucially, in adoptive transfer from the lungs of mice overexpressing IL-10 into RAG–/– mice, effector CD4+ T cells primed in the IL-10–enriched environment maintained their vasculature phenotype and were unable to restrain M. tuberculosis infection. Together our data support a model wherein IL-10 compromises the protective function of CD4+ T cells by promoting the differentiation of a vasculature-associated phenotype with reduced ability to translocate into the lung parenchyma and infiltrate the infected lesion.
Early but not late IL-10 production impairs the control of airborne M. tuberculosis infection. The progression and severity of TB has been associated with increased levels of IL-10 in both humans (27, 28, 30, 31, 36) and mice (24, 25, 33). Despite this, the immunological consequences of IL-10 overexpression following aerogenic M. tuberculosis infection have not been fully defined. We used the recently described pMT-10 transgenic mouse line (35) wherein IL-10 overexpression can be induced at different stages of infection by feeding the mice a 2% sucrose solution with 50 mM zinc sulfate. Inducing IL-10 overexpression allowed us to define the temporal role and mechanisms by which IL-10 antagonizes the protective immune response and control of M. tuberculosis infection. After aerogenically infecting pMT-10 mice with M. tuberculosis, in one group we induced IL-10 overexpression starting at day 5 after infection to determine the early-phase impact of this cytokine in the ability of mice to control M. tuberculosis bacterial burdens. To determine the cytokine's chronic-phase impact, in another group we induced IL-10 overexpression starting at day 30 after infection. B6 mice used as controls were also fed with the 2% sucrose solution with 50 mM zinc sulfate used to induce IL-10 overexpression in pMT-10 mice.
We began by testing the impact of IL-10 overexpression during the early stages of infection. After aerosol infection, we found similar bacterial burdens in both groups of mice until day 27 after infection (Figure 1A). While B6 mice controlled M. tuberculosis growth from this point onward, bacterial growth continued to increase in pMT-10 mice (Figure 1A). Consequently, pMT-10 mice displayed significantly lower survival compared with B6 mice (Figure 1B). Gross histological examination revealed that, shortly before they succumbed to infection, there were larger lesions in the lungs of pMT-10 mice than in B6 mice (Figure 1C). As expected, the increased susceptibility of pMT-10 mice was associated with increased IL-10 levels in the lungs (Figure 1D), and the blockade of IL-10R activity reverted the susceptible phenotype of pMT-10 mice that now displayed similar bacterial burdens (Figure 1E) and lesion sizes compared with B6 mice (Figure 1F).
Early, but not late, IL-10 overexpression affects the outcome of aerogenic M. tuberculosis infection. B6 and pMT-10 mice were infected with M. tuberculosis H37Rv via aerosol route and IL-10 overexpression induced after day 5 after infection (A–F). (A) Lung bacterial burdens and (B) survival of B6 and pMT-10 mice following M. tuberculosis infection. (C) Representative hematoxylin and eosin–stained lung sections and percentage of infiltrated area in the lungs at day 40 after infection. Individual data points represent individual animals. Lung bacterial burdens and survival and histology data are representative of 3 independent experiments with 4–5 mice per group. (D) IL-10 concentration in lung supernatants at day 30 after infection, determined by ELISA. Data represent a composite of 5 independent experiments with 4–5 mice per group. **P < 0.01; ***P < 0.001; ***P < 0.0001 using Student's t test. (E) Lung bacterial burden and (F) percentage of lung infiltrated area at day 40 after infection in B6 and pMT-10 mice that were injected weekly with an anti–IL-10R or control Ab starting 1 day before infection. (E and F) Data are representative of at least 3 independent experiments with 5 mice per group. **P < 0.01 by 1-way ANOVA followed by Tukey's test. B6 and pMT-10 mice were infected with M. tuberculosis H37Rv via aerosol route and IL-10 overexpression induced at day 30 after infection (G and H). (G) Lung bacterial burden of B6 and pMT-10 mice at days 65 and 90 after infection. (H) Representative hematoxylin and eosin–stained lung sections and percentage of infiltrated area in lungs at 65 days after infection. Individual data points represent individual animals. Data represent 3 independent experiments with 4–5 mice per group. Data area shown as the mean ± SD. **P < 0.01; ***P < 0.001; ***P < 0.0001 by Student's t test.
We next sought to determine the impact of IL-10 overexpression during the chronic phase of M. tuberculosis infection. To do this, we quantified bacterial loads after inducing IL-10 overexpression from day 30 to day 60 or 90 after infection. Compared with B6 controls, pMT-10 mice showed no marked differences in lung bacterial loads (Figure 1G) or lung pathology (Figure 1H).
Taken together, these data showed that IL-10 overexpression during the early stages of the M. tuberculosis infection promoted progression of infection and resulted in the hosts' rapid death. On the other hand, IL-10 overexpression induced during the chronic phase of infection significantly altered neither progression nor control of M. tuberculosis.
Early IL-10 production delays the accumulation of IFN-γ–producing CD4+ T cells in the lungs. The above data warranted further detailed analysis of IL-10's role during the early stages of M. tuberculosis infection. As IL-10–mediated susceptibility to M. tuberculosis infection had been associated with reduced Th1 responses in both humans and mice (22, 23, 26, 29, 32, 34), we first asked if M. tuberculosis–infected pMT-10 mice displayed impaired CD4+ T cell responses to M. tuberculosis infection. To do this, we infected mice and induced IL-10 overexpression starting at day 5 after infection (as above) and analyzed the kinetics of the CD4+ T cell response in the lungs. Compared with B6 mice, pMT-10 mice showed a delayed accumulation of activated CD4+ T cells expressing the activation marker CD44 in their lungs (Figure 2A). To further explore the impact of IL-10 environment in the Ag-specific IFN-γ response, we determined the frequency and number of M. tuberculosis–specific CD4+ T cells and their ability to produce IFN-γ. To do this, we restimulated lung single-cell suspensions with the early-secreted antigenic target-6 (ESAT-6)1–20 epitope and determined IFN-γ producers by intracellular cytokine staining (18). In accordance with the above data, we found a delayed kinetics of M. tuberculosis–specific IFN-γ–producing CD4+ T cell accumulation in the lungs of pMT-10 mice compared with B6 mice (Figure 2B). Interestingly however, despite the high levels of IL-10, IFN-γ–producing CD4+ T cells increased rapidly in the lungs of pMT-10 mice after day 30 after infection. The kinetics of IFN-γ protein in the lungs of infected mice followed a similar pattern to that of the Ag-specific response determined by flow cytometry (Figure 2C). These data showed that IL-10 production early during infection would delay the accumulation of IFN-γ–producing CD4+ T cells required to control M. tuberculosis infection.
IL-10 overexpression delays the onset of the protective immune CD4+ T cell response. B6 and pMT-10 mice were infected with M. tuberculosis H37Rv via aerosol route and IL-10 overexpression induced after day 5 after infection. B6 mice maintained in the same conditions were used as controls. (A) Frequency and number of CD4+ T cells in the lungs of mice throughout infection. (B) Frequency and number of IFN-γ–producing CD4+ T cells after in vitro restimulation with the immunodominant ESAT-61–20 peptide. (C) IFN-γ protein in lung supernatants determined by ELISA. Data represent at least 3 independent experiments with 5 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 by Student's t test. NS: not significant.
IL-10 impairs the priming and effector differentiation of recently activated CD4+ T cells. Reduced frequencies of Ag-specific CD4+ T cells in the lungs can result from defective priming of Ag-specific cells in the lung-draining lymph nodes or defective recruitment to the lungs. Taking into consideration that (a) M. tuberculosis dissemination to the MLNs is concomitant with CD4+ T cell priming (37, 38) and (b) IL-10 impairs dendritic cell trafficking to the MLNs (39), we first determined the impact of IL-10 in the transport of M. tuberculosis bacteria to the MLNs. To do this, we quantified the bacterial load in the MLNs at a time when CD4+ T cells would first encounter Ag (37). We found similar bacterial loads in B6 and pMT-10 MLNs at days 12 and 16 after infection, showing that IL-10 did not impair the transport of the bacteria from the lung to the MLN (Figure 3A). To determine if IL-10 expression altered the MLNs' inflammatory context, we aerogenically infected B6 and pMT-10 mice with M. tuberculosis–mCherry and characterized M. tuberculosis–infected myeloid populations at day 14 after infection. For gating strategy, see Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.150060DS1
Early IL-10 overexpression does not significantly impair CD4+ T cell priming and differentiation in MLN. B6 and pMT-10 mice were infected via aerosol route with M. tuberculosis–mCherry and IL-10 overexpression induced after day 5 after infection. B6 mice maintained in the same conditions were used as controls. (A) MLN bacterial burdens at days 12 and 16 after infection. (B) Representative FACS plots and frequency of M. tuberculosis–mCherry–infected myeloid cells in the MLNs of B6 and pMT-10 mice at day 14 after infection. (C) Representative distribution of M. tuberculosis–mCherry-infected myeloid cell subsets in the MLNs of B6 and pMT-10 mice at day 14 after infection. (D) Mean fluorescence intensity of CD86 and (E) MHC-II in M. tuberculosis–mCherry-infected myeloid cells from the MLNs of B6 and pMT-10 mice at day 14 after infection. (F) Frequency of CFSE-labeled CD4+ T cells expressing CD69 and (G) CD62L at different points following aerosol infection with M. tuberculosis. (H) IFN-γ quantification in supernatants of ESAT-61–20-stimulated single-cell suspensions prepared from lung-draining lymph nodes of B6 and pMT-10 mice infected for 14 or 16 days. Data represent 2 independent experiments with 4–6 mice per group. Data are shown as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 using Student's t test.
We found that the MLNs from B6 and pMT-10 mice presented similar frequencies of infected cells (Figure 3B). Furthermore, the two strains' profiles of infected cell populations were similar (Figure 3C), the majority of infected cells being CD11c-CD11bhi and CD11c-CD11blo. We additionally analyzed the expression of MHC-II and CD86 and found that, in M. tuberculosis–infected cells, IL-10 overexpression did not affect the expression of CD86 (Figure 3D) but it decreased the expression of MHC-II (Figure 3E). These data showed that IL-10 overexpression would not alter the profile of M. tuberculosis–infected cells within the MLNs of pMT-10 mice but it impaired MHC-II expression as previously described (40).
We next investigated if these effects of IL-10 had an impact in CD4+ T cell priming. To do this, we adoptively transferred CFSE-labeled P25 T cell receptor–transgenic cells (P25Tg, specific for Ag85b presented in the context of I-Ab) into B6 or pMT-10 mice at different times after aerogenic M. tuberculosis infection and, 24 hours later, determined their activation status (CD69 and CD62L expression) in the MLN (37). We detected CD69 expression on similar proportions of cells transferred into B6 and pMT-10 mice, up to day 16 after infection, when there was a reduced expression of CD69 evident in P25Tg cells transferred into pMT-10 mice (Figure 3F). We also measured the expression of CD62L in transferred cells and found that, in B6 mice, transferred P25Tg cells lost the expression of CD62L faster than in pMT-10 mice (Figure 3G). This suggested a delay in differentiation of newly activated CD4+ T cells due to the IL-10 environment.
To clarify this issue, we restimulated MLN cells from B6 and pMT-10 mice with ESAT-61–20 to measure IFN-γ production by Ag-specific CD4+ T cells. We found that, at day 14 after infection, there was a reduced production of IFN-γ by MLN cells in pMT-10 mice compared with B6 mice (Figure 3H). Nevertheless, at day 16 after infection, the levels of IFN-γ production were similar in B6 and pMT-10 (Figure 3H).
Together, these data suggested that IL-10 would not significantly impair the kinetics of CD4+ T cell priming although it would delay the differentiation of newly activated CD4+ T cells. Nevertheless, it was unlikely that the delay differentiation before day 16 after infection accounted for the delayed accumulation of IFN-γ–producing CD4+ T cells in the lungs (Figure 2).
Recent data suggested that IL-10 may inhibit the expression of chemokines that guide Th1 cells to M. tuberculosis infection sites (32). Therefore, we next asked if IL-10 downregulated the production of chemokines that guide newly differentiated cells to the infected lungs. We quantified CXCL9, CXCL10, and CXCL11 in the lungs of infected mice and found similar protein and expression levels of these chemokines in B6 and pMT-10 mice (Figure 4, A–C). Previous data showed that, in the absence of IL-10, there was an upregulation of CXCL10 production in the lungs of M. tuberculosis–infected mice (32). However, the high levels of IL-10 in our model did not impair chemokine production in the lungs following M. tuberculosis infection. This response was likely a consequence of the elevated bacterial loads seen in our model. Indeed, we saw enhanced CXCL9 responses in pMT-10 mice after day 25 of infection, the time at which these mice begin displaying higher M. tuberculosis loads than B6 mice. As such, the data showed that the delayed accumulation of IFN-γ–producing CD4+ T cells was not caused by altered chemokine expression in the lungs.
IL-10 overexpression does not impair chemokine production in M. tuberculosis–infected lungs. B6 and pMT-10 mice were infected with M. tuberculosis H37Rv via aerosol route and IL-10 overexpression induced after day 5 after infection. (A) Relative expression and protein levels of CXCL9, (B) CXCL10, and (C) CXCL11 in the lungs of infected mice. Protein levels of CXCL11 were below detection limit at every time point assessed. (D) Frequency of CXCR3-expressing I-Ab ESAT-61–20–specific CD4+ T cells in the lungs of infected mice over the course of M. tuberculosis infection. Data represent 2 independent experiments with 4–5 mice per group. Data are shown as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 using Student's t test.
We then asked if Ag-specific CD4+ T cells from B6 and pMT-10 mice would express similar levels of CXCR3, the chemokine receptor for CXCL9, CXCL10, and CXCL11. Compared with B6 mice, we found in pMT-10 mice that ESAT-61–20–specific CD4+ T cells from the lungs displayed reduced expression of CXCR3 at days 24 and 31 after infection (Figure 4D). Together, these data showed that IL-10 would not impair the production of chemokines that guided CD4+ T cells to the M. tuberculosis–infected lungs but it would inhibit the expression of chemokine receptors essential for CD4+ T cells to enter the lung parenchyma and induce control of infection (16, 41).
IL-10 impairs the migration of CD4+ T cells into the lung parenchyma. Our results showed that M. tuberculosis–specific CD4+ T cells present in the lungs of pMT-10 mice expressed reduced levels of CXCR3. We therefore hypothesized that IL-10 would antagonize control of M. tuberculosis infection by impairing the translocation of CD4+ T cells into the lung parenchyma, where they could interact with infected phagocytes to induce control of infection. In accordance with this hypothesis, the histological examination of lung sections from M. tuberculosis–infected mice 30 days after infection revealed the formation of perivascular cuffs in pMT-10 mice whereas in B6 mice lymphocytes localized within the granuloma (Figure 5A). Therefore, we performed intravital flow cytometry to determine the location at days 23, 27, and 33 after infection of CD4+ T cells in the lungs of B6 and pMT-10 mice. To do this, we intravenously injected mice with a fluorochrome-labeled CD45.2 antibody, and mice were culled 3 minutes after injection. Lungs were then collected and processed for flow cytometry analysis as previously described (16). Our results showed that pMT-10 mice presented increased frequencies of intravascular CD4+ T cells at all time points analyzed (Figure 5B). They also presented intravascular CD4+ T cells capable of producing IFN-γ in response to ESAT-61–20 at day 33 after infection (Figure 5C).
IL-10 overexpression limits CD4+ T cell migration into lung parenchyma and impairs antigen recognition and proliferation. B6 and pMT-10 mice were infected with M. tuberculosis H37Rv via aerosol route and IL-10 overexpression induced after day 5 after infection. (A) Representative immunofluorescence of CD3+ cells in lungs of mice at day 30 after infection. Scale bar: 200 μm. (B) Flow cytometry analysis and frequency of intravascular (CD45+) CD4+ T cells in lungs of mice throughout infection. (C) Flow cytometry analysis and frequency of intravascular (CD45+) IFN-γ–producing CD4+ T cells in lungs of mice at day 30 after infection. (D) Frequencies and numbers of CD69+CD4+ T cells from the lungs of mice throughout infection. (E) Frequencies and numbers of Ki-67+CD4+ T cells from lungs of mice throughout infection. Data represent 2 independent experiments with 4–5 mice per group. Data are shown as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 using Student's t test.
The reduced migration of CD4+ T cells to the lung parenchyma of pMT-10 mice was associated with reduced frequencies of CD4+ T cells expressing the early activation marker CD69 at day 23 after infection (Figure 5D). It was also associated with reduced proliferation of CD4+ T cells, as determined by the expression of Ki-67, until day 30 after infection (Figure 5E). Nevertheless, from day 30 after infection onward, pMT-10 mice displayed an increase in the frequency and number of CD4+ T cells expressing CD69 (Figure 5D) and Ki-67 (Figure 5E). This increase was associated with the increased Ag-specific IFN-γ response described above (Figure 2C). Despite this increase, bacterial growth was not curbed by the antigen-specific IFN-γ response. Therefore, we questioned the ability of effector CD4+ T cells generated in a high IL-10 environment to control M. tuberculosis infection. To measure this ability, we purified CD4+ T cells from the lungs of B6 or pMT-10 mice at day 30 after infection and adoptively transferred them intravenously into recipient RAG–/– mice infected for 15 days. We then analyzed the accumulation of these cells in the vasculature and their ability to control infection (Figure 6A).
CD4+ T cells differentiated in IL-10–rich environment maintain their vasculature phenotype in an environment with normal levels of IL-10. (A) B6 and pMT-10 mice were infected with M. tuberculosis H37Rv via aerosol route and IL-10 overexpression was induced after day 5 after infection. At day 30 after infection, B6 and pMT-10 mice were sacrificed and 5 × 105 CD4+ T cells, purified from their lungs, were adoptively transferred into RAG-deficient mice infected 15 days earlier. Frequencies of (B) CD4+ T cells and (C) CD4+ T cells capable of producing IFN-γ in response to ESAT-61–20 at day 15 after adoptive transfer in lungs of infected mice. (D) Representative flow cytometry analysis and frequencies of CD4+ T cells capable of producing IFN-γ in response to ESAT-61–20 in lung vasculature of infected mice. Each flow plot represents 1 animal per group. (E) Lung bacterial burdens in RAG–/– mice that received B6 or pMT-10 effector cells at day 30 after infection (15 days after adoptive transfer). Data represent 2 independent experiments with 4–5 mice per group. Data are shown as the mean ± SD. *P < 0.05; **P < 0.01 using Student's t test.
At day 30 after infection, we confirmed that RAG–/–:B6 and RAG–/–:pMT-10 mice presented similar frequencies of total CD4+ T cells (Figure 6B) and total M. tuberculosis–specific IFN-γ–producing CD4+ T cells (Figure 6C) in the lungs. Interestingly, however, we found increased frequencies of intravascular IFN-γ–producing CD4+ T cells in the lungs of recipient mice populated by pMT-10 CD4+ T cells (Figure 6D). These data showed the intrinsic impact of IL-10 in CD4+ T cells' ability to locate within the lungs, independent of the lung microenvironment. Importantly, we found that this phenotype was associated with increased lung bacterial burdens (Figure 6E), highlighting the association between CD4+ T cells' defective spatial distribution in the lungs and susceptibility to infection.
Overall, our data supported a model wherein IL-10 would have a detrimental impact in the development of the acquired immune response. This effect would be mediated by the differentiation of CD4+ T cells that display a vasculature phenotype unable to interact with infected phagocytes to induce control of M. tuberculosis growth.
Although IL-10 has been associated with increased susceptibility to M. tuberculosis infection in humans (27, 28, 30, 31, 36) and in mice (24, 25, 39), the immunological mechanisms underlying this effect are not completely understood. According to the data we obtained in this study, IL-10 overexpression during the early stages of the M. tuberculosis infection promotes uncontrolled bacterial proliferation in the lungs and has severe immunopathological consequences as well as reduced survival of the host. In stark contrast with these data, IL-10 overexpression after the onset of the T cell response did not impact the outcome of infection. During the early stages of infection, IL-10 overexpression altered neither DC populations in MLN nor the kinetics of CD4+ T cell activation. Instead, CD4+ T cells primed in the IL-10–enriched environment were recruited to the lungs but accumulated in the vasculature and did not migrate into the parenchyma. This migratory deficit impaired antigen sensing, which limited the proliferation and effector cytokine production of recently activated effector CD4+ T cells. Crucially, the adoptive transfer of CD4+ T cells from the lungs of M. tuberculosis–infected mice into RAG–/– mice revealed that CD4+ T cells primed and differentiated in the IL-10–enriched environment maintain their vasculature-associated phenotype and are unable to restrain M. tuberculosis infection. Together our data support a model wherein IL-10 compromises the protective function of CD4+ T cells by promoting the differentiation of a vasculature-associated phenotype with reduced ability to translocate into the lung parenchyma and infiltrate the infected lesion.
Previous published data show that IL-10 inhibits cytokine production by monocytes (42) and macrophages (43) while downregulating MHC-II expression (40, 42). These effects of IL-10 are consistent with the reduced Th1 responses following M. tuberculosis infection observed in genetic (24, 26) and transgenic murine models of IL-10 overexpression (25). Our data show that this reduced Th1 response is a consequence of IL-10 production during the onset of the T cell response. This is consistent with a recent study showing that blockade of IL-10R signaling during the first 20 days after aerogenic M. tuberculosis infection reverts susceptibility in CBA/J mice (26).
Moreover, our data show that CD4+ T cells generated in an IL-10–enriched environment display an impaired parenchymal migratory potential. Likely associated with the reduced expression of CXCR3, this impaired potential ultimately hampers CD4+ T cells' protective function by compromising their ability to engage with infected phagocytes. Indeed, because T cells are better able to reduce bacterial growth if they are in direct contact with infected phagocytes (19), the parenchyma location of CD4+ T cells is crucial to control M. tuberculosis infection.
Early data on CXCR3–/– mice suggested a redundant role for this chemokine receptor in the recruitment of CD4+ T cells to the lungs of M. tuberculosis–infected mice (44). However, more recent data show that CXCR3 is crucial for CD4+ T cells to populate the lung parenchyma and induce control of M. tuberculosis growth (16–18). Indeed, deficiency in this chemokine receptor decreases by half the rate at which Th1 cells enter the lung parenchyma (41). Therefore, the reduced expression of CXCR3 seen on Ag-specific CD4+ T cells from pMT-10 mice overexpressing IL-10 in early stages of infection likely plays a crucial role in the delayed migration of these cells into the lung parenchyma.
In this regard, CXCR3 expression is rapidly upregulated early during differentiation of Th1 cells under Ag stimulation whereas the cytokine milieu has a minimal impact (45, 46). As such, the inhibition of cytokine production by IL-10 and, particularly, IL-12 (47), is likely to play a minor role in CXCR3 expression while the reduced expression of MHC-II during priming, as seen in this study, may have a more significant contribution. However, we anticipate that other chemokine receptors may also contribute to the migration of Ag-specific CD4+ T cells into the lung parenchyma, particularly in high IL-10 environments. Specifically, the expression of CCR2 and CXCR5 (and, to a lesser degree, CCR5 and CXCR6) has recently been shown to participate in Th1 cell recruitment to the lungs of M. tuberculosis–infected mice (41). Particularly important is the expression of the chemokine CXCL13 (48, 49) and that of its receptor CXCR5 (17, 50); both are crucially required for T cells to efficiently migrate into the lung parenchyma and into the infected lesion. While we did not see a negative impact of IL-10 in CXCL13, we did see a reduced expression of CXCR5 as well as B cell areas in the lungs of pMT-10 mice (B6 = 7.79 ± 3.50 vs. pMT-10 = 0.76 ± 0.22, P ≤ 0.05).
The expression of indoleamine 2,3-dioxygenase (IDO1) has been shown to hamper the development of inducible bronchus-associated lymphoid tissue and to prevent the optimal homing of CD4+ T cells as well as their interaction with M. tuberculosis–infected phagocytes (51, 52). As the expression of IL-10 and that of IDO1 are linked (53), we questioned the impact of IDO1 in our model. However, we did not find increased expression of IDO1 in pMT-10 mice except at late time points after infection (Supplemental Figure 2). This suggests the activity of IDO1 is unlikely to contribute to the development of vasculature-associated CD4+ T cells seen in pMT-10 mice. On the other hand, IDO1 may contribute to the uncontrolled proliferation of M. tuberculosis at late stages of infection in pMT-10 mice with high levels of IFN-γ (Figure 2B) and with necrotic lesions (Figure 1C). However, this needs to be further addressed.
The developing consensus that protective CD4+ T cells locate in the lung parenchyma stems from studies showing that nonprotective antigen-specific T cells are associated with vasculature and express high KLRG1 and T-bet during chronic M. tuberculosis infection (16). KLRG1 expression marks a population of high cytokine-producing CD4+ T cells, particularly IFN-γ, during chronic M. tuberculosis infection, but not a self-renewing population. By contrast, PD-1 expression marks a population of self-renewing parenchymal CD4+ T cells (15). As the retention of CD4+ T cells in the vasculature of mice expressing high IL-10 is not a consequence of chronic antigen stimulation, we do not see increased KLRG1 expression. However, because progression of infection is associated with vasculature-associated CD4+ T cells, our data further support parenchymal migratory potential as a key feature of protective CD4+ T cells.
The fact that we see CD4+ T cells expanding in the lungs only after extensive bacterial proliferation occurs further reinforces the crucial role of the parenchymal migratory capacity of T cells for their response and protective function. As such, the data show that one of the crucial antagonistic effects of IL-10 in the control of M. tuberculosis infection is impeding the differentiation of effector CD4+ T cells with parenchymal migratory capacity. Accordingly, IL-10 overexpression after the onset of T cell responses does not influence the host's overall bacterial burden or disease pathology. We saw reduced frequency of CD3+CD4+ T cells (B6 = 80.33 ± 4.07 vs. pMT-10 = 73.09 ± 4.52, P ≤ 0.01) and CD4+ T cells capable of producing IFN-γ in response to ESAT61–20 (B6 = 3.47 ± 0.53 vs. pMT-10 = 2.9 ± 0.74, not significant) at day 65 after infection. However, this T cell reduction is likely a consequence of the IL-10 influence in the local environment of the lung lesion, as recently investigated (54). Moreover, it supports the limited contribution of IFN-γ–producing CD4+ T cells in the control of M. tuberculosis infection in the lungs (55).
Although we do not see a negative impact of IL-10 during chronic infection, we do not exclude the possibility that further extending the overexpression of IL-10 throughout chronic infection would result in increased bacterial burdens, as other studies have suggested (25, 33, 39, 54, 56). However, the most likely explanation for this discrepancy is that IL-10 overexpression in our model is controlled by zinc supplementation; indeed, the levels of IL-10 are normal before zinc supplementation whereas, in other models, IL-10 is likely overexpressed throughout infection (25, 33). As such, these models' effects of IL-10 during the early stages of infection will affect bacterial burdens during chronic infection. Accordingly, the susceptible phenotype of CBA/J to M. tuberculosis infection is more clearly seen during chronic infection (24, 57); however, the blockade of IL-10 signaling during the first 21 days of infection was sufficient to revert the susceptible phenotype of CBA/J mice (26). These data further highlight the crucial role of IL-10 during the onset of the T cell response.
Previous data have shown that the early control of M. tuberculosis infection in BCG-vaccinated mice is not mediated by recently activated effectors, but likely by memory cells colonizing the lungs (58). Furthermore, blockade of IL-10R signaling during BCG vaccination has been shown to significantly increase protection against aerogenic challenge with M. tuberculosis (34). One key challenge in the rational design of new TB vaccines is to overcome the delayed activation and expression of antigen-specific responses in the lungs following aerosol challenge (59, 60). Therefore, together with results from earlier studies, our data suggest that targeting IL-10 or the IL-10 pathway during vaccination may help overcome the delayed T cell response and enhance vaccine-induced protection.
Together our data demonstrate that IL-10 antagonizes the control of pulmonary M. tuberculosis infection by impeding the differentiation of T cells with capacity to migrate into lung parenchyma. These data, when taken together with other recent data showing enhanced vaccine-induced protection through IL-10 blockade during BCG vaccination and after M. tuberculosis infection (34), suggest that IL-10 may be an impeding factor to the rapid expression of T cell immunity in the lungs. Future vaccines should promote the development of T cells capable of rapidly migrating, of persisting within the lung parenchyma, and of colocating with infected phagocytes.
Mice. C57BL/6 and B6.129S7-Rag1tm1Mom/J (RAG–/–) mice were bred at the ICVS animal facility from stock purchased from Charles River Laboratories and the Jackson Laboratory, respectively. Breeders of P25 TCR transgenic mice that recognize Peptide-25 of the immunodominant antigen Ag85b of M. tuberculosis, presented in the context of I-Ab, were originally obtained from Anne O'Garra's lab at the Francis Crick Institute, London, United Kingdom; these were previously described (61). pMT-10 mice on a C57BL/6 background were produced by António G. Castro and Paulo Vieira, as previously described (35). Briefly, mouse IL-10 cDNA sequence was cloned in the p169ZT vector, which carries the sheep metallothionein (MT) Ia promotor, a β-globin splice site and the SV40 polyadenylation signal. The resulting vector was injected in C57BL/6 eggs and transgenic founders were identified by PCR using MT-specific primers. IL-10 overexpression in pMT-10 mice was induced by supplementing their drinking water with 2% sucrose solution containing 50 mM of zinc sulphate, as previously described (35). The resulting rapid increase in circulating levels of IL-10 were maintained until zinc sulphate was withdrawn (35). B6 mice used as controls were maintained in the same condition as pMT-10 mice, including drinking water supplemented with 2% sucrose solution containing 50 mM of zinc sulphate. Both male and female mice between the ages of 6 and 12 weeks were used for experimental procedures.
M. tuberculosis aerosol infection and bacterial load determination. The H37Rv strain of M. tuberculosis, originally from the Trudeau Institute, was used in this study. To generate M. tuberculosis–mCherry, the parental M. tuberculosis strain was transformed using the plasmid pBP10 provided by David G. Russel, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA, as described previously (62, 63). Both M. tuberculosis and M. tuberculosis–mCherry were grown to log phase in Middlebrook 7H9 broth supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.2% glycerol, 0.05% Tween 80, and 50 μg/ml hygromycin B (for M. tuberculosis–mCherry), and subcultured in Proskauer Beck medium with 0.05% Tween 80 to mid-log phase before frozen at −80°C. These frozen stocks were quantified and used to infect mice with a low dose of bacteria (~75 CFUs) using a Glas-Col airborne infection system, as previously described (18). At selected time points after challenge, mice were killed by CO2 asphyxiation; organs were aseptically excised and individually homogenized in saline. Organ homogenates were then 10-fold serial-diluted and plated on nutrient 7H11 agar (BD Biosciences) for 3 weeks at 37°C, at which point CFUs were counted.
Sample collection and preparation of lung single-cell suspensions. Aseptically excised lungs were sectioned and incubated at 37°C for 30 minutes with collagenase D (0.7 mg/mL, Sigma-Aldrich). Lungs were then disrupted into a single-cell suspension by passage through a 70 μm nylon cell strainer (BD Biosciences). After centrifugation, the cell-free suspensions were aliquoted and frozen at −80°C until their concentrations of cytokines were determined using ELISA kits (Thermo Fisher Scientific), following the manufacturer's instructions. Lung single-cell suspensions were then treated with erythrocyte lysis buffer (0.87% of NH4Cl). To remove cell debris and nonhematopoietic cell interference, lung cells were further processed over a 40:80% Percoll (GE Healthcare). The resulting cell suspension was washed twice and counted. A single-cell suspension was prepared from the MLNs by passing the organs through a 70 μm nylon cell strainer (BD Biosciences), followed by treatment with erythrocyte lysis buffer.
For intravital flow cytometry analysis, mice received APC-labeled CD45 antibody intravenously 3 minutes before euthanasia (16). Lung or MLN single-cell suspensions were then stained with fluorochrome-conjugated antibodies for 30 minutes on ice. For tetramer staining, cells were incubated in the dark for 1 hour at 37°C with the Brilliant Violet 421–conjugated I-Ab ESAT-64–17 tetramer provided by the NIH Tetramer Core Facility. For intracellular cytokine detection, cells were cultured in 5 μg/ml of ESAT-61–20 peptide for 1.5 hours before 10 μg/ml Brefeldin A (Sigma-Aldrich) was added to the culture for an additional 3.5 hours. The cells were phenotyped using the following antibodies: CD3 Brilliant Violet 605 (145-2C11, BioLegend), CD4 APC-Cy7 (GK1.5, BioLegend), CD11b PE-Cy7 (M1/70, BioLegend), CD11c Brilliant Violet 650 (N418, BioLegend), Ly-6C PerCPCy5.5 (HK1.4, BioLegend), MHC-II FITC (M5/114.15.2, BioLegend), CD86 APC (GL-1, BioLegend), CD44 PerCPCy5.5 (IM7, BioLegend), CD69 PE (H1.2F3, eBioscience), CD62L PE/Cy7 (MEL-14, BioLegend), CD45 Brilliant Violet 510 (30-F11, BioLegend), CD45.2 APC (104, BioLegend), Ki-67 PE/Cy7 (SoIA15, eBioscience) and IFN-γ PE/Cy7 (XMG1.2, BioLegend). Data were acquired on an LSR II flow cytometer (BD Biosciences) using Diva software and analyzed using FlowJo software (BD Biosciences). The total number of cells for each population was determined based on the percentage of cells measured by flow cytometry and the total number of cells per lung.
CD4+ T cell adoptive transfers. For adoptive transfers of P25 TCR-Tg CD4+ T cells, the spleens of P25 mice were collected and processed into single-cell suspensions. Naive CD4+ T cells were negatively selected using the Naive CD4+ T Cell Isolation Kit from Miltenyi Biotec, following the manufacturer's instructions. Purified cells were then stained with 5 μM CFSE (Molecular Probes) for 10 minutes at 37°C and adoptively transferred into recipient B6 and pMT-10 mice (1 × 106 total CD4+ T cells per mouse). The following day, B6 and pMT-10 recipients were sacrificed and their MLNs analyzed for the presence of CFSE+ (P25TCR-tg) transferred cells.
For adoptive transfer of effector CD4+ T cells into RAG–/– mice, the lungs of B6 and pMT-10 mice were harvested at day 28 after M. tuberculosis infection and processed into single-cell suspensions, as described above. CD4+ T cells were magnetically labeled with CD4 (L3T4) microbeads (Miltenyi Biotec) and purified following the manufacturer's instructions. Positively selected lung CD4+ T cells were then counted and injected intravenously into RAG–/– mice infected with M. tuberculosis 15 days earlier (5 × 105 cells per mouse). At day 30 after infection (15 days after adoptive transfers), the lungs were aseptically excised to determine bacterial loads.
Anti–IL-10R mAb treatment. One day prior to M. tuberculosis infection, mice were injected i.p. with 1 mg of either anti–IL-10 receptor (IL-10R) mAb (CD210) or IgG1 isotype control mAb (both from Bio X Cell), as previously described (64). Mice were then infected with M. tuberculosis through the aerosol route. To maintain IL-10R blockade, mice received 0.35 mg of the respective mAb i.p. at weekly intervals until the designated experimental times.
Histology and immunohistochemistry. The upper right lobe of each lung was inflated with 4% PFA and processed routinely for light microscopy with hematoxylin and eosin stain. Morphometric analysis was performed in a blinded manner using ImageJ software (version 1.50e; NIH). The percentage of total lung area involved with inflammation was calculated by dividing the cumulative area of inflammation by the total lung surface area for each sample.
Immunofluorescence was performed on formalin-fixed lung sections as described previously (65). Sections were probed using a purified rabbit polyclonal anti-CD3e (1:100; ab185811, Abcam), and visualized by adding Alexa Fluor 568 goat anti-rabbit (1:500; A-11011, Invitrogen). SlowFade Gold Antifade Mountant with DAPI (Invitrogen) was used to counterstain tissues and to detect nuclei. Representative images were obtained using an Olympus BX61 microscope and were recorded using a digital camera (DP70, Olympus) using the Olympus cell^P software.
Quantitative RT-PCR analysis. Quantitative RT-PCR (qRT-PCR) was performed as previously described (66). Total RNA from whole lungs was extracted using TRIzol (Invitrogen) from which cDNA was generated using the GRS cDNA Synthesis Mastermix (Grisp), following the manufacturer's instructions. The resultant cDNA template was used to quantify the expression of target genes by qRT-PCR (CFX96 Real-Time System with C1000 Thermal Cycler, Bio-Rad) and normalized to ubiquitin mRNA levels using the ΔCt method. Target gene mRNA expression was quantified using SYBR Green (Thermo Fisher Scientific) and specific oligonucleotides (Invitrogen).
Statistics. Differences between groups were analyzed using a 2-tailed unpaired Student's t test or 1-way ANOVA as appropriate. Survival curves were analyzed using the log-rank test. Data were displayed as mean ± SD. Differences were considered significant when P ≤ 0.05.
Study approval. All procedures involving live animals were performed in accordance with the European Directive 86/609/EEC and approved by the Subcomissão de Ética para as Ciências da Vida e da Saúde (SECVS 074/2016) (University of Minho, Braga, Portugal) and the Portuguese National Authority Direcção Geral de Alimentação e Veterinária (DGAV 014072) (Lisbon, Portugal).
CMF, AGC, and ET conceived and designed the study. CMF, AMB, and PBS performed the experimental work and data analysis. RS, CC, AC, FR, and MCN provided technical and/or material support. CMF, AGC, and ET drafted the manuscript. AGC and ET acquired funding. All authors critically revised and approved the manuscript and accepted accountability.
View Supplemental data
We thank the personnel of the ICVS animal facility and histology core for all their technical assistance. This work was supported by national funds through the Foundation for Science and Technology (FCT) projects PTDC/SAU-INF/28463/2017, PTDC/MED-ONC/28658/2017, UIDB/50026/2020, and UIDP/50026/2020 and by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023. ET and RS were supported by the FCT Investigator grants IF/01390/2014 and IF/00021/2014, respectively; AC and CC by Estímulo Individual ao Emprego Científico CEECIND/03628/2017 and CEECIND/04058/2018, respectively; and CMF and AMB by FCT PhD fellowships PD/BD/137447/2018 and SFRH/BD/120371/2016, respectively.
Conflict of interest: The authors have declared that no conflict of interest exists.
Copyright: © 2021, Ferreira et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License.
Reference information: JCI Insight. 2021;6(21):e150060.https://doi.org/10.1172/jci.insight.150060.
Dye C, et al. Prospects for tuberculosis elimination. Annu Rev Public Health. 2013;34:271–286.
View this article via: CrossRef PubMed Google Scholar
Trunz BB, et al. Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet. 2006;367(9517):1173–1180.
Colditz GA, et al. Efficacy of BCG vaccine in the prevention of tuberculosis: meta-analysis of the published literature. JAMA. 1994;271(9):698–702.
Mangtani P, et al. Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials. Clin Infect Dis. 2014;58(4):470–480.
Havlir DV, Barnes PF. Tuberculosis in patients with human immunodeficiency virus infection. N Engl J Med. 1999;340(5):367–373.
Casanova J-L, Abel L. Genetic dissection of immunity to mycobacteria: the human model. Annu Rev Immunol. 2002;20:581–620.
Filipe-Santos O, et al. Inborn errors of IL-12/23- and IFN-gamma-mediated immunity: molecular, cellular, and clinical features. Semin Immunol. 2006;18(6):347–361.
Geldmacher C, et al. Interaction between HIV and Mycobacterium tuberculosis: HIV-1-induced CD4 T-cell depletion and the development of active tuberculosis. Curr Opin HIV AIDS. 2012;7(3):268–275.
Boisson-Dupuis S, et al. Inherited and acquired immunodeficiencies underlying tuberculosis in childhood. Immunol Rev. 2015;264(1):103–120.
Elias D, et al. PPD induced in vitro interferon gamma production is not a reliable correlate of protection against Mycobacterium tuberculosis. Trans R Soc Trop Med Hyg. 2004;99(5):363–368.
Kagina BMN, et al. Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis after bacillus Calmette-Guérin vaccination of newborns. Am J Respir Crit Care Med. 2010;182(8):1073–1079.
Lu LL, et al. IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure. Nat Med. 2019;25(6):977–987.
Tameris MD, et al. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet. 2013;381(9871):1021–1028.
Scriba TJ, et al. Key recent advances in TB vaccine development and understanding of protective immune responses against Mycobacterium tuberculosis. Semin Immunol. 2020;50:101431.
Reiley WW, et al. Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection. Proc Natl Acad Sci U.S.A. 2010;107(45):19408–19413.
Sakai S, et al. Cutting edge: control of Mycobacterium tuberculosis infection by a subset of lung parenchyma-homing CD4 T cells. J Immunol. 2014;192(7):2965–2969.
Moguche AO, et al. ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis. J Exp Med. 2015;212(5):715–728.
Torrado E, et al. Interleukin 27R regulates CD4+ T cell phenotype and impacts protective immunity during Mycobacterium tuberculosis infection. J Exp Med. 2015;212(9):1449–1463.
Srivastava S, Ernst JD. Cutting edge: direct recognition of infected cells by CD4 T cells is required for control of intracellular Mycobacterium tuberculosis in vivo. J Immunol. 2013;191(3):1016–1020.
Boussiotis VA, et al. IL-10–producing T cells suppress immune responses in anergic tuberculosis patients. J Clin Invest. 2000;105(9):1317–1325.
View this article via: JCI CrossRef PubMed Google Scholar
Gao X, et al. Interleukin-10 promoter gene polymorphisms and susceptibility to tuberculosis: a meta-analysis. PLoS One. 2015;10(6):1–16.
Gong JH, et al. Interleukin-10 downregulates Mycobacterium tuberculosis-induced Th1 responses and CTLA-4 expression. Infect Immun. 1996;64(3):913–918.
Zhang M, et al. T cell cytokine responses in persons with tuberculosis and human immunodeficiency virus infection. J Clin Invest. 1994;94(6):2435–2442.
Beamer GL, et al. Interleukin-10 promotes Mycobacterium tuberculosis disease progression in CBA/J mice. J Immunol. 2008;181(8):5545–5550.
Turner J, et al. In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice. J Immunol. 2002;169(11):6343–6351.
Cyktor JC, et al. IL-10 inhibits mature fibrotic granuloma formation during Mycobacterium tuberculosis infection. J Immunol. 2013;190(6):2778–2790.
Barnes PF, et al. Cytokine production at the site of disease in human tuberculosis. Infect Immun. 1993;61(8):3482–3489.
Bonecini-Almeida MG, et al. Down-modulation of lung immune responses by interleukin-10 and transforming growth factor beta (TGF-beta) and analysis of TGF-beta receptors I and II in active tuberculosis. Infect Immun. 2004;72(5):2628–2634.
Almeida AS, et al. Tuberculosis is associated with a down-modulatory lung immune response that impairs Th1-type immunity. J Immunol. 2009;183(1):718–731.
Verbon A, et al. Serum concentrations of cytokines in patients with active tuberculosis (TB) and after treatment. Clin Exp Immunol. 1999;115(1):110–113.
Olobo JO, et al. Circulating TNF-alpha, TGF-beta, and IL-10 in tuberculosis patients and healthy contacts. Scand J Immunol. 2001;53(1):85–91.
Redford PS, et al. Enhanced protection to Mycobacterium tuberculosis infection in IL-10–deficient mice is accompanied by early and enhanced Th1 responses in the lung. Eur J Immunol. 2010;40(8):2200–2210.
Schreiber T, et al. Autocrine IL-10 induces hallmarks of alternative activation in macrophages and suppresses antituberculosis effector mechanisms without compromising T cell immunity. J Immunol. 2009;183(2):1301–1312.
Pitt JM, et al. Blockade of IL-10 signaling during bacillus Calmette-Guérin vaccination enhances and sustains Th1, Th17, and innate lymphoid IFN-γ and IL-17 responses and increases protection to Mycobacterium tuberculosis infection. J Immunol. 2012;189(8):4079–4087.
Cardoso A, et al. The dynamics of interleukin-10-afforded protection during dextran sulfate sodium-induced colitis. Front Immunol. 2018;9:400.
Jamil B, et al. Interferon gamma/IL10 ratio defines the disease severity in pulmonary and extra pulmonary tuberculosis. Tuberculosis (Edinb). 2007;87(4):279–287.
Reiley WW, et al. ESAT-6-specific CD4 T cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes. Proc Natl Acad Sci U.S.A. 2008;105(31):10961–10966.
Chackerian AA, et al. Dissemination of Mycobacterium tuberculosis is influenced by host factors and precedes the initiation of T-cell immunity. Infect Immun. 2002;70(8):4501–4509.
Demangel C, et al. Autocrine IL-10 impairs dendritic cell (DC)-derived immune responses to mycobacterial infection by suppressing DC trafficking to draining lymph nodes and local IL-12 production. Eur J Immunol. 2002;32(4):994–1002.
Koppelman B, et al. Interleukin-10 down-regulates MHC class II αβ peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling. Immunity. 1997;7(6):861–871.
Hoft SG, et al. The rate of CD4 T cell entry into the lungs during Mycobacterium tuberculosis infection is determined by partial and opposing effects of multiple chemokine receptors. Infect Immun. 2019;87(6):e00841.
de Waal Malefyt R, et al. Interleukin-10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med. 1991;174(5):1209–1220.
Fiorentino DF, et al. IL-10 inhibits cytokine production by activated macrophages. J Immunol. 1991;147(11):3815–3822.
Chakravarty SD, et al. The chemokine receptor CXCR3 attenuates the control of chronic Mycobacterium tuberculosis infection in BALB/c mice. J Immunol. 2007;178(3):1723–1735.
Rabin RL, et al. Chemokine receptor responses on T cells are achieved through regulation of both receptor expression and signaling. J Immunol. 1999;162(7):3840–3850.
Rabin RL, et al. CXCR3 is induced early on the pathway of CD4+ T cell differentiation and bridges central and peripheral functions. J Immunol. 2003;171(6):2812–2824.
Boonstra A, et al. Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, and TLR-independent signals. J Immunol. 2006;177(11):7551–7558.
Khader SA, et al. In a murine tuberculosis model, the absence of homeostatic chemokines delays granuloma formation and protective immunity. J Immunol. 2009;183(12):8004–8014.
Khader SA, et al. IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung. J Immunol. 2011;187(10):5402–5407.
Slight SR, et al. CXCR5+ T helper cells mediate protective immunity against tuberculosis. J Clin Invest. 2013;123(2):712–726.
View this article via: JCI PubMed Google Scholar
Ganatra SR, et al. Antiretroviral therapy does not reduce tuberculosis reactivation in a tuberculosis-HIV coinfection model. J Clin Invest. 2020;130(10):5171–5179.
Gautam US, et al. In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2018;115(1):E62–E71.
Cole JE, et al. Indoleamine 2,3-dioxygenase-1 is protective in atherosclerosis and its metabolites provide new opportunities for drug development. Proc Natl Acad Sci U.S.A. 2015;112(42):13033–13038.
Wong EA, et al. IL-10 impairs local immune response in lung granulomas and lymph nodes during early Mycobacterium tuberculosis infection. J Immunol. 2020;204(3):644–659.
Sakai S, et al. CD4 T cell-derived IFN-γ plays a minimal role in control of pulmonary Mycobacterium tuberculosis infection and must be actively repressed by PD-1 to prevent lethal disease. PLoS Pathog. 2016;12(5):e1005667.
Moreira-Teixeira L, et al. T cell-derived IL-10 impairs host resistance to Mycobacterium tuberculosis infection. J Immunol. 2017;199(2):613–623.
Turner J, et al. Immunological basis for reactivation of tuberculosis in mice. Infect Immun. 2001;69(5):3264–3270.
Connor LM, et al. A key role for lung-resident memory lymphocytes in protective immune responses after BCG vaccination. Eur J Immunol. 2010;40(9):2482–2492.
Robinson RT, et al. The onset of adaptive immunity in the mouse model of tuberculosis and the factors that compromise its expression. Immunol Rev. 2015;264(1):46–59.
Orme IM, et al. The balance between protective and pathogenic immune responses in the TB-infected lung. Nat Immunol. 2015;16(1):57–63.
Tamura T, et al. The role of antigenic peptide in CD4+ T helper phenotype development in a T cell receptor transgenic model. Int Immunol. 2004;16(12):1691–1699.
Gill WP, et al. A replication clock for Mycobacterium tuberculosis. Nat Med. 2009;15(2):211–214.
Huang L, et al. Growth of Mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny. J Exp Med. 2018;215(4):1135–1152.
Lin C-C, et al. Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation. Nat Commun. 2014;5:3551.
Khader SA, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol. 2007;8(4):369–377.
Resende M, et al. Myeloid HIF-1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection. Immunology. 2020;159(1):121–129.
Version 1 (September 23, 2021): In-Press Preview
Version 2 (November 8, 2021): Electronic publication


text:
2000AD's big, blue, genetically engineered clone soldier carves a swath of destruction on PS2.
Publisher: SCi
Nu Earth: a poisoned planet where a seemingly endless war rages between the Norts and the Southers. A futile struggle on a hostile planet with no clear end in sight.
But there are tales of a lone warrior. A man who can appear out of thin air and take out whole platoons, then disappear back into the shadows to which he belongs. A man who knows no allegiance but to his own mission and is hell-bent on revenge. He is the ultimate soldier, the last survivor of the massacred Genetic Infantrymen.... the Rogue Trooper!
One of 2000AD comic's longest running and most popular characters targets PlayStation 2 for his own brand of war-torn revenge on a poisonous planet.
Use Rogue's fallen comrades, Helm, Gunnar and Bagman, (now implanted as Bio-chips in his equipment) to bring about the maximum amount of destruction
Truly freeform gameplay, incorporating an unlimited amount of routes and tactics with which to progress
Intense split-screen and online multiplayer action
1 Player Types


The Senate narrowly confirmed President Donald Trump's pick for the nation's top energy regulator, with Democrats voting en bloc against the nominee over concerns that he would use his position to promote fossil-fuel interests.
Bernard McNamee, a Republican Energy Department staffer who played a role in Energy Secretary Rick Perry's ill-fated plan to subsidize coal and nuclear plants, came under fresh fire when a video surfaced in which he equated the environmental movement with tyranny and criticized renewable energy.
Those comments were enough to dissuade even coal-state Democrat Joe Manchin of West Virginia from supporting him. Still, the Senate approved his nomination to join the Federal Energy Regulatory Commission in a 50-49 vote.
'Throughout his career, Mr. McNamee has been manifestly biased in favor of the fossil fuel industry, and biased against renewable energy sources, so much so that one cannot believe he would be a fair arbiter on these issues,' Senate Minority Leader Chuck Schumer said during a procedural vote Wednesday.
Other Senate Democrats raised concerns about McNamee's role in Perry's coal bail-out effort, which was ultimately rejected by the energy commission early this year. The independent panel, which is charged with overseeing the U.S. electric grid, is supposed to be fuel-neutral.
McNamee has responded to those concerns by pledging to be an "independent arbiter" who will arrive at decisions based on the law and facts. He also promised to consult with the agency's ethics official.
Still, critics have called on McNamee to recuse himself from any commission matters in which there might be a conflict of interest. Harvard University's Electricity Law Initiative plans to file a brief with the energy regulator recommending that McNamee be disqualified from any matters concerning rates for "fuel secure" generators.
"FERC Commissioners must be impartial adjudicators, but as a matter of law, Mr. McNamee is incapable of meeting that standard with regard to matters that he worked on while serving as an attorney at the Department of Energy," Ari Peskoe, director of the Electricity Law Initiative, said in a statement.
At Trump's direction, Energy Department officials have proposed a new plan to extend the life of money-losing power plants, which they argue are essential to national security. Under the strategy outlined in a May agency memo, the president would use sweeping authority under the 68-year-old Defense Production Act to force grid operators to buy electricity from specific power plants at risk of closing. Perry in September said that the agency was awaiting a final decision.
Depending on how the plan is structured, it may or may not come before the energy commission. Still, the degree of controversy over the proposal has drawn more attention than usual to the political leanings of the agency's members.
The Senate narrowly confirmed President Donald Trump's pick for the nation's top energy regulator, with Democrats voting en bloc against the nominee over concerns that he would use his position to promote fossil-fuel interests.


These days, many men and women are looking for a better alternative to a traditional facelift. A thread lift is a safe and non-surgical procedure that can get you similar results without having to deal with surgery and the extensive recovery time. It can be used to reduce sagging around your neck, jawline and cheeks.
Basically, a thread lift is the process of using special threads to hold up facial tissue and skin in place. Generally, a thread lift can be completely discreet, especially when it is performed by a skilled surgeon like Dr. Gilmer. After the procedure, the results are immediate; however, they only improve in time.
At Restored Images in Nashville, we are proud to offer the NovaThread thread lift procedure. This increasingly popular treatment may be just what you've been looking for to achieve your aesthetic goals.
An ideal candidate for a thread lift is one showing early signs of skin aging. This non-surgical procedure can be completed in the comfort of our office in Nashville. During the procedure, our skilled surgeon will place threads in certain key areas and attach them to the skin tissue. The surgeon will skillfully pull back the threads to lift and smooth the skin.
After your thread lift, you'll want to follow all of the aftercare instructions. It may be a good idea to have someone drive you home from your procedure and assist you for 24 hours or so. In the days and weeks following the procedure, you'll start to see the full outcome of the thread lift.
Why Choose a Thread Lift?
While dermal fillers have become increasingly popular, many patients prefer to have a thread lift procedure. A traditional facelift isn't right for everyone, as some people don't want to undergo surgery, and others aren't able to have a surgical procedure done.
If you are considering having a thread lift yourself, you should only contact a board-certified plastic surgeon for a consultation. Look no further than Dr. Gilmer at Restored Images in Nashville. As a talented and qualified plastic surgeon, he will be able to educate you on the types of techniques used during the procedure and answer all of your questions. Contact us today to schedule an appointment and get started!


The exclusive MrSteam® F1 Plus-Controller responds with speed and precision to temperature fluctuations . . . providing accurate, consistent heat levels -- even during peak hours. Pre-wired and factory installed so the owner/operator is in control.
Durable and capable of 1 degree F control at point of measurement, the sensor responds accurately and rapidly to room temperature changes, sending a precise signal to the F1 Plus control.
AromaFlo® Essential Oil Injection System for use with MrSteam® aromatic oils.


Request to the server "boul.nl"
boul.nl. 86400 IN MX 20 ALT1.ASPMX.L.GOOGLE.COM.
boul.nl. 86400 IN MX 20 ALT2.ASPMX.L.GOOGLE.COM.
boul.nl. 86400 IN MX 10 ASPMX.L.GOOGLE.COM.
boul.nl. 86400 IN MX 30 ASPMX4.GOOGLEMAIL.COM.
boul.nl. 86400 IN MX 30 ASPMX3.GOOGLEMAIL.COM.
boul.nl. 86400 IN MX 50 relay.transip.nl.
boul.nl. 86400 IN MX 30 ASPMX2.GOOGLEMAIL.COM.
boul.nl. 86400 IN MX 30 ASPMX5.GOOGLEMAIL.COM.
boul.nl. 86400 IN NS ns1.transip.net.
boul.nl. 86400 IN NS ns2.transip.net.
boul.nl. 86400 IN NS ns0.transip.net.
boul.nl. 86400 IN TXT "v=spf1 a mx include:_spf.google.com ~all"
keywords: Roeland & Ilse met een rugzak. blog, Roeland & Ilse met een rugzak.


Beef Wellington Roast Dinner. I just wanted to say those words first to let it sink in. It's a thing, and they're serving it up at The Lygon Arms in Broadway, and it's... Beef Wellington Roast Dinner. I just wanted to say those words first to let it sink in. It's a thing, and they're serving it up at The Lygon Arms in Broadway, and it's truly delicious.
Not only can you eat dinner in the most beautiful setting – trust me, it's stunning – but... Beef Wellington Roast Dinner. I just wanted to say those words first to let it sink in. It's a thing, and they're serving it up at The Lygon Arms in Broadway, and it's truly delicious.