yeah well it's a real pleasure to be here I thank uh Su very much for the invitation and finally I was able to accept and be here um it's been a few years in the making is a little bit of a history to me standing here um but I it's really is a pleasure to be here and today I'm going to talk about something that is not necessarily the bread and butter of my lab but it's a project brought to me by a post-doctoral fellow in my lab and it turned into be a really interesting project um so that is what I'm going to show you today hopefully I can work this okay so as we're thinking of CF we obviously all all striving to get to the Cure and that for many people we think about Gene replacement Gene correction or even Cell Therapy which is kind of what we've been interested in looking at the feasibility of a Cell Therapy becoming a reality and what that would take so when we thinking of regenerative medicine approaches this think we need to think about things like what Sellar we targe does this even matter which cell we're targeting how many cells do we need to Target can replacement cells functionally engraft and persist in the lung for how long and how does stem cells behave in a diseased environment and it's that last point that I'm kind of going to hone in on we think about the epithelium and how the epithelium is functioning what it's doing and Performing its barrier but that epithelium receives signals from the environment that it sits in and the stem cell which is the main cell that regenerates an airway epithelium when it becomes injured receives cues and signals from the environment that it sits in and it's often forgotten about when we studying disease in a dish um so we all know that CF is associated with inflammation and inflammation has a significant impact on Airway function in fact in CF abnormal Airway inflammation is present very early um within the first few weeks of life life even in the absence of infection and this PR is over the years of life um and and remains abnormal throughout the life of someone with CF people with CF are obviously susceptible to repeated and chronic infections leading to elevated Airway inflammation and this inflammation results in a significant change in that stem cell Niche and this is just an image here seat pointer this is an image here of an airway in a CF lung the airway is full of mucus we can see that in this area here we've got what we call B basil cell hypoplasia basil cells are the main stem cells in the airway epithelium we've got excessive mucus in the submucosal glands and we've got huge infiltration of inflammatory cells it not only happens in the lung it happens in other tissues impacted by CF here is an example from a oops wrong button I'll get the hang of this in a second um this is an example from a pig model of cystic fibrosis looking at the pancreas and again you can see a huge amount of immune cell infiltration associated with the exocrine pancreatic insufficiency and pancreatitis which occur in cystic fibrosis these um processes of inflammation and pancreatitis are also commonly linked to pancreatic cancer and as people with CF and now aging we're seeing an increase in the incidence of cancers and other things that the lung dysfunction used to kind of um overshadow um so inflammation is not only just related to the pancreas and the lungs many of the other organs affected in CF such as the GI tract and kidney also are impaired by inflammatory processes so now we're in this postm modulat era we've seen obviously a significant impact on the quality of life of patients with CF generally getting improved f1's overall Improvement in lung function we seen a reduction in the exacerbations improved quality of life well-being and significant improvements in life expect however there are still challenges remain it's not the end of the road we still got things that need to be worked on and and there are patients um who still get chronic infections inflammation seems to persist in the lung and other tissues patients some patients have chronic and ongoing GI challenges the incidence of CF Associated diseases so the CF related diabetes liver disease kidney disease is increasing as the population with CF is now aging and now we're seeing more complications in this area obesity is actually something that is becoming more prevalent among the CF population um CF patients I'm sure you're all aware have the lack of nutritional value so they've been taught to eat more calories and now with the modulators that is a a challenge that is difficult to now change and we're seeing an increase in s obesity the reason I've highlighted these here is that a lot of these complications are associated with inflammation so my talk today I've split it into three parts and it's not necessarily in the order that I want to tell you it in but it's in the order of what I believe is the most important order um we're going to start with I am not getting hang of this thing um start with the impact of cystic fibrosis transmembrane regulator on maccrage functions so macres is one of the key um immune cells in the in the body we're then going to move on to how does a CF epithelium talk differently to immune cells and then the final part is is developing more physiological or Pao physiological models using human cells in a dish so this first part as I mentioned earlier was um really a project brought into lab by postop Ben Calver and um he was really interested in looking at how immune cells interact with the area epithelium and how there's cross talk between the two and how the epithelium signals to the immune cells and his fellowships kept getting rejected and one of the biggest uh criticisms and it was a constructive criticism well taken is that each donor is different so when we're looking at primary patient cells we have significant donor to donor heterogeneity so interpreting that in a dish is incredibly challenging to find out what is significant and what is related to CF when you've got such donor variability between your controls and your CFS so he went away and thought about how he could tackle this and this is the story that evolved so we know that CF mcroof fages are are different we know that the hyperinflammatory and has been research done in the past that has shown that um there's increased mcage numbers in the airway Airways showing fetal development and this correlates with elevated levels of of monotic chemot Trant in the bronchol Alva lavage fluids we also see that patients have elevated inter lucans Incan 4 and 13 um response to camonis in um infection this suggests that macras is in CF perhaps tolerant to these Incans um which these Incans typically will promote a phenotype of a maccrage that is anti-inflammatory or an M2 maccrage um these are not as prevalent in CF patients we also know from a number of studies that CF macres tend to be suboptimal as fos sites um so fosy Tois and key fosic markers such as cd11b are suppressed in CF fyes um markers that would be typical on your M2 macro fages the anti-inflammatory maccro fages are also suppressed in CF and there's been studies showing that acidification of the flyone which is critical for killing the bacteria um are decreased in CF is this decreased in CF so we know that there's there's an impact on phagocytosis and here's just a number of papers that have tried to show that this is related to the cystic fibrosis transmembrane regulator however it has been very challenging to really definitively State the role of the cftr in these processes largely due to the cellular hyrogen and the Dona variability and really defining that this is a cftr related process and this was a nice um a figure from a nice paper from Ben cops Lab at Emory um that showed that when you have a non-cf ma fagee and treat it with spacia here you can see that um cftr which is in green is is on the cell membrane over your microage and you get functional bacterial killing where you can see the red has the red bacteria inside the cell is not so bright not coming through so great on the screen but when you have a CF maccrage here and infected with um sapaa you can see that the bacteria persist and there's no CF expression here and here they're showing that if you treat it with some of the um modulators that you can see an improvement in cftr expression and some level of bacterial killing though you can probably appreciate there still a significant amount of bacteria present here so it's not fully restoring fos cytosis so this is where the challenges that really confirming cftr expression and its precise function in tissue resident alveola macras is in the lung has been really challenging due to these reasons that I've mentioned the cellular heterogenity the Don donor variability and the capacity that macras have is their ability to rapidly adapt to changes in their environment so when you take them out of the body and manipulate them in a dish they've already changed so are you looking at the ca phenotype anymore so it it poses many challenges when trying to really Define what what is going on with macro fages and CF so this was Ben's solution he went away and thought about how he could do this in a controlled way and he decided to take advantage of this thp1 monoy human cell line um this cell type can be stimulated with PMA to differentiate the monocytes into macro fages in a controlled manner what he decided to do is capitalize on the gene edian technology using the Cris past 9 where he could go into these normal thp1 cells and actually insert a Delta f8 mutation so we did the gene editin we clonely expanded these cells and generated a Pure Line of cells that are now genetically identical to the parent except for they contain a Delta f508 mutation we also created um CFR Knockouts um and have a line of those two he then did basic characterization of both the Delta f508 mutants and the wild types and showed that their phenotype was very similar in response to PMA he could differentiate both lines into macrofagos and when he looked at the cell surface profile which included these markers and a larger panel there was no difference in the phenotype of his monoy and macrophages but then the question is are they functionally different does having a Delta f508 mutation in your in your macrophages um change their function and he does this by stimulating them with lipopolysaccharide to stimulate the inflammatory response and what he saw was that there are three cyto kindes here so we're looking at interlukin One beta tnf Alpha and interlukin 2 the red is the wild types the blue is your CF the left column of both bars is the vehicle control and the right column is the LPS stimulated and what you can see is that um in the f508 mutants there is a significant increase in the interlukin 1 beta in the tnf alpha and in interlan 2 and interestingly there is a significant decrease in the anti-inflammatory in Tucan 10 all suggesting a pro-inflammatory phenotype has been induced in these cells by adding the Delta F5 mutation we also looked um at phagocytosis uh we did this using a pH rodo um assay so this flues is red Upon A cerification and we monitored this over time and again the wild types are always in red the CFS are in blue what you can see is that there is impaired phagocytosis in the CF macroasia compared to the wild types over time this is summarizing the graph here and we use cyoc and D here to maximize this process and again on the right hand side this is just a visualization of it we have our Delta F5s on the top and our controls on the bottom you can see the red signals we're getting from the acidification of the pH Roto but what I hope you can appreciate here is the significant bacteria which is kind of the grainy background you can see here compared to the bacteria quantity that is in our wild type so you can see a significant increase in the existing bacteria indicating a failure of Phagocytosis in the Delta f58 mutants we also collaborated with Chang yong's Lab at Oklahoma State University he's been working with these sell lines as well and he did a bacterial killing essay for us um using stail ccus orus and again you can see here over 24 hours and at 72 hours there is a significant impairment in the bacterial killing due to the Delta F5 mutation in these cells so Ben I wanted to ask you know can we restore this using the modulator therapies are the macras is responsive to modulators and can we restore um phagocytosis and and reduce the pro-inflammatory phenotype um and as I mentioned earlier this paper here suggested that yes some of these modulators can do this so Ben went in and started looking at um draa so he used alexaa iaca and tacter in combination and he looked at our inflammatory cyto production and again uh wild types are red CF is the Delta Delta fic mutant are in blue you can see that actually in both wild types and in the CF there is a reduction in interin One beta production and response to a dose dependent increase in the um ETI drugs almost bringing the CF back down to the levels of the wild types so we thought this is great they're responsive um but then we looked at tnf Al and the story was a little bit different here what you can see in the wild types with increasing doses at the lower doses not much happens but when you get up to the higher doses which we believe are still in the clinical range here um you're seeing a significant increase in fact this is is a huge increase in tnf Alpha production and the same same happens in the CF well there's a very little um effect at the lower doses that at a higher dose so in a dose dependent manner we' seen an increase in pro-inflammatory tnf production so then Ben wanted to ask the question is this due to a specific modulator in the triple therapy or is it due to the combination of all three being given together so he broke it down and looked at each modulator individually and we're looking at the same sidey of Kinder into Lucan One beta on the left tnf Alp on the right and again interlukin One beta we seem to see some reduction here however the tnf alpha production in both the wild types and the Delta f508 is increasing in response to um iaca moving on to tacter um we see very little change um in in response of inuk One beta to Tesa treatment however similarly the tnf alpha is increasing with in a do dependent manner in both the wild types and the CFS and then finally we looked at alexaa and again in the um inuk One beta really isn't changing but there are some slightly smaller changes in tnf Alpha and response to alaca so it seems like all the modulators in some way are impacting tnf Alpha production in these um in these uh macro fages so looking at the individual drugs on phagocytosis again we've got time across the um bottom axis and your concentration of um bacteria on the on the Y AIS here red is your wild types and um blue is your CFS and shades of it are different doers which are highlighted on the right which I appreciate are probably very difficult for you to see out there but top um bottom of the story is that the there is no change in phagocytosis in response to the uh three drugs that we tested here at any of the doses that we gave when we put them in combination so it's combining the ETI together we do see that at the um there is some suppression of um fago so rather than Improvement that we would expect to see there is actually suppression at the higher dois in fagal cytosis um and this also happens in the wild type as well as the CF so this is not specific to CFT dysfunction um but the high doses of the modulators are leading to a decreased phagocytosis so Ben then went in and started to profile the macro fages and see what is is there anything changing in the cell surface profile of these macro fages and he did a huge panel that I'm not going to show you of self Surface markers indicative of the different types of macrofagos and um with this is just after 1 hour of exposure to the modulator so this is a very quick response not time to get transcription translation changes here but what he sees is that cd14 on the cell surface is significantly increasing after one hour in response to the modulators um in both the wild type and the Delta f508 cd14 on the cell surface is pro-inflammatory and is also tied to tnf Alpha production and regulations so we've kind of seen a little bit of a cycle here that maybe there is something going on that is leading to um a a change in a pro-inflammatory status of these cells that is is independent of CFT he then has um this is actually very recent data and it doesn't look as clean because he's pulled a lot of experiments together here but you can still appreciate that alexaa is actually seems to be the drug that is driving this at regulation of cd14 if you look at iaca and tter that's very little difference um but alexaa or the drugs in combination seems to be what is driving this um so I will come to what Ben's hypothesis is here this is not a closed book story it's still an investigation that we're continuing with um but what we see is that cftr does directly influence the inflammatory and functional responses of macro Fades I think we've shown that now where everything is the same but that Delta FID mutation we've generated paired while typing Delta f58 thp1 cells that can be a very useful model for looking at maccrage function in people with CF we've shown that ETI combination therapy is not necessarily improving the function of mcroof fages and at higher ETI combinations are actually driving its pro-inflammatory cocine release and suppressing pathogen clearance and that alexaa seems to be the one that is contributing to potentially trafficking other proteins to the cell membrane including cd14 as as we just showed you so this has led to BS hypothesis right now that alexic is actually driving the trafficking of cd14 to the macroasia surface um and at CFT it's actually he his hypothesis that cfdr expression is actually critical on the fagal lome and not on the maccrage cell surface there's data out there showing that its expression on the fagal lome is is regulating the acidification which is then critical for bacterial killing and what he thinks is happening in response to modulat is a modulat are doing their job they're trafficking C CFT to the cell membrane and away from the fago lysosome so he's thinking that the modulators may be having an adverse effect on bacterial killing through this impact on cftr expression in the fagal lome and that we're also getting this concurrent traffick in of cd14 to the cell surface which is increasing TL tlr4 induced inflammation and regulated by tnf Alpha so he's going to keep working on this to prove the mechanism here and hopefully that will give us some potential targets that may help to reduce inflammation in patients that are a modulators with sustained inflammation so that was data that I thought was incredibly exciting um and and potentially quite important but all this project came around because of Ben's desire to really understand how the epithelium and the macro fages interact and talk to each other so that is what I'm going to move on to tell you about so just to remind you that this is the CF Niche and this is um a cross-section of an airway we've got the airway which really is not looking very healthy on the apical surface here and you can see all of these green cells underlying these are all nutrifil um so this this is not typical of an airway this is typical of a of a CF Airway where we have neutrophilic inflammation changing this micro environment and in addition to the inflammatory cells that are in this micro environment we also have extracellular Matrix and we also have other cells other neighboring cells and often when we create a cellular model in a dish we look at the epithelium or we look at a maccrage we look at exxtra solu Matrix we don't necessarily put them all in combination and think of this as a tissue level model and that is one of the challenges of research the more complex the model gets the more hard it is to interpret it um but it often means that we're overlooking some specific phenotypes in our cells and things that we're looking at so Ben took these traditional air liquid interface culture models that we use to study our Airway epithelium and he's done a few things with them he's worked on creating different types of integrated co-culture models using macro fages and then we've evolved these to use um emulate biosciences microf flued chips and developed an airway epithelium model on this which we published uh a couple of years ago now the original model but we're now using it to study maccrage epithelial interactions so this was some of the earliest work that Ben did he did co- cultures in submerged culture where you can see green macro fages with red epithelial um Airway epithelial basil stem cells here this is called his submerged model and then he has the air liquid interface lifted culture and a fully differentiated Airway epithelium we has two three model systems one where the macres are integrated within the epithelium this has been the most challenging and and still is a challenge one where the macrofagos are in contact with the epithelium on the basil lateral side of the membrane so there is some contact here and then ones where the macroad is are on the basement me um on the base of the well underneath and are not in direct contact so the whatever the secreting or changing can be interacting with the epithelium and we can use each of these models to ask different questions so these graphs are a little bit complicated I apologize that I haven't broken down but on the left the green and the red bars which I realize I apologize for anyone who's colorblind the left left is the green and then we have red um these are co-culture models and then the right hand bars which are blue and gray over here these are monocultures so by monocultures I mean that we have the epithelium cells alone down here these are the macrophages alone and these are epithelial cells with macrofagos to complicate this we have different combinations of CF and wild type so we have um CF epithelial cells with wild type macr CF with CF and vice versa wild type epithelium CF epithelium so you can get why I'm going here there's a lot of combinations and when you're doing this in a donor to donor way you've got to decide which donors you're piring with which donors so to eliminate some of the complications he used newly iny cells which are cell lines from um wild type ncf um just to eliminate some of that donor heterogeneity so he could really look at how the macro fages were interacting with the epithelium so these macro fages were isolated from donors and and grown in in the co-culture systems using this Underside model and Ben's interested in the inflammatory response so he started by looking at changes in the inflammatory response by taking a washes from the apical side and the basil lateral side here was just seeing the response for tnf Alpha production again and the one thing I want you to take home from this all this data that's shown here is that monocultures of macres so these are wild types these are CF um monocultures and maag is are releasing tnf Alpha when you put them in co- cultures with the epithelium we see a suppression of that tnf Alpha release and there the same quantity of macrophages in each of these assets so there is an interaction with the epithelium that is actually helping to regulate the tnf alpha that is secreted by um macras so Ben looked at other cyto kindes and it wasn't always the same situation here is looking at Incan six production um so another pro-inflammatory cyto kind you can see that in um epithelium alone there's not really any release in macro fages alone there is some release not much difference between the wild types and the CFS but when you put these in the co- cultures you see that there is a substantial increase in in Lucan 6 production and if you look down here there is actually a significant increase between the wild types and the CFS and the CFS with the so this is the CF with the CF macr fages this is the CF with the wild type macro fages and then these are wild type with CF wild type with wild type and wild Ty with CF but the take-home is is that co- cultures of CF with CF have increased into Lucan six and this is um when you stimulate them with LPS you can see that this is significantly elevated in the CFCF co- cultures so again suggesting that there is some interaction between the epithelium and um and the mcroof fages leading to changes in proinflammatory cyto release and finally he saw the same um very similar effect with Incan 8 and I hope we can appreciate that these numbers are very large so where you're seeing this significant increase here in the CF models we can see that there's there's much more Incan 8 being released in co-cultures with CF epithelium and CF macrofagos and some of that is coming from the CF epithelium alone where you can see that the CF epithelium here without stimulation is already increased at the tonal level without having um LPS stimulation so interian a is key because Inter leucan a is is one of the main attractant for um inter for nutrifil if we've got changes in inlan 8 the likelihood is is that you're attracting nutrifil into the vicinity of the epithelium um so this increase in incant Aid in these co cultes could identify a potential a potential Target to help prevent Airway neutrophilia what he also showed was that signals from the this this title oops sorry wrong button um this title's a little misleading actually so this is the statement that actually we need to read so what he did here we haven't done this assay yet with the CF epithelium but we took wild type um so non-cf bronal epithelial cells and cultured those with non-cf macras these are primary macr fages here um CF macras or just use the CF um sorry the non-cf macro fages have to PO it got this WR way M non CF epithelium with CF epithelium and these are all non CF macrophages here so we simplified it by sticking with the non-cf macrofagos and what we can see is that when you have the non-cf macrofagos with the non CF epithelium we can see that there is more phagocytosis happening when you put the non-cfs or the wild type macrofagos with the CF epithelium there is a suppression in phagocytosis this suggests that the CF epithelium is signaling back to the maccrage as an impact in the maccrage function and not typically as you would think about it the other way around where the maccrage functions responding to the infection and influence in the epithelium so this kind of proves Ben's hypothesis that the epithelium and the CF epithelium is is a a key driver in changing immune cell responses in the CF epithelium he also showed here and this is something that I'm we're particularly interested as a lab in in ciliogenesis and mucco clearance but he also showed here that when you grow the macras is in direct contact with the epithelium on the underside so you can see here the outline of a maccrage that is grown on the underside um with the CF epithel um with the epithelium as it differentiate so this is comparing non-cf macro fases to CF patient derived macro fases this is taken after 28 days of air liquid interface culture was typically sufficient to mature and polarize the air epithelium and typically you will see these nice cyan cells which are um ciliated cells and it's nice tight uh Junctions which are marked in red of the epithelium what you can see here when you've got CF macrophages with the exact same donor wild type epithelial cells there is a substantial impairment in multiciliogenesis at this time Point um again suggesting that signals here from the CF macras is uh regulating Airway stem cell function now I do have to say we did follow this out for much longer and took it to three months and by three months we did see that this Cog Genesis had been restored so it seems to be something that can be overcome over time but the initial there is an initial impact and impairment on differentiation of Basil stem cells in the Airways and a presence of CF macres compared to non-cf and the other thing Ben has observed is that when we look at CF macro ages again compared to non-cf with a wild type epithelium here we can see that these CF macres are naturally hyper migratory so over time you can see that these ones on the left here are actually remaining on the underside you can see it's nice and flat and laid out on the underside there's another one up here on in the CF macras you can see that many of these highlighted by the Red Arrows have migrated to an up and through the epithelium and are now appearing on the apical surface so it's it suggests there are functional differences of CF macres when they're cultured in the envir environment of um a wild type epithelium here so we're wanting to pursue this and keep moving on and looking at all the different combinations but hopefully those thp1 um mutant cells that we've now generated will actually Aid Us in being able to interpret this and eliminate some of the Don of variability um in and make it so that we can actually interpret the data in a statistically relevant way so I've got one I'm not going to give you the summary right now I want to move on on and go through the final part um so this is a work that was really led by Jana nth she has recently left my lab and is now running her own Lab at Helm Holtz Munich um in Germany but she was really interested in moving these models into tissue level models and she brought this technology into my lab this is a chip from em like biosciences where we have two channels we have an air interface channel in blue and a blood interface channel in red and these chips have the potential to provide more of a tissue level model because you can have the epithelium you can have air interface you can have stretch you can have physiological function in there you can have a blood interface lined with endothelial cells where you can put uh immune cells through um and you can do realtime um analysis of these chips as as processes you know when you want to evaluate a process so when we brought this into the lab I thought this is great these are going to be awesome awesome they're going to transform what we're doing and two years later we're still working out how to culture the cells on these things um but we finally did get it working and the paper was published in collaboration with huge help from an Vander do who worked in the Netherlands using the same chips for Airway um we were able to eventually get the cells to grow and differentiate and we see very little differences in the phenotype of the cells um from a standard air liquid interface chip um transall culture to the chips um but this was the biggest problem we had to overcome was that these chips that are stretchable so to allow us to have the function and put stretch on them the membranes have to be more flexible and this means having larger pores in them well the larger pores epithelial cells left to migrate through and what we were seeing was the cells migrate through and reside on both sides of the the membrane and don't differentiate and it make a pretty useless model um but after a long time we found um that treatments with an anti-adhesive coating actually was able to prevent this from happening long enough that the cells would polarize and differentiate um so then we did a lot of ceration of these models to make sure that we could really model Airway function on them and we looked at um ciliated self function so these graphs on the left are really showing that there's um not huge difference in ciliated beat frequency so the function of the cells um there was a difference in the area of the chip that was covered multiciliated cells we saw that the chips after 14 days had significantly more diff differentiated and functional ciliated cells than the inserts did we looked at mucco ciller clearance again this is like data you saw yesterday we were tracking bead particles and we can see that the chips actually after 14 days have all great coverage but also great directional flow of the cyia compared to inserts at 14 days where you see very few ciliated cells and very little directional flow this Dynamic flow actually is really key in maturing the a epithelium too and what we saw was that in the chips um markers of PL of cell polarity such as fangle you can see that you're starting to get significant organization and polarization of these cells with expression of vangle on the outside edges of the cells whereas in the inserts these have not yet um organized themselves and developed CER cell polarity um this is something that was significantly um increased in the chips um we'll just skip over the and not particular data the um exciting thing that we what we thought this was exciting was that you know we looked at the um we looked at the epithelial cell types and although we don't see huge differences between transwells what we do think is that the chips are actually leading us to a more smaller Airway F Type which would actually match the level of stretch we were giving um to the Airways and the flow Dynamics we're giving so we think that these may be able to help us model different branching Generations um of the Airways and it's something we're looking into but what was interesting is when we add airf flow in these we have directional air flow the CIA actually Orient themselves and beat against the airflow um so you can see here this is without air flow you can see there's no there's a tendency to go towards the inlets over here but there's no directional um beating when you at airflow you get very organized directional beating um so we believe that these models are actually a really great way to study Airway function um we did look at the impact of air flow and stretch on on extracellular Matrix Gene Matrix gene expression and we saw some significant differences to transwells where you're seeing a reduction in some of these um Matrix proteins and Matrix metalloproteases um and we just validated that at different levels and the reason I highlighted these is that these are some genes and Matrix um components that are actually significantly associated with Airway remodeling that occurs in CF and other Airway diseases so the fact these are different in a functional chip to a an air liquid interface culture may have um important implications for studying studying the role in disease so these are my final couple of slides we've now got to the point where we culture in our CF patient cells on these chips we trust them enough that we're not going to waste our CF precious CF patient cells um and we started to look to see if there are any differences between a CF cells and how they behave when we have air flow and stretch on these chips and we've now got an end of three I don't have statistics on it but this is something that we found quite interesting is that um our wild type chips we get very distinct mucco cery clearance in a directional manner um and you can see that silly emotion is even across the culture when you look at the CF chips we see that although there's good quality beating the beating is not necessarily perfectly aligned and the seems to be some disinisana now repeated this three times and it's it's holding true that we seen some disc skinesis when you have airflow and stretch so it may be indicating a new component to disregulated clearance in CF Airways that we've not seen when we've had a contained trans wall culture and another thing that we noticed in these cultures we added nutrifil to try and memic our Airway neutrophilia here and compared this is a wild type line on the top and a CF line on the bottom again this is a representative example we've now done this in three independent donors seeing similar responses in them all the red is the neutrophilic marker and you can see after 13 minutes of culturing these in the basil lateral blood Channel with just the CF epithelium present you can see that the neutrals are already migrating up to the epithelium and after 90 minutes there's a substantial amount of them comes through the epithelium actually appearing on the apical surface we see Zero of them coming through on the wild types and just to prove the uh nutrifil in the wild types these are the nutrifil remaining in the basil lateral channel so again the CF epithelium here is talking differently than a wild type epithelium to your inflammatory cells so to summarize the talk um you know I think what we've seen here is that there is cross talk between the epithelium and immune cells and this can significantly alter the tissue level inflammatory and responses we've seen that air flow and stretch can alter the properties of the epithelium and the function of the epithelium and we' shown that CF macrophases are functionally impaired due to CFT mutations and that modulators actually seem to be having an increase in PL inflammatory cyto production and further decreases in fago cytosis at higher doses in both non-cf and CF micras indicating that maybe in a patient specific manner or a donor specific manner that ETI may be confounding inflammation and um be part of the reason why we're not seeing resolution of inflammation in patients and that this seems to be due to a mechanism that is maybe driven by alexic trafficking cd14 to the cell surface and also trafficking CFT to the cell surface of microf fages as opposed to the flyone changing bacterial killing properties so hopefully I've given you a little bit of food for thought here it is not a polished story yet we're still following up on these investigations um but this work was largely led by Ben Calver my post do Jana who I said is already got her own lab in Germany now and schuba Eric and Greg are some of the other key players in these specific projects so thank you all for your time