T Cell Role in the Pathophysiology of Heart Failure
December 21, 2021December 16, 2021
Yale Pathology Grand Rounds
MariaPilar Alcaide, PhD
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Transcript
- 00:00So. Good afternoon everyone.
- 00:03Thank you for attending a
- 00:06year pathology gram one.
- 00:07Sending a series.
- 00:09It's our great pleasure to invite Doctor
- 00:12Killer or Katie to speak at our grandma.
- 00:16I met at scientific conferences and I'm
- 00:19impressed by her outstanding mechanistic
- 00:21research in immunology and cardiology.
- 00:25Cloud received her pH D from the Autonomous
- 00:28University of Madrid, Spain, she.
- 00:31Performed her postal research
- 00:33at Brigham Women Hospital,
- 00:36have a medical school.
- 00:37She studied her faculty position
- 00:39as an assistant professor at the
- 00:41Department of Medicine, Tufts University.
- 00:43She has been attending
- 00:45resources professor since 2019.
- 00:47She's the program director of Immunology
- 00:51graduate program is an endowed chemist,
- 00:54and Joann,
- 00:55where professor and the interim
- 00:58vice chair of department
- 01:00immunology at Tufts Class Group,
- 01:03has made important discoveries in the
- 01:05area of mechanism T cell trafficking.
- 01:08Their research efforts are focused
- 01:10on understanding why and how T cell
- 01:14subsets and in gaseous interaction.
- 01:16They also study intrinsic properties of
- 01:19the vascular in the cilium that modulated
- 01:22key cell and leukocyte recruitment
- 01:24in the T cell trafficking and survival.
- 01:27Another exciting line of risk in
- 01:29class group is about inflammation,
- 01:31heart failure to recent research has
- 01:34contributed significantly to your paradigm
- 01:37shift in understanding of heart failure.
- 01:39Putting T cell inflammation as a measure
- 01:42of player in this heart failure disease.
- 01:46So exciting research program have
- 01:49led to multiple impactful papers
- 01:52such as circulation journal,
- 01:53experimental medicine,
- 01:54JCR inside a TVP and etc.
- 01:58Pillars research has been has
- 02:00been funded by NIH one brand.
- 02:03She has also showed great leadership in
- 02:06science by serving our editorial board
- 02:08such as Junior Clinic investigation,
- 02:10Fast, GMC and etc.
- 02:14Additionally,
- 02:15she has been serving the American Heart
- 02:18Association during past many years.
- 02:20She has been the HCA basic cardiovascular
- 02:22Science program chair since last year.
- 02:26Without further ado,
- 02:27let's welcome Killer.
- 02:28To give us her seminar entitled T
- 02:31Cell Role in the passive Physiology
- 02:33of heart failure, can I thank you?
- 02:37Thank you very much.
- 02:38You ring for the invitation.
- 02:40It's really nice to be here and also
- 02:44thank you to all those of you who
- 02:48I've met with this morning because
- 02:50I've returned your science because I
- 02:52I've been really enjoying, you know,
- 02:54all the things that you're doing here.
- 02:56So as giving said,
- 02:58I'm going to focus today's talk on the
- 03:01aspect in the lab where we study the
- 03:04role of T cells in the pathophysiology.
- 03:07Of heart failure I have no disclosures
- 03:11and basically this is a cartoon that
- 03:14summarizes the general theme of our lab,
- 03:17which is how the moon system impacts
- 03:19cardiac and vascular health.
- 03:21So as you being said,
- 03:22I train in immunology and then I
- 03:25further train in vascular biology,
- 03:27but we know that immune cells
- 03:31they really need to traffic into
- 03:33tissues to do their functions.
- 03:36But then once.
- 03:37In addition,
- 03:38they need to interact or crosstalk with
- 03:42all the different resident cells in order
- 03:46to modulate homeostasis or pathology.
- 03:49In the case of injury.
- 03:51So for today's talk,
- 03:52I will focus on what we've been
- 03:54learning recently in the lab from
- 03:56our work and also from the work
- 03:58of others of how this interaction
- 04:01between adapted and innate immunity
- 04:03contributes to cardiac remodeling.
- 04:06In hard and I I place a circle
- 04:10here because this is mainly where
- 04:12these interactions between adapted
- 04:14and it made immune cells happen.
- 04:17This is what T cell antigen
- 04:19recognition starts in the lymph nodes.
- 04:22But towards the end of the talk I
- 04:24will show some new data is still
- 04:26unpublished where we really find that
- 04:28they're very similar interactions
- 04:29that are also happening in the
- 04:31heart and that they might modulate
- 04:34correct Physiology this way.
- 04:36So as many of you probably know,
- 04:38heart failure is very complex
- 04:40and it's multifactorial.
- 04:41So to tackle mechanisms,
- 04:42we need to start in a simplistic way.
- 04:46But we also need to understand
- 04:48the full complexity.
- 04:49So what do we know is that
- 04:51regardless of the etiology,
- 04:53whether it was triggered
- 04:54by any ischemic event,
- 04:56such as a myocardial infarct
- 04:59or non ischemic event.
- 05:01The heart remodels and the characteristics
- 05:04of the failing heart are increased.
- 05:07High level curricular pressures and
- 05:11then a hypertrophic cardiomyocytes
- 05:14fibrosis and these results in
- 05:17systolic and diastolic dysfunction.
- 05:19And we've known since the 50s that
- 05:22systemic chronic inflammation
- 05:23is associated with pretty much
- 05:25all of the causes of all of the
- 05:28etiologies of heart failure.
- 05:30I'm just going to set up my timer
- 05:32here to make sure that we're.
- 05:33Runtime here.
- 05:35But unfortunately this by this knowledge,
- 05:37for many,
- 05:38many years today none of the
- 05:40anti-inflammatory therapies for clinical
- 05:42trials that were initially launched
- 05:44to tackle a pro inflammatory cytokines,
- 05:47such as TNF.
- 05:48And more recently,
- 05:49with the counters trial
- 05:51island bed and none of them,
- 05:53this is the anti TNF therapies
- 05:55that are very efficient in treating
- 05:57out immune diseases and chronic
- 05:59inflammatory diseases did not
- 06:01work in heart failure and there
- 06:04are more recent
- 06:05promising data with the Cantor's trial,
- 06:08although it's still early to tell
- 06:10whether it has really benefited in in
- 06:13some of the outcomes of heart failure.
- 06:16So what we know is that there are no
- 06:19anti-inflammatory antifibrotic therapies
- 06:21that have been successful today,
- 06:23and we know from many organ systems
- 06:26that inflammation or immune cell
- 06:29activation and fibrosis go together or
- 06:32have some overlapping functions as well.
- 06:35So the first question that we
- 06:37asked several years ago was is,
- 06:39is there cardiac information?
- 06:41Besides systemic chronic inflammation
- 06:43again going with the concept that if the
- 06:46immune cells traffic to an inflamed issue,
- 06:48do they exert their functions by
- 06:51communicating with the tissue
- 06:53or stroma cells?
- 06:55And then if that was the case,
- 06:57do the cardiac infiltrated muscles
- 06:59contribute to the hallmarks
- 07:00that we see of heart failure,
- 07:02such as correct fibrosis?
- 07:03And does that have any impact
- 07:05on cardiac dysfunction?
- 07:06And obviously we're very interested as
- 07:10basic scientists in understanding how.
- 07:13So the first experiment that we did
- 07:15this this is this was published.
- 07:16This is back in 2015,
- 07:19but we wanted to say whether we
- 07:22could see cardiac inflammation
- 07:24in patients with heart failure,
- 07:25but we wanted to look at patients
- 07:28with non ischemic heart failure.
- 07:29Antonio Barish group at VCU in Virginia.
- 07:33He had elegantly demonstrated
- 07:34years before this that in response
- 07:36to myocardial infarction,
- 07:38there was decent infiltration in
- 07:40the human heart and interestingly.
- 07:42The diesels were infiltrated in the
- 07:46in the scar zone in the infarct zone,
- 07:49but also,
- 07:50so that goes along with a roll of
- 07:53the immune system during evolution
- 07:55to help healing.
- 07:57But interestingly,
- 07:58what they had found in inference
- 08:00was that there were also a lot of T
- 08:02cells infiltrated and remote zones.
- 08:04So we thought if we chose a patients
- 08:06that did not have any impact that have
- 08:08sort of like low chronic inflammation,
- 08:11where we see these as infiltrated
- 08:12in the heart.
- 08:13And this is exactly what we found
- 08:16here in Brown that they end stage
- 08:18nonischemic heart failure and
- 08:20heart samples which were taken as
- 08:22stated here from from there had
- 08:26significant diesel infiltration.
- 08:28Compared to non heart failure controls.
- 08:31And later on we did some experiments
- 08:33where we also wanted to look at what
- 08:36kind of pistols were infiltrated there,
- 08:38and we found that many of those
- 08:41teachers expressed the chemo keen
- 08:43receptor CXCR 3 and this is shown here
- 08:46by Red Arrows and quantified here.
- 08:49So this this made the basis to
- 08:52wanted to understand mechanistically
- 08:54what is thesis that expressed these
- 08:57receptors are doing within the heart.
- 09:01So our very broad hypothesis.
- 09:03110 and Evers joined my lab as
- 09:06postdoc was asking the question,
- 09:08is this purely an association or
- 09:10are they actually contributing or
- 09:12doing something in the heart and her
- 09:15hypothesis was that they would be
- 09:16doing something there in the failing heart?
- 09:20And to start doing this we have to
- 09:23choose a preclinical model and knowing
- 09:25that heart failure is very complex,
- 09:27there's no optimal or perfect.
- 09:31The clinical model that mimics
- 09:33all the symptoms of heart failure
- 09:36or how the disease develops,
- 09:38but we found that for nonischemic
- 09:40heart failure tack or transverse or
- 09:43reconstruction was a one where we could time,
- 09:47and for certain reasons,
- 09:48that we wanted to do.
- 09:49It was really a very good model to do so.
- 09:52Why? Because it induces pressure
- 09:54that mimics the pressure that heart
- 09:56failure patients have in the heart.
- 09:58Although the downside is that here.
- 10:01It induces a sudden pressure that then
- 10:04is restrained versus impatience as
- 10:06we know they developed progressively,
- 10:08but importantly,
- 10:09in this model we can basically track very
- 10:13nicely how cardiac hypertrophy develops,
- 10:15how cardiac fibrosis develops,
- 10:17and whether we can check in per cardiac
- 10:20function and at those time points.
- 10:22We could also look for diesel immune
- 10:25responses and correct diesel infiltration.
- 10:27So using this model I'm showing data
- 10:30from 4 weeks. Play time points.
- 10:32That is all summarizing schematics.
- 10:35Because we published this already.
- 10:38But what we found was that it
- 10:40specifically one type of thesis
- 10:42that are CD 4 positive T cells
- 10:45were infiltrated in the heart as
- 10:46early as two weeks post stack.
- 10:48And this is four weeks after Tak.
- 10:51And then,
- 10:51before they infiltrated in the heart,
- 10:54we saw a significant expansion of the
- 10:56medicinale draining lymph nodes that are
- 10:58the lymph nodes that drain the heart.
- 11:00And most of those T cells express
- 11:03interferon gamma so they were type
- 11:06one TT cells T H1 cells which also
- 11:09expressed the chemokine receptor.
- 11:116 year 3.
- 11:13And then we found that these
- 11:16infiltration was associated with at the
- 11:18time where mice developed fibrosis.
- 11:20As you can see here in pink,
- 11:21the collagen deposition and enlargement
- 11:24of the cardiac myocytes by H&E.
- 11:27And what we found using this mouse
- 11:30model was that if my eyes were
- 11:33genetically deficient in D zones
- 11:34and we use different models,
- 11:36diesel receptor alpha Nokia or MHC 2
- 11:39knockout or right to knock out what
- 11:42we found was that all the mice that
- 11:44did not have decent genetically they
- 11:47did not develop a cardiac fibrosis.
- 11:49We cannot see any College in
- 11:51the position here.
- 11:52And then when these mice were reconstituted,
- 11:54we see Excel 3 positive there
- 11:56from gamma positive.
- 11:57Keystones we could partially
- 12:00reconstitute the fibrosis.
- 12:01Certainly the provascular fibrosis.
- 12:03As you can see here,
- 12:05and we could reconstitute and or
- 12:07a lot of the cardiac dysfunction.
- 12:09Although this data also suggested
- 12:12that there had to be some cardiac
- 12:15antigen specificity involved to
- 12:17to induce the full induction of
- 12:19cardiac fibrosis and dysfunction.
- 12:21In these experiments, as I said,
- 12:23we reconstituted fibrosis,
- 12:25some parameters of systolic
- 12:27and diastolic dysfunction.
- 12:29But these cells that we put
- 12:31back into these mice,
- 12:33they were highly painful.
- 12:35Amatory but not antigen specific and
- 12:37this will link to the second part of my
- 12:41talk and why that might be important.
- 12:43How we can use that in in in to
- 12:47understand this complex syndrome.
- 12:50So the other thing that I needed
- 12:52was well now
- 12:52that we know that these T cells that express
- 12:55in there from gamma are increasing the
- 12:58lymph nodes under infiltrated in the heart.
- 13:02How can we see if they actually cross
- 13:05communicate with a cardiac residents?
- 13:07Because we saw that massive
- 13:09effect on cardiac fibrosis.
- 13:11We see it was a simple
- 13:13experiment to start with,
- 13:14which was isolating primary,
- 13:16correct fiberglass from Adele mites
- 13:18and then see isolated T cells.
- 13:21From the mediastinal lymph nodes of
- 13:23mice that were subjected to either sham,
- 13:25so control surgery,
- 13:27I should say that some might have the
- 13:30the open chest surgery and everything
- 13:33except for the construction to
- 13:36account for possible inflammation
- 13:38that happens during surgery.
- 13:40I know what you did is the Chico
- 13:43culture this and then she she
- 13:45coculture this indirect cultures or
- 13:47entrance once and the idea was that
- 13:49first he was going to see whether
- 13:51these teasers could adhere to the
- 13:54fibroblast and whether these fiberglass
- 13:56transform to myofibroblast and to
- 13:58do the readout for myofibroblast.
- 14:01We looked at alpha small muscle
- 14:04acting which is expressed upon
- 14:07fiber restaurants formation.
- 14:09So in those experiments,
- 14:11this is a representative image.
- 14:13You can see that a direct contact was
- 14:15required for these transformation,
- 14:17so these are two examples of
- 14:20transform my fiberglass that have
- 14:21a lot of T cells bound to them.
- 14:24The T cells were labeled in in green here
- 14:27and this is the quantification of the
- 14:31of of the red of alpha small muscle acting.
- 14:35So the conclusion from these
- 14:37experiments was that there was
- 14:38these communications between.
- 14:40Pieces of fiberglass that was required
- 14:43to induce transformation and then a
- 14:46mechanistically what we found was
- 14:48that this was TGF beta dependent,
- 14:50so it will block TGF beta.
- 14:52We inhibited this transformation that
- 14:54was not too surprising because we know
- 14:57that PDF better was it's a classic
- 15:00profit garlic cytokine that induces.
- 15:03This transformation and that is
- 15:05highly made by my fiberglass as well,
- 15:07but what was more interesting is
- 15:09that we found that T cells bound to
- 15:12the fiber rest through A4 integrin,
- 15:14and we come one in the fiberglass
- 15:16and then once bound,
- 15:18the diesels were induced and DJ better
- 15:21released by the myofibrils are not
- 15:23the other way around and this was also
- 15:26published so I wouldn't show a lot
- 15:28of the data there so I can focus on
- 15:30more recent data in in our lab as well.
- 15:33We're currently working on on
- 15:36further mechanistic insight
- 15:37into how we can prevent this.
- 15:40They released induced by the FIBERLESS
- 15:43upon the contact with the discus.
- 15:46So as a summary of background of
- 15:48why we became interested in this,
- 15:50we can see that in this transformation
- 15:53from the healthy heart to the failing
- 15:55heart using an experimental model,
- 15:57in this case of transverse article
- 16:00struction visit this activation,
- 16:02particularly of this T cell subset.
- 16:05And then we say that these are traffic
- 16:07to the heart and once in the heart.
- 16:09They crossed off with a fiberglass
- 16:11and induced cardiac fibrosis.
- 16:13We block these by either using
- 16:16agent mice that don't have T cells,
- 16:19or by using depletion diesel
- 16:21antibodies in wild I'm eyes.
- 16:23I didn't show the data by the way.
- 16:25We also did those studies.
- 16:27We prevent this transformation.
- 16:30We also found that the characters
- 16:35themselves they in response to
- 16:37pressure in response to duck even
- 16:39before the T cells get there,
- 16:42they can actually make chemokines that
- 16:45Kim attracts positive T cells and we
- 16:47did this using a reported mice for
- 16:50these schemes and and doing a time course.
- 16:53So we actually found that the
- 16:56fiberglass are actually functioning
- 16:58as a semi immune cell because
- 17:00they're releasing chemokines.
- 17:01Then they end up attracting first my
- 17:04load cells and then T cells to the heart,
- 17:07and then as I just showed,
- 17:09we found that they can regulate
- 17:12correct for groceries.
- 17:13So then when I question that we asked
- 17:16was well how and where this is being
- 17:19activated in the heart and this work
- 17:22was done by Jay Wanyama who was a
- 17:25graduate student in the lab and he
- 17:27was really interested in knowing
- 17:29this because he found he said well,
- 17:31if we found a specific antigens that might
- 17:34be relevant for the T cell immune response.
- 17:37Then one could think about in
- 17:39this in the future.
- 17:40Potentially immunizing for heart failure,
- 17:43right?
- 17:44There will be a long term goal or at
- 17:47least understanding whether this is
- 17:50what was this this activation happening
- 17:53and where over time it could be prevented?
- 17:57So to study that we use a reporter
- 18:00mice for T cell activation or
- 18:03T cell receptor engagement.
- 18:05So these are in order to be activated
- 18:07by antigen presenting cells,
- 18:09they need to recognize antigen,
- 18:11and in the case of CD 4 positive T
- 18:13cells they express the diesel receptor
- 18:16here and then dendritic cells.
- 18:18Are they the main antigen presenting
- 18:21cells express MHC two and they
- 18:24can capture antigen and induce T
- 18:27cell receptor signals and these
- 18:30reporter mice mimic.
- 18:32They're basically reporters
- 18:33of diesel receptor engagement.
- 18:35So the green are the cells are because
- 18:39they express N 77 which is downstream.
- 18:42The diesel receptor bound to GFP.
- 18:45So the greener the cells are.
- 18:47That's telling you that
- 18:48they're recognizing antigen.
- 18:49This expression is also transient,
- 18:53so if we see green cells,
- 18:56it means that at the time where
- 18:58we're harvesting those cells,
- 18:59they're recognizing antigen.
- 19:00But it might be that they recognize
- 19:03antigen and then they're not recognizing.
- 19:06Antigen at that point and then
- 19:08they lose expression.
- 19:09So in order to look at this,
- 19:11we basically did the time course
- 19:13of tag again early on,
- 19:15where is compensatory changes and then
- 19:18once is systolic dysfunction is established,
- 19:21and then we kept them for longer.
- 19:23That will mimic more chronic heart
- 19:26failure and what we did is we harvested
- 19:29the hearts and the medicinal influence.
- 19:32And the first thing that we did here.
- 19:35And we found that was very,
- 19:37very surprised,
- 19:38surprising,
- 19:39and the most interesting finding.
- 19:41I I I felt from from this story that we
- 19:44recently published was that we saw
- 19:46this T cell receptor engagement not
- 19:48only in the cardiac lymph nodes,
- 19:50but also within the heart.
- 19:53And as you can see here,
- 19:54you said the very bright GFP
- 19:57cells that increase overtime.
- 19:59So that's telling us that once
- 20:01the T cells infiltrate the heart,
- 20:04they must be vendrick cells.
- 20:06And potentially other cells that
- 20:08capture the antigen and induce
- 20:10decent expansion within the heart
- 20:12and that would be bypassing the the
- 20:14final trafficking that you need from
- 20:16the lymph node into into the heart.
- 20:19So we quantify this and as you can see,
- 20:22there's a significant increase of
- 20:25GFP positive active T cells in
- 20:28the heart that correlates with
- 20:30decline in systolic function.
- 20:32Measure here with fractions.
- 20:35So if we go back to how this teaser
- 20:38activation happened in the timers,
- 20:41we have a lot of high T cell
- 20:45receptor clonal diversity because our
- 20:48diesels are deciding what you know,
- 20:52depleting what's against self antigens
- 20:54and selecting for what we might
- 20:57need in the future if we get closer
- 20:59to the high in the Middle East and
- 21:01lymph node we will have the selected
- 21:03a pool of clones that will get expanded.
- 21:05If there is any immune response
- 21:07and then the question was what
- 21:09we were seeing in the heart,
- 21:10so our data using these reporter
- 21:13found that indicated that there was
- 21:15this expansion in the correct running
- 21:18lymph nodes as well as in the heart,
- 21:20and then we decided to do this
- 21:22receptor sequencing to get a closer
- 21:24look at whether these T cells or
- 21:25whether it might be recognizing.
- 21:27So this is the structure of the
- 21:29T cell receptor.
- 21:29Many of you probably know,
- 21:31but just as a brief reminder
- 21:32it has two Chainz,
- 21:34the alpha and the beta chain.
- 21:36And then they recombine in many
- 21:38different ways to form a specificity.
- 21:41Or these pocket to many different antigens.
- 21:43So by sequencing this Cdr three region,
- 21:47which is where these two chains
- 21:50get closer and form the pocket
- 21:53for antigen recognition,
- 21:54we could get a sense of whether we
- 21:56get in many different clones which
- 21:59will indicate high clonality and not
- 22:01not really an antigen specificity.
- 22:03There was anything in the heart,
- 22:05or whether we get.
- 22:08Enrichment so the results that
- 22:10we found was as expected.
- 22:11There was a lot of clones,
- 22:13many different diesel receptors
- 22:15sequences in the timelines.
- 22:18These are the inguinal lymph nodes and
- 22:20again we have a high clonal diversity
- 22:22and what we found was that the
- 22:24closer that we were getting to the heart.
- 22:27There more the decreased number
- 22:29of total clothes that we found,
- 22:32and the highest enrichment of the
- 22:35top 20 most represented groups.
- 22:37So today's my zaveri complicated
- 22:40analysis and we found that there was
- 22:43a restricted clonal pool in the heart
- 22:45and that the majority of the cells
- 22:47were represented by top 20 clones.
- 22:49So we started diving more into
- 22:51what this could be.
- 22:53So what are these enrich
- 22:55clones responded to in the
- 22:56hunt and this will be the working month.
- 22:59We know that this is happening and
- 23:01then we know that there's this close
- 23:04being expanded and what could those be?
- 23:06So we ended up focusing on Russ and then
- 23:10again because of the time limitation.
- 23:13I'm not going through
- 23:14everything that we went through,
- 23:16but it basically one hypothesis was
- 23:18that you could have a cardiomyocyte
- 23:20proteins right that the myocytes die,
- 23:23and then the fragments are picked
- 23:26up by adding percentage selves.
- 23:29But it turns out that in the attack
- 23:30model and like in response to my
- 23:32kardelen function where you see a lot
- 23:34of cell damage in the attack model.
- 23:37We don't see significant salvage early
- 23:39on and even later on at four weeks.
- 23:42But what we do see is high increases of
- 23:46intramyocardial reactive oxygen oxygen
- 23:49species which are labeled here in green.
- 23:53So we went back to literature and we
- 23:56hypothesized that maybe Rose could modify
- 23:58correct proteins that then form new
- 24:01antigens that are listed AT cell response.
- 24:04Why do we?
- 24:05Why would do we think that
- 24:07there was a hypothesis?
- 24:09It was because similar mechanisms
- 24:11had been described in the vasculature
- 24:13in the context of hypertension,
- 24:15where there was this formation of ice level.
- 24:19Glenda Lynn's,
- 24:20which are highly reactive intermediates by.
- 24:24Lipid peroxidation.
- 24:25That then can adapt to self proteins
- 24:27and create these new antigens.
- 24:29So we contacted the people who had
- 24:32done the scientists that had done this
- 24:36very interesting research hypertension
- 24:38David Harrison and Annette Kirabo,
- 24:40and this started a beautiful
- 24:42collaboration in which we were able
- 24:45to test this hypothesis in the heart.
- 24:48So basically we obtain a lot
- 24:50of reagents from their labs,
- 24:51and the first thing that we wanted
- 24:53to know is whether this matter
- 24:55in the human heart.
- 24:56So we went back or human heart failure
- 24:59sections and we use this one day 11 which
- 25:02is an antibody that recognizes proteins.
- 25:05Modified biologist was given to us
- 25:08by David and Annette and as you
- 25:11can see here we saw significant
- 25:13recognition in three different
- 25:16heart failure patient samples.
- 25:18And no signals in a non
- 25:21heart failure patient.
- 25:22We went back to the marsh model and in
- 25:24the marsh model we went back to these
- 25:27reporter my cells and T cell recognition.
- 25:31And we used the D1DD11 antibody
- 25:35that recognizes proteins
- 25:37modified by strategies in mouse.
- 25:39This was a different version of the antibody.
- 25:42And then they also send us some ISO
- 25:45so they can generate and I soils LG
- 25:50scavengers that could be used in in mice.
- 25:54So how does it work with experiments in tack?
- 25:57And then this is the structure
- 25:59of the eye surgeons calendar,
- 26:00and this is the control peptide.
- 26:02So the two Joba that I'll be
- 26:04showing is one with coverage.
- 26:06Those rose reactive proteins and then
- 26:09we tracked this activation and T
- 26:13cell receptor engagement over time.
- 26:15In some experiments we use tempo
- 26:18because it's an antioxidant,
- 26:20so it works upstream of the of the.
- 26:24Draws formation so just to make it
- 26:27simpler in for understanding the idea,
- 26:31this is a question that we were trying
- 26:33to ask and the idea is that in response
- 26:36to diagnose Ross and then this Ross
- 26:38induces the formation of this lipid
- 26:41peroxidation and this ISO, geez,
- 26:44that then they adapt to a cardiac protein.
- 26:47I'm from Disneyland pigeons that could
- 26:49be taken by the dreaded cells and
- 26:52being presented these cells to induce
- 26:55T cell activation and proliferation.
- 26:57So we could block these with temple perhaps.
- 27:00And if we block this, if this was true,
- 27:03maybe we would see less this
- 27:05activation or proliferation in heart.
- 27:07And if you block this with the
- 27:10ISO G specific scavenger,
- 27:12we could potentially block this and
- 27:13block this activation in the heart,
- 27:15and then of course the final question was,
- 27:17will this have any impact incorrect function?
- 27:22So the first thing that we did is we
- 27:24did those experiments in mice and then
- 27:27we isolated and Rick cells from mice
- 27:29treated with these AI soldiers scavengers.
- 27:32And as you can see here,
- 27:34this is the antibody that detects
- 27:36the isolated protein adducts.
- 27:38And as you can see with the four coma,
- 27:41which is the control and the
- 27:44control compound,
- 27:45we see that in drink cells
- 27:47express this and take a protein.
- 27:49But this is significantly
- 27:51inhibited when we scavenged.
- 27:55And then when we look directly in the House
- 27:57looking for teachers using the reporter mice,
- 27:59we found that only in those miles that
- 28:02were treated with the iceberg scavengers,
- 28:05we were able to significantly decrease
- 28:08this teaser activation within the heart.
- 28:11This is again GFP,
- 28:12because this underreported mines and
- 28:14I don't think I mentioned it earlier,
- 28:16but this is this receptor beta
- 28:18to make sure that we're focusing
- 28:20on the right distal population.
- 28:23So then how can OK?
- 28:25So now we know that under excels pick it up,
- 28:28but added functional inducing
- 28:30diesel proliferation.
- 28:32Here we found that there's
- 28:35less this activation,
- 28:36so the hypothesis is that when
- 28:38these are become activated,
- 28:39then they have to proliferate.
- 28:42So we tested this hypothesis ex vivo
- 28:45and we took the genetic cells from
- 28:48control mice and loaded them with either
- 28:51ice or GS or with a correct license
- 28:54that were taken from the tag mice.
- 28:58And then we coculture this and Rick
- 29:00cells that were incubated with these
- 29:02characteristics with teachers that
- 29:04came from medicinal lymph nodes
- 29:06from either sham or tag mouse.
- 29:09And again we were trying to
- 29:11mimic these response.
- 29:12And after four days we can
- 29:14look at T cell proliferation.
- 29:17So the way we look at T cell proliferation
- 29:19is because the teachers are labeled
- 29:21with a membrane guide that is CFC.
- 29:23And if they proliferate as you can
- 29:25see here to see if you see a die is
- 29:29diluted and this is just one example.
- 29:32If we combine some T cells with sham lysates,
- 29:37there's no proliferation,
- 29:38and if you put tactics so there's
- 29:41significant proliferation.
- 29:43But basically what we found here,
- 29:45this is only a representative experiment.
- 29:47Everything is quantified in the manuscript.
- 29:50But what we found was that only when
- 29:53the teachers came from mice with
- 29:56experimental heart failure and the
- 29:58proteins that cells were loaded with
- 30:01cardiac proteins that came from Tak
- 30:04only this combination is when we
- 30:06were able to see these new antigen
- 30:10presentation and decent proliferation.
- 30:12So did this have any impact in
- 30:15cardiac function?
- 30:15And these are echoes done
- 30:17by our collaborators.
- 30:19That Medical Center Rob Landon,
- 30:21who is a cardiologist that whom I've
- 30:24I've been collaborating with for many,
- 30:27many years,
- 30:27and what we found is that
- 30:29these attack animals.
- 30:31You can see the flat line here and
- 30:34decrease historic function that is,
- 30:37is quantified here.
- 30:39Fractional shortening and
- 30:40the harsh from the two hobas.
- 30:42For them I struggle with this.
- 30:43Lavender it were very healthy
- 30:46compared to to the control.
- 30:51So to summarize this part,
- 30:53what we found was that in response
- 30:55to high level trickler pressure.
- 30:58There was a significant induction of
- 31:01intramyocardial rose in the heart
- 31:03and ended in Derrick cells that are
- 31:06here are picking up some of the
- 31:09of the proteins that are modified
- 31:11by brass induced eye surgeries.
- 31:14And then in the lymph node,
- 31:15we saw that these T cells respond
- 31:18to antigen and expand and then
- 31:20they can go back to the heart.
- 31:22And traffic to the heart.
- 31:23But perhaps what was more intriguing
- 31:26to us was that once in the heart.
- 31:29You sometimes bypass this later on
- 31:32within the heart because they can
- 31:34actually recognize antigens within the
- 31:37heart and be expanded there under.
- 31:40This has significant effects
- 31:42on cardiac fibrosis.
- 31:46And I I didn't show the kind of
- 31:49fibroglandular also had significantly
- 31:51decreased fibrosis and this is going
- 31:53to become more relevant for the next
- 31:56part of the talk where I will be
- 31:59talking about this critical antigen
- 32:01recognition that happens in heart.
- 32:04So for the last part of the talk,
- 32:08then we I will focus about these kids
- 32:12are correct fibroblast crosstalk.
- 32:15So as I showed in the first part of the talk,
- 32:17when the diesels Infiltrator hi,
- 32:19this is an image ex vivo.
- 32:21So these are fiberglass and culture
- 32:23with T cells and you can see the
- 32:26green cells are here and the blue
- 32:29little nuclei of the diesels and
- 32:31this is a large nuclei of fiberglass.
- 32:33So we had found that with the diesels,
- 32:37either DH one cells that were
- 32:39generated extra evil or T cells
- 32:42isolated directly from TAC mice.
- 32:44They bound to the fiberglass
- 32:46and once they bound they induce
- 32:48the transformation to alphasim
- 32:518 producing correct fiberglass.
- 32:53And then in in the second part of
- 32:55the talk I just recently showed you
- 32:58that I didn't present themselves
- 32:59and particularly in lyrics.
- 33:01Elves present antigen to T cells
- 33:04and there is this intramyocardial
- 33:07diesel receptor engagement.
- 33:10So then we we thought,
- 33:11well,
- 33:11they're not that many dendritic cells
- 33:13in the heart as compared to other cells,
- 33:16right?
- 33:16Is it possible that during this T cell,
- 33:20fiberglass crosstalk not only the T cells
- 33:23are telling the fiberglass to to induce TGF,
- 33:27beta and transform?
- 33:28But maybe the fiberglass because diesels
- 33:31are firmly adhered to the fiberglass.
- 33:34Maybe the fiberglass may be functioning
- 33:37as also an antigen presenting
- 33:39cell that is semi professional.
- 33:42So we went back to literature,
- 33:44and in this there's this growing
- 33:47field of struggle immunology,
- 33:48where the concept is that antigen
- 33:51presentation is no longer an
- 33:53exclusive domain for the lyrics.
- 33:55Also obviously then Derek cells
- 33:57are antigen professional antigen
- 34:00presenting cells,
- 34:01but they're also evidence that
- 34:03a stronger cells that support
- 34:05tissue architecture can serve as
- 34:08antigen presenting cells.
- 34:09Depending on the context.
- 34:11So this is an.
- 34:13In an example of fibroblastic
- 34:15particular cells in the lymph nodes.
- 34:18Timing is stomach cells can do that as well.
- 34:21There.
- 34:21There are many reports that
- 34:22show that two more infiltrated
- 34:24fiber blasts do that as well,
- 34:26and it's also a recent report in
- 34:28the Lang where Lang epithelial cells
- 34:30in the context of inflammation,
- 34:32can actually present antigen
- 34:35to certain diesels.
- 34:37So we hypothesize that cardiac
- 34:39fibroblasts may be functioning as antigen
- 34:42presenting cells, and that these.
- 34:45T cell receptor engagement that we
- 34:47were seeing in the heart was not
- 34:50exclusively due to the dirt excels,
- 34:52but also to cut fiber breads.
- 34:54And a wind.
- 34:55Emma let this work and then called it cower.
- 34:58In my lab was also contributed
- 35:01significantly to this project.
- 35:03So to remind you what an antigen
- 35:05presenting cell in order to be an
- 35:07antigen presenting cell as poor
- 35:09as an indirect cell.
- 35:10You need to efficiently internalize
- 35:13and process antigens.
- 35:15You need to display them
- 35:17bound to MHC 2 molecules.
- 35:20And then you have to present
- 35:22that at the cell surface and
- 35:24professional Apcs and Rick cells
- 35:26constitutively express all of these.
- 35:29MHC do is constantly expressed and then
- 35:33these costimulatory molecules CD80 or
- 35:36CD86 that are induced upon stimulation.
- 35:41So we started investigating whether correct,
- 35:44fabulous may fit into this category.
- 35:47So this is a way that we select correct
- 35:50fibrous in the heart with digest the hearts,
- 35:53and then then we acquire this
- 35:56and all the non fraction.
- 35:58We're having the filial cells.
- 36:01We'll have local sides and we have
- 36:04a correct fiberglass here here.
- 36:06Sorry so we have leukocytes here.
- 36:093045 positives City 31 positive and
- 36:12killer cells within the local sides
- 36:15you could look for any local side that
- 36:18you're interested in and within the
- 36:20double negatives not in the filling.
- 36:22No leukocytes.
- 36:23We use these marker to detect
- 36:26a cardiac fiberless mask 4.
- 36:29We also do this in Linux reporter mice,
- 36:33and that's where is indicated here.
- 36:35So these are Linux tracing mice
- 36:38where we could more more definitely
- 36:40get into the cardiac fiberless.
- 36:43So the first thing that we did is do
- 36:45they express MHC two and a baseline?
- 36:48They don't.
- 36:48But as soon as you activate them with
- 36:52interferon gamma you induce expression
- 36:55of MHC two and actually in the filling
- 36:57search for instance by people here
- 36:59at nearly the department of Pathology,
- 37:01German Barber,
- 37:02and others found that endothelial cells
- 37:05can present antigens to T cells as well,
- 37:09and they respond and express MHC two
- 37:11in response to interferon gamma.
- 37:12So this would be a.
- 37:14It's similar mechanism of expression.
- 37:17And then what we found it was
- 37:19that they do express customer
- 37:20little molecules that you need to
- 37:22trigger that diesel activation.
- 37:24They do express CD 80 and
- 37:27is not further inducible,
- 37:29induced by different comma but
- 37:32they don't express 86.
- 37:34We collaborated with Jenn Davies and
- 37:36their impact grad student in her
- 37:38lab at the University of Washington
- 37:40and exactly the same experiments
- 37:42using the Linux trace in mice.
- 37:44That is a reporter for correct fiberglass,
- 37:47as shown in here.
- 37:49And as you can see here,
- 37:50all the correct fibers that are shown here.
- 37:53The majority of them in response to it there,
- 37:55from gamma, they express MHC 2 here in red,
- 38:00so this is GFP and this is no inter
- 38:03from gamma. With their from them.
- 38:07Does this matter in vivo?
- 38:08So in vivo we did pack and we found
- 38:11that carrot fiberglass isolated from
- 38:14from this report device expressed
- 38:18MHC 2 and you can see it here.
- 38:21You can focus here and this is zoom vision.
- 38:24So this will be all in green.
- 38:27Are cardiac fibrosis and as you can
- 38:29see there are also other cells that
- 38:31could be in the filler cells in a
- 38:33small kappel Aries or the drink cells
- 38:35as we previously shown that Expressen EC2.
- 38:37I've been here.
- 38:39Definitely,
- 38:39the correct fiberglass are expressing MHC
- 38:43two in response to tech as well.
- 38:45And sometimes if you look you can find
- 38:48that T cells seem very close to this
- 38:52MHC 2 expressing correct fiberglass.
- 38:54We use all the models of cardiomyopathy
- 38:58and cardiac inflammation to see
- 39:00whether this was unique or not,
- 39:02and we used the tickers eye infection
- 39:05model of myopathy because we know
- 39:07that because I is a parasite,
- 39:10I didn't use his highly strong
- 39:12in there from gamma responses,
- 39:14and as you can see here in this model,
- 39:16the correct fabulous also expressed
- 39:18any todo and more more of them
- 39:21express MHC do and then at the MFA.
- 39:25The prizes and densities also higher.
- 39:29So then the next question was,
- 39:31well, let's see if they can.
- 39:34Take up the antigen processor and
- 39:36present that induce T cell proliferation
- 39:39and to do that we use a reagent that
- 39:42is do DQ of album and so this is a
- 39:45novel women protein that can be taken
- 39:48up by proteins and if it goes in the
- 39:51lysosomes with acidic lysosome pH,
- 39:53which is what you're required to
- 39:56process antigens it costs related
- 39:58degradation and becomes for S.
- 40:00And as you can see here,
- 40:02regardless of the interference.
- 40:04Treatment DQ over is processed
- 40:06by cardiac fibroblast.
- 40:08This is just one example of correct
- 40:11fiber rest treated with equal woman,
- 40:13but you can see here the comparison
- 40:15of a large correct fiberglass
- 40:17and obviously a smaller in size.
- 40:19Here the bone marrow derived
- 40:21cells that a process.
- 40:24So that was very exciting too,
- 40:26because then that means that
- 40:28if they're able to process it,
- 40:30they might be able to load it into MHC
- 40:33two and induce decent proliferation.
- 40:35So we did similar studies as
- 40:37what I showed before to to look
- 40:40for diesel proliferation,
- 40:41and we use in this case we use
- 40:44transgenic mice that are what they do,
- 40:46so these mice all the T cell
- 40:48receptors in the details express
- 40:50a receptor for available mean.
- 40:53And then we took this specific piece
- 40:56of argument and then on the other hand,
- 40:59we took it a wild diaper.
- 41:00Makes you do knockout.
- 41:01Correct fiber rest.
- 41:03Three of them within their front comma
- 41:05and treated them with normal woman.
- 41:07So in this Co cultures,
- 41:09if they carry fiberglass,
- 41:10are processing available mean?
- 41:11As I I recently showed with Valve woman.
- 41:15All these diesels with a receptor
- 41:18for Valve women should be able
- 41:20to proliferate and we did other
- 41:23experiments in which we use E.
- 41:24Coli,
- 41:25a bacteria that over expresses
- 41:27about women as well.
- 41:29And again,
- 41:29these are the readout of proliferation.
- 41:31If there is prolific,
- 41:33there's no proliferation.
- 41:34These teachers that I label with
- 41:36CFC will not dilute the die,
- 41:38so we wouldn't see any peaks any dilution,
- 41:42but if there is proliferation we will
- 41:44see this dilution of the membrane dye,
- 41:47but that's exactly what we saw here,
- 41:50so this is overwhelming protein.
- 41:52But here the fiberglass haven't
- 41:55been treated with interferon gamma,
- 41:57so they don't express MHC 2.
- 41:59Very little proliferation,
- 42:00but here what you can see is that
- 42:03when they you induce expression
- 42:04and you treat with over protein,
- 42:06there is a significant proliferation
- 42:09of diesel,
- 42:10suggesting that the fiberglass
- 42:12can induce diesel proliferation.
- 42:14And here's a demonstration of
- 42:16dendritic cells as a positive control,
- 42:18where we see a proliferation
- 42:20and as I said before,
- 42:22these are the professional
- 42:24antigen presenting cells,
- 42:25so they don't need to be pre
- 42:27treated within their from grammar.
- 42:28They express MHC 2. Constitutively.
- 42:31We also did these experiments,
- 42:33obviously with the MMC to knockout.
- 42:35Correct fiberglass to to show
- 42:37that this was a specific.
- 42:41So just to be.
- 42:44Over a convenience with this
- 42:47we do particularly proteins.
- 42:48So instead of putting a
- 42:50soluble of argument there,
- 42:52we collaborated with Carolyn Genco and
- 42:54Robert in our floor in order Department
- 42:57and they just happen to have these E.
- 43:00Coli that over expresses,
- 43:02so we try to correct fiberglass with E.
- 43:04Coli that had an empty vehicle
- 43:07or expressing involvement,
- 43:09and we saw that only when when E.
- 43:12Coli was expressing about woman we saw.
- 43:14Diesel is specific for ovum proliferate,
- 43:17and this is all quantified here,
- 43:20and this is the positive
- 43:22control with the sender excels.
- 43:25So going back to the cardiac pathophysiology,
- 43:29does this make?
- 43:30Does this have any effect on correct
- 43:33dysfunction or cardiac fibrosis?
- 43:36So in collaboration with Jenn Davies,
- 43:38the University of Washington,
- 43:40we obtain the TCF 21 Mercury
- 43:44Mirror mice decree driver.
- 43:46Please inducible and we
- 43:48crushed it with MHC to flux.
- 43:51And we generated the correct
- 43:54fiberglass specific deficient in MHC
- 43:57do and these mice are only deficient
- 43:59if you treat them with tamoxifen
- 44:01because it's an inducible line.
- 44:03So as you can see here,
- 44:05we corroborated that it when
- 44:07we treated with tamoxifen,
- 44:09we decrease the expression of MHC 2.
- 44:13Here, so this this is what we will be
- 44:15looking at and we decrease expression.
- 44:18Incorrect.
- 44:18Fabulous, but not in bone marrow Dr.
- 44:20Dendritic cells where MHC 2 levels
- 44:23remain compatible in the treated
- 44:26and not treated with oxygen.
- 44:28And then we looked at fibrosis and as
- 44:30you can see here there was significant
- 44:33fibrosis in the TACK control group
- 44:36than non democracies untreated,
- 44:38but when we treated with tamoxifen we
- 44:41reduce fibrosis significantly and this
- 44:44has an impact in fractional shortening.
- 44:46So here's that mouse with flattened.
- 44:50You know contraction here
- 44:52and this is the most mice,
- 44:55so again these are the ones that don't
- 44:58have image do in the fiberglass and
- 45:01they have preserved systolic function.
- 45:04So we looked in the lymph nodes
- 45:07to right because we wanted to
- 45:09see whether this was where.
- 45:11Remember that we're eliminating this in
- 45:14the in the cardiac fibroblast and they
- 45:17might be other cells that express TCF 21,
- 45:20although it is,
- 45:21it was described to be a
- 45:23driver for collect fibers.
- 45:24And what we find is that the T cell
- 45:27immune response in the lymph node
- 45:29is not altered by by this intact.
- 45:32And we also see.
- 45:34Similar infiltration of character
- 45:36in these mice with tamoxifen.
- 45:39So working hypothesis now is that
- 45:42these are the conclusions right that
- 45:44in these crosstalk we have the T cells
- 45:48and the fiberglass communicating.
- 45:50And in this two week crosstalk
- 45:52we think there correct fibrils
- 45:53are Sentinel cells that can sense
- 45:56correct insults and directly boost
- 45:58the adaptive immune response.
- 46:00We think that there's a potential of
- 46:03moderating decent immune responses
- 46:04without impairing systemic diesel
- 46:06activation by the cells which could
- 46:09have undecided major suppressive effect.
- 46:11So the fact that we see.
- 46:14Similar activation in the in the
- 46:16lymph nodes that that tells us to
- 46:19think that these this is critical in
- 46:22the heart for the correct fibers.
- 46:25And then the overall summary,
- 46:26just to wrap up and leave some time
- 46:30for questions is that responses.
- 46:32I think we're pretty convinced with
- 46:34our work and a lot of the work,
- 46:36that of others that I've cited and
- 46:40that we we always site in in our papers,
- 46:42is the diesel immune responses
- 46:44contribute to the pathophysiology
- 46:46of nonischemic heart failure in
- 46:47many different ways, right?
- 46:49So we think that eleven took blood pressure,
- 46:52induces a significant levels of
- 46:56drugs and the formation of new
- 46:59antigens that trigger this activation
- 47:02in the heart. Within that limited repertoire
- 47:05of those T cells respond to ISO LGS.
- 47:08Modified cardiac new antigens and contributes
- 47:11to cardiac fibrosis and dysfunction.
- 47:13But we don't think these are the early
- 47:16antigens that diesels are recognizing,
- 47:18because if you recall from our data,
- 47:20even when we scavenge those icebergs
- 47:24modified proteins, we still see
- 47:26some decent activation in the heart.
- 47:29So we are doing a lot of more in
- 47:31depth analysis of single cell.
- 47:33He's service center sequencing and
- 47:34trying to get to what are those other
- 47:37antigens that might be induced response?
- 47:39And we also see that they're not the same.
- 47:43Backgrounds overtime,
- 47:44which might be very relevant to
- 47:46see how heart failure progresses,
- 47:48at least pretty nicley.
- 47:51And then the last conclusion from this
- 47:53is that these bidirectional actions
- 47:55between correct resident cells,
- 47:58in this case fiber rise and T cells
- 48:01contribute to correct this activation.
- 48:03My fibrous transformation and
- 48:05dysfunction under the correct
- 48:08fabulous expression of MHC 2 molecules
- 48:11is central for these response.
- 48:15So with that, I'd like to thank my lab.
- 48:19I think I've mentioned everyone who's
- 48:22done the work who's now moved on to new
- 48:26exciting research leading positions,
- 48:28and then this is the new members of
- 48:30the lab that are trying to pick up
- 48:32on all the good work that previous
- 48:35former members did in the lab.
- 48:37Our collaborators at the University
- 48:38of Washington,
- 48:39Vanderbilt or collaborators at absent as
- 48:43Medical Center and our funding sources from.
- 48:4718 and also from Dallas University.
- 48:51With that,
- 48:52I'll be happy to answer questions,
- 48:54but before that I'll make an announcement
- 48:56of a very exciting conference,
- 48:59which will hopefully happen in
- 49:01person is scheduled to be in person
- 49:04in Chicago and there will be a
- 49:06lot of interest in science,
- 49:07not only in information but a lot of
- 49:10cardiology and basic and traditional science.
- 49:14So I'll be happy to take any questions.
- 49:16Thank you for your time.
- 49:21Thank you Paula for the wonderful talk.
- 49:24So now we are open to questions.
- 49:28I can maybe stop sharing and.
- 49:33Either way, would you like me?
- 49:34Or maybe I can leave it open
- 49:35in case I need to go back to
- 49:37good? Yeah, good idea Harold, please?
- 49:40Yeah hi, I really enjoyed the talk.
- 49:44Wonderful stuff.
- 49:45A very simplistic question.
- 49:47So at autopsy when we see
- 49:49hearts from patients who have
- 49:52really horrible heart failure.
- 49:54I don't ever recall seeing a
- 49:56striking infiltrate of lymphocytes.
- 49:58Is it that we just get them
- 49:59at the end stage or or?
- 50:03I think so compared to other cells
- 50:05T cells they you don't see massive
- 50:08infiltration as you would see.
- 50:10For instance post MI in the
- 50:12in the in fact zone, right?
- 50:15And I think I think you don't
- 50:18need that many of them,
- 50:20so they're definitely sparse
- 50:22compared to other major cells.
- 50:24And then the other thing that I would
- 50:26say that we've seen when we take
- 50:28samples from Elbert issue is that
- 50:30the human heart is very large, right?
- 50:32So it it also depends where you take
- 50:36the the piece from, and we've seen.
- 50:40We've seen some samples that
- 50:43have more than others.
- 50:46I don't think it's the timing
- 50:48because we've done. I mean,
- 50:50we haven't looked at human hearts early on,
- 50:52other than those of those healthy,
- 50:54right, and those have noticed.
- 50:57But if there was a way to
- 50:59look inhuman in mice,
- 51:00you can track that very easily, right?
- 51:03And one thing that we don't see is that.
- 51:06And in the chronic phase.
- 51:08So if you take the mice way longer,
- 51:10we see that the diesels are more
- 51:12activated when we use the reporter mice.
- 51:15But we don't see more details
- 51:17of our own per say.
- 51:19So I think that's that's
- 51:21maybe why inhuman is tricky,
- 51:23but definitely is not a
- 51:24dominant cell in the heart.
- 51:26You have to.
- 51:27You'll have to find them,
- 51:29but not being dominant doesn't mean
- 51:31that they don't do a lot right,
- 51:34because if they're highly
- 51:35activated they can release a lot
- 51:37of factors and do do other things.
- 51:39But that's a great point, thank you.
- 51:41Thank you for your question.
- 51:44I have a question actually 2 questions.
- 51:49So wonderful talk again.
- 51:52So do you think this fibroblast
- 51:57CD4T cell interaction may also
- 51:59induce macrophage to invade or
- 52:03activate resident macrophage
- 52:06to contribute to the fibrosis?
- 52:09That's the first question.
- 52:10Second question is related
- 52:13with hardwood question,
- 52:14have you considered utilizing
- 52:17genetic heart failure model such
- 52:19as meising heavy chain mutation?
- 52:22Our foes reach you genetic HTM mice
- 52:25as well as dilated cardiomyopathy like
- 52:28muscle Lim protein knockout mouse.
- 52:32They have natural heart failure.
- 52:34Whether your hack information could
- 52:36be extended to genetic heart failure,
- 52:40which may mimic human heart failure
- 52:42more closely. What do you think?
- 52:44Yeah, those are two great questions,
- 52:46so I'll ask for the first one.
- 52:47The first one, you're totally right.
- 52:50We've seen that cardiac fibroblast.
- 52:52Really, ski machines that
- 52:54not only attract diesels,
- 52:55but they attract monocytes.
- 52:58And we we did find that actually
- 53:02before Sumanth Prabhu had now in
- 53:05Washington University of Saint Louis.
- 53:08He found that early on,
- 53:11and we've corroborated that the Maya
- 53:14size the CCR 2 positive Milo itself,
- 53:17so the haematopoietic Lee derived monocytes.
- 53:20They infiltrate the hard
- 53:22before the diesels do,
- 53:24and we found following following up on that,
- 53:27we found that the correct fiberglass
- 53:29they make CXCL 9 and 10 that are
- 53:33chemoattractants for diesels,
- 53:35but they also make a lot of C, CL two.
- 53:37So that makes a lot of sense
- 53:39that when they sense pressure.
- 53:41And they release the chemo kids.
- 53:43The second thing in that related
- 53:46to that question is that.
- 53:51Once they infiltrate,
- 53:52so we,
- 53:53we've found that the major source of the
- 53:56diesel chemoattractant proteins is not.
- 53:58The fiberglass is actually the Milo itself.
- 54:02So those I think it's all orchestrated.
- 54:05Basically they fibroblasts release
- 54:07chemo treatments for innate cells.
- 54:09Then they adapted cells come
- 54:12because there's also an interaction
- 54:13between a myeloid cells and the
- 54:16fibers that we cannot ignore.
- 54:17I didn't do your second question.
- 54:19I would love to look at these
- 54:22models of carry myopathy.
- 54:25We haven't looked because we we've never.
- 54:28We don't have the tools or or the mice,
- 54:31but I would love to to do it because
- 54:34I think it's it's very important
- 54:37and especially in those mutations
- 54:39that the myocytes are are working.
- 54:42And I did,
- 54:44at dysfunctional from very early
- 54:46on it spontaneously right.
- 54:47You could really track and I'm sure that
- 54:50there will be other antigens involved, right?
- 54:53So it might be that we will need to find
- 54:56out whether inflammation plays a role.
- 54:58It might be that it has
- 54:59nothing to do with information,
- 55:01but if it did, if it did,
- 55:03it would be easier to track whether the T
- 55:07cells might be recognizing proteins that are.
- 55:11You know that may be misfolded,
- 55:14or that their mutated due to
- 55:16the mutation in the myocyte,
- 55:17so that that would be a yeah, that would be.
- 55:20That's that would be an excellent Ave.
- 55:23Thank you any other questions?
- 55:33So yeah, I I,
- 55:35I'm very excited about the talk.
- 55:37I have another follow-up question.
- 55:40What do you think about
- 55:42stressed cardiomyocytes?
- 55:44They may release new entity to the
- 55:48to the identical cells or two so
- 55:50they they catch up the antigen and
- 55:53present to CD 4T cells because we
- 55:56have cardiac troponin T troponin I.
- 55:58Fragmented release in the injured heart
- 56:01and and also cardio my side when stressed.
- 56:05They may secrete teacher
- 56:07Beta 2 instead of beta one.
- 56:09We adapt be interesting avenues for you to
- 56:12yes so that so first hypothesis.
- 56:15When we started going after the antigen
- 56:18when J join my lab and he really wanted
- 56:21to look at this diesel drones right
- 56:24and our first hypothesis was I had
- 56:27just written a commentary on a paper.
- 56:30Looking at all these in altimmune
- 56:33myocarditis you know how character
- 56:35planning and myosin binding protein
- 56:38C and all these proteins that
- 56:42that are fighting people, right?
- 56:45So we thought that that that those
- 56:47who were going to be the ones but
- 56:50in the tag model because we didn't
- 56:53see death of Carrie myocytes,
- 56:55we didn't focus on that.
- 56:56But you're right, they might be.
- 56:58They might be that the stretch.
- 57:00Induces, as you could do that
- 57:02nicely with your model, right?
- 57:03Because you can stretch all these cells we.
- 57:06We don't have the ability to do that,
- 57:07but I think it's also possible because
- 57:10the myocytes see the fiberglass
- 57:12seed in between the myocytes, right?
- 57:14So there's all these literature that,
- 57:18and a huge field of research that
- 57:20people study kind of fiber as
- 57:23kind of myocyte communication.
- 57:25So it might be that those fragments
- 57:28are actually picked up by you?
- 57:30Know the Mayo side doesn't
- 57:31really need to die.
- 57:32It might be that it's a stretch
- 57:34and the fiber rest pick it up.
- 57:37And then the fiberglass percent,
- 57:38but that's purely an in speculation.
- 57:41We haven't.
- 57:41We haven't looked at that,
- 57:44but I think it's not only.
- 57:46I think this is very complex and
- 57:48it's not only limited to the
- 57:50T cell binding to fibroblast.
- 57:52I think there is a.
- 57:54Cross communication with like an
- 57:57orchestrated response there with my
- 58:00insides fiberglass and immune cells.
- 58:03Yeah, so we have resident go ahead.
- 58:07Yeah this is Jeff Squire.
- 58:10I just wonder you made a comment
- 58:12on your introduction that said
- 58:14that no immune intervention.
- 58:16No trial has produced any
- 58:18effect on cardiac failure.
- 58:22And I wonder whether there any
- 58:24observations in patients who receive.
- 58:27Chronic immunosuppressive therapy
- 58:29with any number of different drugs,
- 58:32whether there's any effect
- 58:34on cardiac failure.
- 58:36Yeah, so we did actually have to look at
- 58:40that because we made a long table of.
- 58:44Exactly looking at that right of,
- 58:46you know, these were the TNF blockers
- 58:49and these are other immunosuppressive
- 58:51agents and we didn't find any.
- 58:54I don't think there's been.
- 58:56There's been a small trials looking at that,
- 58:59and I think people have looked
- 59:02at method tracks and other drugs,
- 59:05but I don't think there's a detail
- 59:07investigation of what having the
- 59:09expectation would be that if you
- 59:11suppress inflammation it be good, right?
- 59:13But those drugs also have a lot of side
- 59:17effects that may be in patients with.
- 59:20Cardiac failure are no quick right,
- 59:24so I think we really need to dive into
- 59:27into not blocking inflammation generally
- 59:32and try to find a smaller pathways.
- 59:37I certainly agree, but I just
- 59:39wonder whether there's any
- 59:40evidence that you know what the
- 59:42effect of the immune system is
- 59:44on in clinically and inpatient.
- 59:46A lot of patients who get,
- 59:48you know steroids and get
- 59:50cyclosporine and other.
- 59:53I I don't recall all the details,
- 59:55but there is a very elegant review by dogmen.
- 59:59And Luigi Adamo that they published recently?
- 01:00:02Maybe? Maybe not.
- 01:00:03That recently, maybe a year ago in Nature,
- 01:00:06reviews, cardiology, and they have they.
- 01:00:09They did exactly that,
- 01:00:11and they reviewed all the literature
- 01:00:14in large trials, small trials,
- 01:00:16directly tackling immune mediators,
- 01:00:20or general immuno suppressors. And I,
- 01:00:23I recall that the conclusion is what I said,
- 01:00:26but maybe you know, maybe I mean.
- 01:00:29But I. I think yes, if you have time.
- 01:00:32That review was very detailed and it was.
- 01:00:35It was very nice to to read and they had the
- 01:00:38they reviewed the mechanistic part of it,
- 01:00:40but then they review all the patient trials.
- 01:00:43At the end.
- 01:00:45I believe it was in nature reviews,
- 01:00:47cardiology for sure and I don't
- 01:00:49know if it was 2020 or 2021.
- 01:00:52But
- 01:00:53thank you yeah. Yeah.
- 01:00:59So if if there are no additional
- 01:01:01questions so thank you so much
- 01:01:04Paula for this exciting talk.
- 01:01:05We learn a lot cardiology immunology,
- 01:01:08so thank you very much.
- 01:01:09Have a nice afternoon.
- 01:01:11Thank you for the invitation.
- 01:01:13I thank you all for attending bye.
- 01:01:15Bye bye thank you.