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[Music] hello and welcome back to immunology the war is over episode 8 the compliment regulators this episode follows on from the compliment deep dive that we did back in episode seven so if you haven't watched episode seven you should definitely go back and do that now because this tutorial will make a lot more sense okay so in this episode we're gonna get to know the compliment regulators what they do and where they work and then we're going to connect these concepts to some key clinical conditions for your exams namely atypical hus pnh and hereditary angioedema

and just like episode 7 this tutorial will also be handwritten so grab your worksheets and other supplies and let's jump right in the complement regulators can roughly be divided into two groups those that whizz around in our circulation the so-called soluble regulators and those which are located on the surface of our cells the circulating complement regulators include factor h factor i c4 binding protein factor h like protein 1 veteranectin and clusterin the majority of these are made in the liver although clustering is expressed by various tissues the membrane-bound regulators include membrane cofactor protein or cd46

complement regulator 1 decay accelerating factor daf or cd55 and cd59 also known as the mac inhibitory protein and let's not forget thrombomodulin so that's all the characters let's find out where they fit now we saw in episode seven that the alternative pathway requires the most policing for a couple of reasons one because it's always ticking over in the background so being spontaneously activated and two because it's responsible for the amplification loop which is sparked every time complement is in full swing and so needless to say mother nature knew she had to install the most potent

complement regulators at this stage of the cascade within the alternative pathway now we know from episode 7 that this amplification loop involves c3b making c3 converters which in turn makes c3b which makes more c3 converters and round and round we go and so it follows that in order to inhibit the amplification loop we need to do two things prevent the assembly of the c3 converters and promote the breakdown of the c3 converters and the complement regulators have this down to a fine art first up preventing assembly this is predominantly performed by factor i which is

responsible for inactivating c3b and c4b for that matter now before we dive into the magic of factor i there's something you should know factor i is talented and powerful but sadly lacks initiative factor i f left to its own devices would do nothing at all the whole complement cascade could be in full swing and factor i would just whiz on by but f explicitly told what to do and where factor i is incredibly helpful and will spring into action so factor i needs a bit of encouragement to become activated and do its thing and this

encouragement is performed by cofactors and actually the majority of complement regulators have the ability to act as a cofactor for factor i including cr1 mcp daf factor h factor h like protein 1 and c4 binding protein so whilst factor i requires encouragement there are multiple sources of encouragement available and so factor i in the presence of these cofactors will deactivate c3b into inactivated c3b now although it's inactive this molecule will still function as an opsonin encouraging phagocytosis and lowering the threshold for b cell activation but it's not going to generate any more c3 converters okay

so that's c3b out of the way now let's see how we break down c3 converters the breakdown of the c3 converters in the alternative pathway is promoted by factor h fhl1 and daf dk accelerating factor now i think it's worthwhile just taking a moment to point out the potency of factor h factor h binds to c3b preventing the formation of c3 converters it is also capable of breaking down any established alternative pathway c3 converters and it acts as a cofactor for factor i which then inactivates c3b and what's more factor h unlike factor i can

do this without any encouragement at all it doesn't need cofactors it just gets the job done and because factor h is circulating in our plasma it can do this anywhere in the body and this is absolutely necessary because the expression of those membrane-bound cofactors i mentioned is variable in different tissues around our body so in any one tissue these cell membrane regulators are going to benefit from the help of factor h so factor h is the ultimate complement off switch because it can turn off the alternative pathway amplification loop from any location factor h is

the most potent and capable complement regulator we have and factor h like protein one as the name suggests looks a lot like factor h and this regulator has pretty much the same capabilities as factor h so everything i just said about factor h tends to apply to factor h like protein one as well and some of you might be wondering if factor h is so good at switching off complement could that ever be problematic does factor h ever get in the way and block our immune response to infection and that is a great question but

the answer is no factor h is able to recognize structures which are specific to host cells such as cialic acid which pathogens don't tend to express so factor h knows when complement is handling a pathogen and when complement is spilling over onto healthy tissue so factor h is not going to get in the way of an immune response to infection but it does protect our healthy cells from complement-mediated damage so clever okay so now we've covered the regulators of the amplification loop but this only tackles the c3 converters within the alternative pathway c3 bbp and

we know that we have another c3 converters that can be generated via the lectin and classical pathways c4b 2b so let's shift our attention to see how regulators handle this side of the complement cascade and helpfully this is very similar to what we just saw factor i also inactivates c4b into c4d so prevents the assembly of c3 converters but again factor i will only do this with encouragement from cofactors and on this side of complement the membrane-bound regulators such as mcp and cr1 can help factor i just like they did in the alternative pathway but

on this side factor h doesn't help at all factor h is the strongest regulator we have and so it needs to focus all of its attention on the amplification loop and it does not leave its station but standing in for factor h on this side of complement is c4 binding protein a soluble regulator that travels in our plasma to help factor i do its thing and lastly we have daf daf promotes the breakdown of c3 converters in any form so regardless of whether this is created in the lectin classical or alternative pathways daf will break

this enzyme down in a jiffy so daf doesn't let any c3 converters get comfortable but again because factor h is only dealing with the amplification loop this means that daf is the key regulator of the c3 converters in the classical and lectin pathways okay so we can see that mother nature poured a lot of resources into dealing with c3 converters in any form but she also installed some other gadgets further down the cascade which inhibit the formation of the membrane attack complex or mac mac is composed of c5b 6 7 8 and 9 which all

come together to form a pore in the cell membrane the pore doesn't really start to form until c8 and 9 come along and there's a particular complement regulator designed to block these and this regulator is cd59 cd59 goes by many different names mac inhibitory protein membrane inhibitor of reactive lysis and protecting i'm not sure why we needed so many names for the one molecule but there you have it anywho this regulator binds to c8 and 9 thus preventing them from establishing a hole in the cell wall which makes this the most important mac inhibitor we

have but it's not the only one we also have clustering and veternectin these plasma proteins bind to the building blocks of mac thereby preventing them from coming together as a complex and as if that wasn't enough our body cells love to show mac the door when healthy cells detect mac on their surface they have a tendency to wall it off and get rid of it they may do this via exocytosis sort of chopping mac away or alternatively they may choose to internalize and degrade mac inside the cell incredible okay so now we've sorted c3 converters

and mac but what about those pesky anaphyla toxins c3a and c5a just whizzing around in our circulation causing havoc these are deactivated by enzymes known as carboxypeptidases into c3 disarg and c5 desart sorted and now that we've covered the major complement regulators there's another one i'd like to throw into the mix thrombomodulin thrombomodulin is located on our endothelial cells with only a small amount in our circulation it has roles in regulating both the complement and coagulation cascades but when it comes to complement thrombomodulin helps out in a couple of areas firstly it enhances the effect

of factor h as a cofactor for factor i so factor h encourages factor i to inactivate c3b but when thrombomodulin is around factor h can do this better than ever the other place that thrombomodulin helps out is in promoting the carboxypeptidase which deactivates c3a and c5a so thrombomodulin doesn't do anything single-handedly but we can see that it adds value to the complement regulators at important points in the cascade helping to combat the amplification loop and those pesky anaphylotoxins so that's the complement regulators in all their glory now let's bring this together by taking a look

at where these show up in our clinical practice arguably the most notable condition when it comes to complement dysregulation is atypical hus in this condition complement gets out of hand and causes tissue damage this manifests clinically as thrombotic microangiopathy or tma which is the combination of microangiopathic hemolytic anemia thrombocytopenia and organ ischemia and so atypical hus is both organ and life threatening and in particular it just loves to attack my favorite organ the kidney if left untreated atypical hus results in end adrenal failure in 70 of cases which is a disaster in anyone's book but

when diagnosed and treated early with ekalizumab the outcomes much improve and so it's really important to know about this condition and be able to diagnose it now if you're sitting there thinking oh my goodness i would not know tma if it was sitting right in front of me you need to check out our tma tutorial on our website this covers ttp atypical hus and shiga toxin hos including how to diagnose and manage these conditions i also teach the complement regulators in a different way than i have here and i also go into echolusimab in a

bit of detail so that tutorial will reinforce the concept we've covered here but it's more comprehensive and shouldn't double up so definitely check that out if you want to become a tma ninja anywho back to atypical hus this condition arises because of impaired regulation of the alternative pathway and this impaired regulation occurs in the setting of a genetic predisposition if we regard the amplification loop as the on switch and the regulators of the amplification loop as the off switch we can quickly appreciate the factors which are relevant to atypical hus all of these factors are

encoded for by their respective genes so we have genes which are important for the on switch and genes that are important for the off switch atypical hus can occur via two scenarios the first is a loss of function mutation in the genes which code for the complement regulators thereby impairing the off switch the second mechanism is via a gain of function mutation in genes which code for complement factors such as c3 or factor b resulting in an overactive on switch and whilst the genetic predisposition is thought to be important in all cases only 50 of

patients will have an identifiable mutation in the genes which contribute to the on and off switch of complement and the severity of disease will depend on both the severity of the underlying mutation as well as the particular gene affected you can appreciate from what we've learned so far that a genetic mutation affecting factor h or factor i would lead to a severe phenotype whilst mutations in mcp would be associated with milder disease so people with atypical hus have an impaired ability to regulate the alternative pathway of complement and so when complement is triggered by say

an infection or pregnancy things can quickly get out of hand leading to complement-mediated damage and tma okay so those are the key points about atypical hus now let's shift our attention to another complement-mediated condition which is also treated with echolusimab namely paroxysmal nocturnal hemoglobin urea or pnh the clinical manifestations of this condition are centered around hemolysis and that hemolysis is happening because of the destruction of red cells by complement now this really sets pnh apart from other types of hemolytic anemia because the anemia part is not the worst part of pnh sure patients can experience

hemolytic anemia but they can also experience abdominal pain or chest pain due to transient ischemia in those vascular beds the ischemia arises because free heme in the circulation scavenges nitric oxide which leads to vasoconstriction but that's not even the most dreaded complication of pnh the worst part is the risk of thromboembolic disease which can be life-threatening and these clots may occur in unusual places leading to things like bud chiari syndrome or cerebral vein thrombosis for example so again although this is a rare condition it is potentially life-threatening and so we should know a little bit

about it okay so this hemolysis in pnh is happening because of destruction of red cells by complement and the red cells are vulnerable to this because they have a deficiency of complement regulators on their surface namely cd55 or daf and cd59 the mac inhibitor now what if i told you that this condition is underpinned by a genetic mutation but this mutation only affects blood cells and even then it doesn't affect every single blood cell we have rather we have a population of blood cells which are affected so why might that be the case and this

is where it gets interesting cd55 and cd59 both require something known as gpi anchors to exist on the cell surface without these anchors they will simply not be there and these anchors are encoded by the pig a gene in p h there is an acquired mutation of the pig a gene this occurs exclusively in the bone marrow so for people with pnh it's not a problem they're born with rather they have the peggy gene working just fine until one day one of those little hematopoetic stem cells chucks a genetic mutation into the mix so there

in the bone marrow early in the production line there's a cell with a pig peg-a-mutation and every cell which is derived from that cell will also have the peg-a mutation and these affected cells will have difficulty placing any gpi anchored proteins such as cd55 or cd59 on their surface and this problem will affect both the white cells and the red cells but red cells are particularly vulnerable to hemolysis for a couple of reasons we saw before that there are a number of complement regulators which may exist on the cell surface and you'll have different regulators

in different quantities in different tissues a healthy red cell would have cd55 cd59 and cr1 but red cells don't express mcp so if you knock out cd55 and cd59 you're only left with cr1 a cofactor for factor i and even then cr1 is only sparsely present on red cells as compared to their white cell counterparts and so in pnh the red cell defense against complement is diminished and on top of this we know how osmotically sensitive red cells are they do not respond well to having holes in their cell membrane so pnh happens when there

is an acquired mutation in the pig a gene within the bone marrow production line this generates a population of red cells which are extremely vulnerable to hemolysis at the hands of complement fascinating right so fascinating in fact that i've decided to dedicate an entire bonus episode to this condition sometime soon so stay tuned for that now we have covered a lot in this session in terms of the complement regulators and diseases where these show up or don't show up as the case may be but there is one complement regulator that i have not yet mentioned

the c1 inhibitor and i've left this until last with good reason you'll see why in a second the c1 inhibitor works by inhibiting the proteases within both the classical and lectin pathways so it inhibits cr1 and s in the classical pathway and inhibits mass in the lectin pathway now this sounds like an important job but the truth is that when it comes to switching off the complement cascade at large the c1 inhibitor is not a game changer and when this enzyme is deficient we don't necessarily have problems shutting down complement because we have so many

other potent regulators downstream which can compensate for this so c1 inhibitor deficiency doesn't lead to complement mediated disease per se but c1 inhibitor deficiency does lead to a potentially life-threatening disease called hereditary angioedema in this condition there is a genetic mutation which results in deficiency of the c1 inhibitor and it turns out the c1 inhibitor has important inhibitory functions not just in the complement cascade but in other proteolytic cascades as well with influence on coagulation and the canon calicrine system now if you have never heard of the canon calacrine system until this moment that makes

two of us but don't worry we can sum this up in a sentence or two the canon calacrine system has only one job making bradykinin bradykinin is a potent inflammatory molecule which causes vasodilation and altered vascular permeability the c1 inhibitor is incredibly important for keeping bradykinin under control and so if the c1 inhibitor is deficient bradykinin production is not well regulated and can lead to angioedema this tends to happen in the setting of a trigger such as infection or a medical procedure at the very minimum this condition is unpleasant and temporarily disfiguring but if the

angioedema affects the airway it can be life-threatening and what's important to be aware of is that because this condition is mediated by bradycainin and not histamine it doesn't necessarily respond to the usual treatment for anaphylaxis it does however respond to a c1 inhibitor infusion that'll help turn off that bradycainin in a jiffy so the c1 inhibitor whilst it does have a role in regulating the complement system it's not essential for keeping complement in check because other regulators can compensate for this but on the contrary c1 inhibitor is essential to regulation of bradykinin and giving this

enzyme in the setting of deficiency can be a lifesaver so that's a wrap for today's episode i hope you've enjoyed the journey through the complement regulators and the clinical conditions which can show up in your exams and i hope to see you again soon for some more high-yield learning bye [Music]