Today, we will be starting off with the second part of innate immunity.
Before we go into that, it might be a good idea to summarize some of the main aspects
of the last lecture. The first is the importance of innate versus adaptive immunity. As mentioned
previously, it is a quick response; it is non-specific; it does not differentiate between
different types of bacteria, but it tells the host that there is some sort of invasion
that has taken place. And, that is the important thing. It is an evolutionary conserved process
and it is present in the lower organism, such as drosophila, horse shoe crabs, etcetera.
So, it would be good idea for you to sort of think about where the drosophila and horse
shoe crab were useful in the previous lecture. For example, the identification of a toll
receptors were shown in drosophila and the measurement of lipopolysaccharides, which
is a potent endotoxin is used using a lysate from the horse shoe crab.
We also talked about physical barriers that are important in innate immunity, for example,
epithelial cells, mucus. So, mucus that is produced, traps these microbes and that is
useful in sort of containing their spread. There was also discussion on different types
of cells, for example, neutrophils, macrophages, NK cells and dendritic cells. So, we will
briefly go a little bit over them. Neutrophils are one of the first cells that the host response
to; and, they are the first ones to arrive at the site of pathogen entry. Subsequently,
they produce chemotactic factors and macrophages are recruited. Macrophages are important in
processing and presenting antigens to T cells; then, you have natural killer cells, which
are important for what is known as antibody dependents or toxicity. Once antibodies are
produced, the eyes target cells; they are also important for tumors. And, dendritic
cells are perhaps the physiologically most important antigen cells, which enter different
antigens to T cells, and so, you can turn on the adaptive immune response.
We had trust about the fact that the innate immunity modulates adaptive responses, and
this is mainly seen in the use of microbial components in adjuvants. So, for example,
complete adjuvants contain killed mycobacteria. And, this was known as the immunologist's
dark secret by Charlie Janeway, who first propounded that the innate components needed
to be activated to get an optimal immune adaptive response. And, subsequent studies resulted
in identification of toll-like receptors that are present in the host, which recognizes
specific microbial components. So, TLRs, for example; they contain this external leucine
rich repeats and this LLRs; or, leucine rich repeats are important for protein-protein
interactions and it contains an internal IL1 receptor signaling domain by which the signal
transduction can be done. We also discussed the ways by which LPS is
detected and the response by host. So, LPS binds to LPS binding protein in the serum,
and this complex is transported and it is recognized by CD14. And, TLR4 is important
for recognition of LPS. And, this complex alone cannot signal; it needs a signaling
molecule known as MD2, which is important in signal transduction. So, you turn on cytokine
response and you turn on an acute innate response, which is often manifested with respect to
cytokine release, increase phagocytosis, killing of target cells or pathogens, etcetera. We
also discussed an important part, which is septic shock. And, septic shock is quite prevalent
especially in hospitals, are post infections. And, this is important, because you have an
acute inflammatory reaction, because of the presence of microbes, and the host responses
is so strong that it often leads to multiorgan failure, low blood pressure and sometimes
even death. So, it is important that these aspects are revised by you before we move
on to the next part.
We had mentioned the role about signaling pattern recognition receptors. And, the ones
that we mainly discussed in the last lecture were toll-like receptors. Now, there are two
main types; there are others too, but the two main signaling pattern recognizing receptors
are the TLRs and the NODs. So, what are the NODs? The NODs are the nucleotide oligomerizing
domain. These are intracellular sensors. If you remember, most of the TLRs - TLR2, TLR4,
etcetera - are present on the surface. Some TLRs are present in endosomes, for example,
TLR9. But, in case of NOD, they are primarily intracellular sensors. So, they sense microbial
entry pathogen within the cells. And, these are characterized by particular domain. So,
NOD proteins contain an N-terminal domain, which contains the CASP domain or the caspase
recruitment domain, which is important apoptosis and activation of NF kappa-B. We will discuss
this important transcription factor, NF kappa-B subsequently.
The middle domain contains the nucleotide binding oligomerizing domain, and hence, the
name NOD, which is important in self oligomerization. The C-terminal domain contains the leucine
rich repeat, which is important for protein-protein interactions. You will remember that TLRs
contain LRR domain, but it is present on the external surface; whereas, in NOD, it is present
in the C-terminal end. The importance of NOD has been shown with its association with Crohn's
disease, which is an inflammatory bowel disease. Now, mutations in NOD2 have been associated
with Crohn's disease, which is an excessive activation of macrophages and T cells in the
bowel. One of the possible reasons for this has been
shown that NOD regulates the TLR2 signaling. So, for example, in TLR2 knockout mice or
mice that lack NOD secrete large amount of IL12 in response to TLR activation. So, it
is possible that NOD displaying the regulator of the TLR activation. And, since the mutations
in NOD are unable to control this, it results in greatly exaggerated activation possibly
resulting in inflammatory bowel disease. There is also a reduction in defensin production,
which are important anti-microbial peptides in NOD2 knockout mice and that may also be
important, because as previously mentioned, that the production of anti-microbial peptides
is important in reducing the number of bacteria that is present in the intestine.
There are other types of pattern recognition receptors and two of the important ones are
endocytic. Now, these do not signal by themselves; they are endocytic, which means they will
bind to complexes and these receptors internalize and clear of these bound ligands. So, one
of the important ones are scavenger receptors. Scavenger receptors are particularly important,
because they bind to modify lipoproteins, for example, LDL, which is important in transport
of cholesterol. So, they also bind to charge polyanionic ligands, bacteria, apoptotic corpses,
etcetera. And, this part is important, because for example, when there is increased cell
death, you want to remove off the dead cells and scavenger receptors may be playing an
important role in these sorts of processes. There are also mannose receptors that are
present on macrophages. And, these recognize high mannose containing proteins, which are
present on surface of microbes and which are then ingested by the macrophages.
We will next be moving on to the complement system. And, the complement system has several
functions. It is important in the control of information. It is most important in the
clearance of immune complexes. So, especially when you have antigen-antibody complexes,
they need to be cleared off and the complement system comes in place over here. They are
important in activation of the antimicrobial defense and we will see parts of that subsequently.
And, it is a major effector of immuno-pathological diseases.
So, there are different ways by which complement can be activated. The most classical and well-studied
is the antigen bound to antibody. So, you have antigen bound to antibody and this activates
complement. And, this is useful in the body and it is also useful for doing in vitro experiments,
where you want to isolate certain population of cells. You have an antibody to a particular
cell type and you can use complement to deplete that particular cell type. So, complement
has a variety of uses. But, in terms of innate immunity, one of the ways that it plays an
important role is to the alternative pathway, where you have the activation of complement
protein (C3b), which binds to certain microbial surfaces, and then, gets activated and remains
activated; and, as a result of which the cascade initiates. The other way by which complement
can be activated is through the mannose binding lectin, which binds to two pathogen surfaces.
And, we will discuss these in slightly greater detail. The important part of the complement
pathway is that the proteins in the system act as an enzyme cascade. So, one protein
gets activated, in turn, activates the other one, and so on until the microbe is lysed.
And, we will see that somewhat later.
What is shown over here is the classical pathway. So, here you have antibodies and these antibodies
have been produced against the microbes. And, these results in what are shown over here
as antigen-antibody complexes. And, these antigen antibody complexes are clumped together
and then complement binds to these, and what it does is, it results in lyses of the microbes.
And, as a result of which, these antigen-antibody complexes and the microbes are lysed.
And, what is shown over here is the cascade as I was talking about. C3b is an opsonin,
which means it enhances the phagocytosis by coating. So, once C3b is coated on microbes,
it enhances phagocytosis. That is the process of opsonization. It also results in activation
of the complement and you can see the cascade leading to the microbial plasma membrane loss
or lyses of microbe. There are other processes involved in here. Complement plays an important
role in inflammation. So, it increases the blood vessel permeability and the chemotactic
attraction during phagocytosis.
And, important disease that is related to this particular pathway is the common opsonic
defect and its relationship with mannose binding lectin, something that we have just discussed
in the previous slide. The mannose binding lectin is the host protein; it binds directly
to mannose, N-acetylglucosamine plus fucose residues, etcetera that are present on microbes,
and directly activates complements. So, this is the third part pathway by which complement
can be activated. What is important is that deficiency in MBP leads to common opsonic
defect, that is, an inability to phagocytose microbes by neutrophils. And, this defect
affects 5 to 6 percent of individuals, which is fairly high and it is commonly detected
in children with recurrent infections. So, how was it discovered? What was found is that
neutrophils from patients, who lack MBL or have mutations in MBL, were unable to phagocytose
yeast, which is saccharomyces cerevisiae, but the defect was reversed when serum from
healthy donors was used in the same assay. So, there was something in the serum that
was missing and subsequently it was identified to be mannose binding elected.
Complement receptors are important over here. Perhaps, the most important one is CR1, which
is present on erythrocytes of CR, stands for complementary receptor. And, it is mainly
responsible for clearance of opsonized immune complexes. CR1 amounts decrease in aged rbcs.
So, as rbcs get old, these amounts decrease. And, they are also low in diseases involving
clearance of antigen-antibody complexes. For example, systemic lupus erythematosus, and
that is again something that will be discussed in the lecture on auto immunity.
CR2 - the complement receptor 2 is present on B cells; and, it allows for enhanced response
to antigens. So, you can imagine a situation with B cells. And, if you have the antigen
with the CR2 complement and bound to antigen-antibody, and is binding to B cells, it is internalized
efficiently. And, this allows for better presentation and activation of B cells. So, CR2 is important
for enhanced B cell responses. CR3, which is shown over here as CD11b and CD18, is present
on neutrophils, NK cells and macrophages. And, it is important for phagocytosis and
destruction of foreign cells. We will see the importance of this particular subunit,
CD18 subsequently.
The other molecules that are important in inflammation are adhesion. During the process
of information, adhesion receptors increase. So, both receptors as well as ligands increase.
And, this adhesion is important especially because neutrophils, macrophages have to leave
the bloods circulatory system and travel into tissues, where the damage has taken place.
So, in order to do that, adhesion plays an important role by which they can go to particular
areas within the part, where tissues are affected. And, adhesion receptors and ligands play a
very important role in this process. One important disease is known as leukocyte-adhesion
deficiency. And, this results due to mutations in CD18, and CD18 is the common beta subunit.
Now, this beta subunit is associated with different types of receptors. For example,
CD18 is important in CR3, which is the complement receptor 3. Here CD18 is associating with
CD11b. And, alternatively, CD18 is also important for LFA, which is an important adhesion receptor.
Here the alpha subunit is different. It is CD11a and which associates with CD18. So,
you can see here that CD18 is common in both these two different types of receptors, but
the alpha subunit is different. So, if you have patients that have mutations in CD18,
what it does is, it affects proper cell surface expression and function of both complement
receptor 3 and LFA molecules.
What happens as a result of this is, it results in defects in adhesion. As a result of which
the cells will not be able to migrate to the affected area. As a result of which diapedesis,
which has this ability to migrate, is affected. And, consequently, patients suffer from recurrent
skin infections, pneumonia, septicemia, gingivitis, which is inflammation of gum, impaired wound
healing, etcetera. So, it shows you clearly, these examples are there to show you the importance
of particular subunits in the innate immune response.
There are other molecules that are important. FC receptors - FC receptors will bind to antigen-antibody
complexes; and, these signal. FC receptors are particularly important, for example, in
signaling during allergies. Then, you have chemokine receptors, which are required for
trafficking to different tissues or sites of inflammation. An important chemokine receptor
is CCR5, which is important for entry of HIV. In the last class, we had talked about CD5
positive B cells present in the peritoneum or live1 B cells in the mouse as the unknown.
And, these are often responsible for production of what is known as natural antibodies. So,
these antibodies are produced in response to different types of microbial pathogens.
So, it is naturally present. And, mice lacking natural antibodies bodies are 10 to 100 fold
more sensitive than the wild type compartments in resisting infections by microbes. So, it
clearly shows you that natural antibodies are also playing an important role in innate
immunity.
An important class of proteins that plays a response in innate immunity is acute phase
response protein. These proteins are produced rapidly in response to information. And, the
liver is responsible for production of several acute phase proteins. An important acute phase
protein is the C-reactive protein. It binds to phosphocholine present on dead or dying
cells and some bacteria in order to activate complement. So, as a consequence of that,
it binds to apoptotic cells in a calcium-dependent manner. So, CRP is important for of dead cells
and it is a way above which you know, once inflammatory reaction is over, you are sort
of down modulating and getting rid of all the debris that is around. It is the way the
body has developed by which dead/dying tissue can be removed efficiently.
Among the soluble factors that are produced during inflammation are cytokines and chemokines.
Some of the important cytokines are IL-1, IL-6, IL-12, TNF. And, in fact, TNF is a marker,
because so rapidly, it is one of the quickest or the fastest produced cytokines during inflammation.
So, it is often thought to be a marker for inflammation. Now, what happens often during
signaling, we are the pattern recognition receptors. One of the downstream consequences
is our production of cytokines, which have a variety of effects. Also important is the
production of chemokines. And, chemokines are especially the example shown, is that
of IL8, which is important in attracting neutrophils during infection. So, the production of IL8
attracts neutrophils to the site of infection.
This slide depicts the main types of chemokines. You have the C-C types shown by MCP, Rantes;
and, the C-X-C shown by IL8, which is a neutrophil attractant. And, as I mentioned to you that
CCR5 is important in playing an important role in HIV infections. So, chemokines have
several different roles. And, at this point, we will not dwell on this further then to
show you that they play a variety of roles.
In cytokines, important cytokines are known as interferons. Interferons originates from
the word interfere; and, interfere, because interferons will discover to interfere with
viral replication. So, in terms of anti-viral immunity, the interferons are known to play
an important role. There are two main types of interferons: type I which is IFN-alpha
beta, IFN-alpha, IFN-beta; or type II, which is interferon gamma. And, the type I interferon,
which is interferon alpha beta is involved primarily in anti-viral immunity. It has other
function, but its main role is very well-known to play an anti-viral role. So, how does the
type I interferon function? There are several mechanisms that are known. One of the important
mechanisms is we are the production of MX GTPases. What these GTPases do is, they inhibit
transcription in one case, but more importantly, they inhibit viral assembly. So, they interfere
with the transport of viral capsids; they also sort them to locations, where they are
not available for assembly. So, the GTPases prevent or slow down a viral assembly.
The other main way is through the production of this enzyme known as 2 5-oligoadenylate
synthetase. What this does, it binds to double-stranded RNA and form this 2 5-oligoadenylates. So,
they adenylate these adenylation and as a result of which, this in turn, activates RNAase
L, which degrades single-stranded RNA. As you will remember, that these are important
during production of viruses and often also for replication of viruses and transcription
viruses. So, this is one way by which it acts in the anti-viral manner.
The other way is, there is phosphorylation of eukaryotic initiation factor-2. As a result
of which, translation is inhibited and viral proteins are not efficiently made. Apart from
their strict anti-viral roles, type I interferons are now shown to be important in other processes
in modulating host immunity. One of which is the in maturation of dendritic cells, generation
of cytotoxic T lymphocytes. And, in some cases, they have been shown to increase the survival
of T cells.
Type I interferons are of use clinically, for example, type I or interferon-alpha along
with the anti-viral drug ribavirin is used to treat liver diseases with chronic hepatitis
B infections. The second case is interferon-beta is used to treat multiple sclerosis. Now,
how does it do? In this particular case, interferon-beta is anti-inflammatory and reduces T cell migration
to affected neurological tissues. Also, it increases the production of anti-inflammatory
cytokines.
The other interferon is the type II interferon: interferon gamma. Now, interferon alpha beta
is produced by all different types of cells; whereas, interferon gamma is produced mainly
by T cells and natural killer cells. And, what interferon gamma does is, it activates
macrophages. And, the way interferon gamma functions is primarily through induction of
expression of several immune genes, for example, transport associated with processing, C II
TA, which is an important transcription factor for MHC class II, Nos2, gp91phox; Nos2 is
important in the production of nitric oxide; gp91phox is important in production of superoxide
radicals. And, these two are one set; we will see a little bit later. Interferon gamma is
a potent inducer of MHC class I and MHC class II expressions. Once the MHC molecules are
increased, the chances of peptides that derived from pathogens are also increased, because
overall, the production of MHC molecules increases. This is especially important during inflammatory
conditions during infections. It is a key cytokine in resisting microbial infections
and it modulates a T helper differentiation. Perhaps, the most important role of interferon
gamma is seen in patients, where inoculation of BCG, which is a live vaccine given to prevent
tuberculosis. And, if this live vaccine is given to children that lack interferon gamma
interferon or its receptor IL12 or IL12 receptor, it results in bacteremia known as BCGosis.
So, one of the primary roles of interferon gamma is in boosting up of immunity against
intercellular pathogens.
There are other types of molecules and we will briefly mention or go over the different
other types of molecules. One of which are the collectins. And, these are calcium-dependent
lectins. What do you mean by lectins? These are sugar binding proteins that recognize
pathogen-associated molecular patterns. And, these are important, because they are involved
in direct opsonization. Opsonization - if you remember, opsinization is the process
by which there is enhanced phagocytosis of opsonized bacteria or microbes; neutralization,
agglutination, complement activation and phagocytosis to curb microbial growth. There are different
collectin members. And, if you remember, the mannose binding lectin is an acute phase protein;
that means it is produced rapidly during the inflammation; and, it is also important for
complement activation, is a member of the collecting family. Two of the member of collectin
family, namely surfactant protein-A and surfactant protein-D are well characterized to play an
important host response in the lungs.
So, these are ways by which these are different proteins that are produced by the host in
order to be able to tackle different types of microbes, because we are constantly under
attack. We have discussed antimicrobial peptides in the previous class and we will again were
some important aspects of it. These represent one of the first line of defense in epithelial
surfaces and this is especially true in the intestine or the gut . And, you will also
remember that TLR and the IMD pathway activation in drosophila results in production of antimicrobial
peptides, for example, drosomycin, which is antifungal, and drosocin and dipterin, which
is antibacterial. TLR activation is thought to increase cytokine production in others,
in mammals, but in case of drosophila, the main role has been shown to be in production
of anti-microbial peptides.
There are different families of antimicrobial peptides. You have defensins, which are small
cationic antimicrobial peptides and defensins are produced by neutrophils, macrophages and
Paneth cells. Paneth cells are present in the intestine and they are potent sources
of defensins. Cathelicidins - they are produced as large precursors, and then, they are trimmed
to produce these antimicrobial peptides; and, they are found in the surface of gastric intestinal
cells. The other one is lysozyme. And, lysozyme as you should know, it is the first enzyme
and the second protein to be crystallized. It hydrolyses N-acetyl glucosamine and N-acetyl
muramic acid bond, which is present in several bacteria. Lysozyme is present in our tear
secretions and other fluids. And, it helps in cleavage of bacteria or lysing of bacteria
in a nonspecific manner.
Once people started working on defensins, what was shown is that these are small peptides
and they are able to insert themselves into microbial membranes and cause their lyses.
What has been shown is that their direct antimicrobial action was well-known. What people are beginning
to appreciate now is that defensins also play a role in host immunity. So, for example,
in anti-viral defensins, they act both on virus as well as on the host cells. They have
been shown to have chemotactic activity for T cells, monocytes, immature dendritic cells,
and they induce cytokine production by monocytes and epithelial cells.
So, this is an example to show you the role of human beta defensins during rhinovirus
infection. So, over here, you have rhinovirus infection. It is affecting this mucosal epithelial
cells and this is produced in response to double-stranded RNA intermediate that is present.
Instead of the double-stranded intermediate, you use poly I:C and activation TLR3. That
also results in the production of human beta defensins 2 and 3. So, essentially that was
shown.
Whatever I said over there is written over here.
But, in case of another viral infection In this case, HIV, the production of human beta
defensins 2 and 3 does not require viral replication. So, you know situations, in which, in some
virus, you need viral replication for production of these defensins; in other virus, you do
not need these. Nevertheless, they play an important role in anti-viral immunity.
As I was saying so, the production of human beta defensin in 2 and 3 in some cases are
the production of an RNA intermediate and in some cases, viral replication; and, whereas,
in other cases, it does not require. Nevertheless, the defensins play an important role in antimicrobial
immunity and this was an example to show you a role of these.
This is a lysozyme. Again, it is highly active against gram-positive species mainly because
of the part that it cleaves the muramil peptide bond, is present primarily in gram-positive.
And, there are other means also; they activate bacterial autolysins. They result in bacterial
aggregation and so on. So, they play important antimicrobial roles.
The other ones are the cathelicidins. Remember, the cathelicidins are produced as larger precursors
and they get trimmed down over here. And, cathelicidins play an important role in the
intestinal lumen and they are present on the surface of gastric intestinal cells.
Lactoferrin is a globular glycoprotein and it is present in secretions of the saliva,
tears, etcetera. It is present in highest amounts in colostrums. So, why is colostrum
so important? Colostrum is important because it is the first milk that babies drink after
birth. And, it is possible that lactoferrin is playing an important antimicrobial role
because it is helping the baby in fighting immunity; because once the baby is born, they
are most susceptible; they do not have immune system of their and they rely a lot in the
mother; their immune response from their mother. So, perhaps, for the initial few months, lactoferrin
may be playing an important role, because it is present in very high amount in colostrums,
which is in the first milk that is produced after birth.
Lactoferrin is also released from neutrophils and respiratory tract epithelium. It has multiple
roles; it is anti-inflammatory, anti-viral, anti-LPS, anti-biofilm. And, biofilm is important
because what happens in some cases, bacteria form from these film-like structures. So,
they form a sort of a colony of their own in different tissues. And, these biofilms
are highly resistant to antimicrobial drugs. So, lactoferrin has this anti-biofilm property,
which is very useful.
This is the list of different viruses that are susceptible to lactoferrin.
And, this slide summarizes the different roles of lactoferrin. So, basically, it has its
fungicidal, which means like for example, shown against candida; it has anti-viral roles;
HIV, CMV shown over. It has anti-inflammatory, because it is anti-LPS. It is also important
for bacterial killing. And, most importantly, it has anti-biofilm properties, which make
it important, because you can see that this is a film-like structure that the microbes
are sort of developing, which makes them very resistant to treatment with antibiotics, and
so on. So, the anti-biofilm property of lactoferrin is very useful.
During the studies on the signal transduction of TLRs, we had shown importantly, the activation
of NF kappa-B. This NF kappa-B and NF kappa-B equivalent in drosophila is the signal transduction
cascade, is almost similar. So, for toll activation and toll-like receptor activation in mammals,
NF kappa-B is playing an important role. So, you can see the conservation of both the receptor,
the signal transaction and especially an important transcription factor like NF kappa-B. And,
it tells you about the conserved signaling and processes during innate immunity in lower
organisms as well as higher organisms, such as mammals. So, NF kappa-B was first described
as a nuclear factor a long time back. At that time, it was shown to be important in transcription
of the immunoglobulin kappa chain in B cells, and hence, the name NF kappa-B. Subsequently,
it has been shown to be important in production of wide variety of molecules, especially those
related to a inflammation. So, for example, in production of cytokines, acute phase proteins,
adhesion proteins, NF kappa-B plays an important role.
What is shown over here is the signal transduction pathway. So, NF kappa-B is usually present
in the cytosol and it is associated with another protein known as inhabited kappa-B, which
is shown over here. So, you have NF kappa B and I kappa B in this complex. Upon signaling,
what happens is that there is ubiquitinization of inhabited kappa-B and degradation of the
inhibitor. So, as a result of which, this degradation is 26 as proteosomal pathway.
And, you have over here, what is shown as the activated NF kappa-B. Once NF kappa-B
is activated, it can now go from the cytosol into the nucleus, where you can bind to its
particular cognate binding sites in front of promoters and turn off transcription of
several genes. And, what is shown over here is apoptotic factors, cytokine, cell cycle
regulators, so on and so forth. So, this pathway of activation of NF kappa-B is important.
And, it is very important for students to understand this particular pathway. Remember,
the NF kappa-B activation pathway is again conserved between drosophila and mammals.
And, the signals are also conserved for this activation, which is the toll and the IMD
pathway resulting in activation of drosophila NF kappa-B; whereas, in mammals, it is the
TLR of the NF kappa-B pathway. And, it results in a wide variety of responses; a very important
concept for students.
So, this is shown over here; NF kappa-B results in transcriptional activation. And, you have
synthesis, are now of several immune related molecules. You have reactive oxygen intermediates,
antimicrobial peptides, cytokines, chemokines, adhesion molecules, acute phase protein and
so on. This is just a partial list, but NF kappa-B is a super transcription factor. It
turns on several different molecules.
One of the ones that was shown over there was the induction of reactivate oxygen species.
And, what I will do here is to discuss the importance of reactive oxygen and nitrogen
species in innate immunity. Over here, ROI and RNI play a very important role in innate
immunity. And, the cells that are mainly responsible for these are the neutrophils, which rely
mainly on the reactive oxygen intermediates and macrophages, which rely mainly on the
reactive nitrogen intermediates. However, the two pathways can converge together
to form a potent oxidant, which is peroxinitrite; and, I will go over this a little bit slowly.
So, you know, you have oxygen, which is converted by NADPH oxidase. Now, NADPH oxidase needs
to be assembled in the membrane upon activation. Once it is assembled, it can form superoxide.
And, the superoxide is highly potent; it can be converted into hydrogen peroxide or it
can combine together with nitric oxide to form peroxinitrite, which is potent oxidant.
And then, the hydrogen peroxides can go on to form hydroxyl radicals and these will be
part of the reactive oxygen intermediates. The other ones are the reactive nitrogen intermediates.
And here, for the production of nitric oxide, arginine is the nitrogen donor; it combines
over here. And, you have nitric oxide synthase, which is responsible for the production of
nitric oxide. Now, in the body, there are three main types of nitric oxide synthase.
You have the e NOS, which is the endothelial NOS; then neuronal NOS (n NOS); and then,
you have the inducible NOS. For the purpose of this class and especially
with respect to the immune system, what we are referring to, is the inducible or immunological
one or the i NOS or commonly known as NOS2, which is shown over here. Now, what happens
with nitric oxide is, it can combine as shown over here with superoxide to form a peroxinitrite,
which is a extreme potent oxidant. But, nitric oxide on its own has a lot of roles too. The
main one that was initially shown was activation of guanylate cyclase resulting in the production
of cyclic GMP; and, this cyclic GMP will have its own roles. And, the activation of guanylate
cyclase is because of nitrosylation of heme, which is present in the active side of this
particular enzyme. The other way by which the nitric oxide function is by S-nitrosylation
of proteins; so, SNO, which is cysteine and then SNO. So, that is how it nitrosylates
different proteins, for example, hemoglobin, glutathione, Ras signaling proteins, so on
and so forth. So, this is an important way by which the reactive nitrogen intermediates
and the reactive oxygen intermediates play an important role.
And, remember, these are potent molecules and they would kill different microbes that
are ingested. And often, the cells also themselves get killed, and which are then subsequently
cleared off by the process of phagocytosis. So, the cells sort of not only kill microbes,
but they might get themselves killed, because of production of these radicals. And then,
ultimately, the body is saved, because of the fact that the microbes are killed. And
then, the debris and all are cleared off later.
And, important molecules in the inflammatory process are prostaglandins. And, they are
derived from fatty acids; and, arachidonic acid is an important one and they play important
physiological roles, for example, pain, fever, inflammation, so on. The enzyme that is responsible
for production of prostaglandins is cyclooxygenase - you have COX1 and COX2; and, which are present
in blood vessels, stomach, kidneys. COX1 is responsible for the basal production of prostaglandins.
And, the increased production due to stimulation is done by COX2. The receptors for prostaglandins
are cell surface G-coupled receptors. Now, the importance of the prostaglandin comes
into focus by the use of non-steroidal anti-inflammatory drugs, which several of us take whenever we
have head ache, pains; we take Aspirin, Ibuprofen, Naproxen, so on. These are belonged to the
category of NSAIDs. And, the way it functions is that they are inhibitors of COX. Now, Aspirin
is particularly important, because not only it is an inhibitor of COX1, it also reduces
platelet aggregation as shown. And, this reduces adverse cardiovascular events. And, for heart
patients, often the doctors prescribe Aspirin. And, now, you know, what the mechanism of
action is, because it reduces platelet aggregation and reduces the risk of adverse cardiovascular
events. So, Aspirin has two roles: one is COX1 inhibitor and also reduction of platelet
aggregation.
Now, once you have the inflammatory process, anything that goes up needs to come down,
because remember, if the activation keeps on in sustained manner, it results in problems
to the cells and to the host. Sepsis is a good example of that, where you have so much
activation of immune cells that it becomes very difficult to control the process, and
ultimately, it leads to multiorgan failure and death. So, in the immune response, whatever
goes up, if the immune response is induced, it needs to have some regulatory mechanisms
by which it can be reduced. And, this brings to us an important aspect, which is downmodulation
of inflammation or downmodulation of the inflammatory response. So, with inflammation, anti-inflammatory
processes are also induced. For example, anti-inflammatory cytokines have TGF-beta, IL-4, IL-10. These
are well-known anti-inflammatory cytokines. Some hormones also play an important role,
for example, glucocorticoids. And, these are produced by the adrenal glands and they are
anti-inflammatory; they are useful in case of allergies, asthma and so on. In case of
heightened immune reaction as observed in case of autoimmune disease, sometimes, glucocorticoids
are also prescribed. The other way - once you have debris, they need to be cleared off,
is apoptosis. So, macrophages are often responsible for recognizing apoptotic cells, because of
the expression of eat me flags. So, these cells that are undergoing apoptosis, express
certain cells of those receptors. These are recognized and then they are phagocytose by
macrophages. One of the eat me flags is phosphatidylserine, which is usually present inside; but, in the
apoptotic cells, it is present on the cell surface. And, this can be recognized using
Annexin V, which binds to phosphatidylserine. Now, this particular assay is useful in detection
of apoptotic cells. So, if you wish to look at cells undergoing apoptosis, often you can
look at the surface expression of Annexin V, which is a marker for apoptotic cells.
Now, in terms of TLR induced responses, again there are different mechanisms in place to
reduce the TLR activation, because microbial infection is often associated with TLR responses.
So, initially I had shown you the role of NOD2 in regulation of TLR responses. And,
it is possible that NOD2 mutations and may explain as to how NOD2 mutations lead to Crohn's
disease. Now, another molecule shown, tollip; now, tollip was shown up in my previous classes
lecture slides on TLR, but I did not explain it, because there was not sufficient time
to explain each and every molecule. But, tollip inhibits IRAK and this IRAK activity is associated
with TLRs. So, you have IRAK activities associated with TLRs; you have a regulator of this particular
activity. So, you can reduce TLR activation. Non-pathogenic bacteria also reduce inflammation,
and in fact, the block inflammation induced by pathogenic bacteria. You will remember
that in our gut, we have lots of non-pathogenic bacteria. And, there are different mechanisms
in the gut to take care of these huge load of bacteria that are living in the gut.
The peroxisome proliferator activated receptor PPAR gamma - now, peroxisome proliferator
activated receptors are nuclear transcription factors, which get activated and they lead
to wide variety of responses. In this particular case, peroxisome proliferator activated receptor,
which results in increased production of peroxisome; and, that is why, the name peroxisome proliferator
activated receptor. And, these might reduce inflammation; these have some role in inflammation.
And, by increasing nuclear export of the RelA subunits of NF kappa-B. So, NF kappa-B goes
into the nucleus once it is activated. And, what this could be going is that it is exporting
this NF kappa-B back into the cytosol. And, by increasing the export of this, you are
reducing activation. So, there are different mechanisms in place by which downmodulation
of responses can occur. The other example is shown by the A20 ubiquitin ligase, which
downmodulates TLR dependent responses. The other molecule, which is well-studied, is
a SOCS-1, which is a suppressor of cytokine signaling. It is an inhibitor of the JAK STAT
pathway, which is important in signaling we are the interferons.
One of the examples of downmodulation of inflammation is the example that is shown over here, is
the expression of SIRP alpha, which is an inhibitory tyrosine motif containing receptor,
on macrophages. Now, usually, binding of this to CD47 results in downmodulation of phagocytosis.
However, as rbcs age, they downregulate CD45 and these aged rbcs are rapidly phagocytosed
by macrophages. So, this is an example again of phagocytosis; about molecules that play
an important role in phagocytosis; and, how you can downmodulate the responses.
I will briefly now talk about the relationship between the innate and the adaptive immune
system. The NK cells produce interferon gamma. These activate macrophages. And, once macrophages
get activated, they will present and process more efficiently to T cells, and consequently,
they will modify your T cell responses, which may be of Th1, Th2. And so, you can see a
role, where NK cells, which play a role in innate immunity, are also modifying the adaptive
immune response. The second one is dendritic cells. Some dendritic cells produce large
amounts of type I interferons in response to viral infections. And, these have been
shown to play not only an important role in reducing viral replication, but they also
result in increased number of viral-specific T cells. So, again you it is a case by which
a classical innate cell is modulating the adaptive response.
A fine example of immunity or this interrelationship between adaptive and innate is seen in the
gut, because in the gut, you have huge numbers of bacteria in there. And, the gut plays an
important role, because you have this gut-associated lymphoid tissue. And, the upper bowel has
lesser number of bacteria, but it reacts to dietary antigens; whereas, the lower ones,
in addition to the dietary antigens, you have huge commensal organism living there.
Over here in the intestine, you have specialized patches of organized lymphoid tissue known
as Peyer's patches. And, among the important cells, which are responsible for uptake of
antigens and then giving it to the dendritic cells and the other cells in the lymphoid
tissue, are known as M cells. And, these are epithelial cells, but which play an important
role in this process. The IgA that is present in large numbers in the gut lumen is also
an important role. In fact, mice that cannot produce IgA have much higher numbers of gut
bacteria. So, the gut immunity or the gut process is very important because of the fine
interaction between innate and adaptive arms.
I will also briefly discuss how plants and innate immunity functions. So, what about
plants? We have been discussing animals so far. So, before ending, I will just briefly
mention the different mechanisms by which the plants also have it. So, plants are also
susceptible to viruses, bacterial pathogens, so on. So, how do they deal with this? First,
is by the production of antimicrobial products, for example, phytoalexins, salicylates, antimicrobial
peptides, enzymes, chitinases. So, they also can shutdown transcriptions and they do so
by the dicer pathway. So, it will turn transcription down.
Then, you have what is known as resistance loci or the R loci, which allows recognition
of specific effectors. Several of the R loci proteins contain LRRs, which are the leucine-rich
repeat domains in their proteins, and which is an important mechanism. So, you can see
that the R factors and the LRRs and the relationships, there is some commonality that one can see
over here. An important response by which plants take
care is through the production of what is known as the hypersensitive response - which
is, upon infection, the cells around that particular infected tissue die off. And, how
they die off is not clear, but it is possible that plants are known to contain caspase related
proteases and whether these are playing an important role in our needs for the studies.
But, the hypersensitive response is clearly an important way by which plants handle the
infection by pathogens.
I will now briefly summaries and just go over some of the main aspects that we have covered
today. First is that what we have tried to do is, study different aspects of the innate
response. You have the complement activation. We have looked at complement activation. There
are different pathways. There are three different pathways: the classical, the alternate and
the lectin pathway by which they can be activated. And, you have receptors that are involved;
you have the TLRs; you have the NODs; you have the complement receptors; you have the
adhesion receptor. We also studied the important roles of interferons
especially the type I, type II interferons. IL8, which is an important chemokine, which
is an important neutrophil attractant; CCR5, which is important for HIV infections. Effectors
- the reactive oxygen intermediates, the reactive nitrogen intermediates; here phox NADPH oxides
is important for it. Reactive nitrogen intermediate - NOS2 is the key enzyme that is responsible
for the production of nitric oxide. We also looked at downmodulation of innate
responses. The production of anti-inflammatory cytokines like TGF beta, IL-10, IL-4; and,
you have further ways by which the production of a glucocorticoids, so that you can suppress
immunity. You have different proteins for example, that will suppress immunity, for
example, A20 ubiquitin ligase; the toll up, which inhibitor; or, SOCS-1, which is an inhibitor
of the JAK STAT signaling pathway, which is important by which interferons signal. We
also studied the link between the innate and the adaptive immunity; especially the role
of NK cells in producing interferon gamma and modulating T cell responses. Some dendritic
cells produce high amounts of type I interferons and which in turn affects the anti-viral activity
and the production of anti-viral T cells. Now, most importantly I would like to conclude
with a study of four diseases; sort of review of these. NOD2 is an intercellular censor.
It has been shown to play a role in... or it is associated with the Crohn's disease,
which is an inflammatory bowel disease. So, mutations are not to result in increased inflammation
of the bowel. It is possible that NOD2 is playing an important regulator of the T cell
responses. CD18 is the common beta subunit. It is important for adhesion, leukocyte adhesion
deficiency. Then, you have mannose binding lectin; common opsonic defect, which is quiet
common and it is important in opsonization and complement activation; phox, which is
NADPH oxidase and the production and its association with chronic granulomatous disease. So, if
you do not have a NADPH oxidase or you have mutations in NADPH oxidase, which are not
functional, you have chronic granulomatous disease, which results in recurrent bacterial
infections. Overall, what these two lectures - innate
immunity, have shed light on the cells, the mechanisms by which our innate cells play
an important role. So, they not only act as the border security force, but they are important
in modulating the adaptive immune response. Thank you.