1. Innate Immunity Guest Lecture: Joel Wertheim

2. Outline General Function Main Cellular Players Toll-like Receptors
Interferon
Complement System (3)
APOBEC3G

4. Innate vs. Adaptive Timeframe
Can take up to a week for a full adaptive immune response to kick in.
Innate immunity can begin acting immediately.
The factors involved in innate immunity do not all dissappear after 4 hours, some stay around a work with the adaptive branch.

5. Importance of Innate Immunity
Lacking adaptive immunity results in a slight increase in pathogen load and a substantial increase in the length of infection
Lacking innate immunity results in uncontrolled infection
Are people more likely to have genetic deficiencies for innate or adaptive immunity? Why?

6. Barriers to Infection Gram-negative bacteria on skin
Human skin pH is between 4 and 6
Infections that occur through the skin often due to wounds/abrasions/burns
In Drosophila, cells in the digestive and genital tracts also produce anti-microbial peptides.
Infectious disease is more common in the very young and very old.
This is because the normal flora is different in an infant. Do not feed infants honey b/c of Clostridium botulinum. They don’t have the normal flora to outcompete it.
Same reason why they get certain types of diarrhea
In patients with AIDS, microorganisms normally found on/in your body can become lethal such as Candida species of yeast, which can cause thrush.
Brock Biology of Microorganisms 10th ed.

7. Microenvironments
Bacteria colonize your body and modify their environment to prevent colonization by other microbes.
Change in pH (skin, genital tract, etc.)
Anaerobic bacteria in your mouth (hence plaque)
Niche-creation
Nancy Moran will talk in much more detail about the interactions between bacteria and hosts.
Ignoring the evolution of resistance, why might unnecessary antibiotic treatments be a bad thing?

8. A Few Cellular Components of Innate Immunity
Neutrophils
Phagocytic, short-lived
Macrophages
APC, long-lived, stimulate innate and adaptive immune responses
NK (Natural Killer) Cells
derived from same lineage as B and T cells
Neutrophils are responsible for pus at the site of infection
All of these are WBC

9. Neutrophils Variable number and shape of nucleus
Not found in healthy tissue
Signalled by macrophages to come to infected site and there become dominant phagocyte
Pathogen Associated Molecular Patterns recognized by neutrophils:
Mannose
LPS (Lipopolysaccharide)
Flagellin
Short lived, only a few hours.
They engulf pathogens and immediately dump a slew of toxis substances on it.
They release internal granules with degrative enzymes;
however, once they use all of these, they cannot make more and die.
They are responsible for pus associated with infection.

10. Macrophages Are covered in surface receptors that recognize PAMPs
Important APCs that coordinate innate and adaptive immune response
Release cytokines to stimulate other cells
Called macrophages because they are bigger than neutrophils (which were once known as microphages) and because they phagocytize pathogens.
They are longer lived than neutrophils and found in most tissues in the body.
Illustrates that bacteria is recognized by the LPS receptor (TLR-4).
Could also recognize something like mannose, a sugar found on bacterial cells (but not host cells), a PAMP.
Bacteria are engulfed and processed to be presented by MHC-II to T-helper cells.
Also important in signaling the adaptive immune system.

11. Bactericidal Agents Produced by Phagocytes
Produced by both macrophages and neutrophils
Mycobacterium tuberculosis prevents fusion of lysosome with the vesicle (in the macrophage) in which it is contained and live in the vesicle system.
Therefore avoiding these chemicals.
Other bacteria escape the vesicle all together and live in the macrophage cytosol. (Listeria monocytogenes). Once in cytosol, CD8 T-cells can find it.
Iron Competition (we will get into this later in the course)

12. TNF stands for Tumor Necrosis Factor
Cytokine = proteins made by cells that affect the behavior of other cells. Bind to a specific receptor on the target cell.

13. Sepsis Results from a loss of blood pressure and vascular integrity
Death occurs from organ failure
An overreaction of the immune system, a prime example of responses to pathogens that can kill the host
Maladaptation.
Caused by systemic bacterial infection (most commonly a gram-negative with LPS to stimulate TNF-alpha) or other viral pathogens.
Dengue hemmorhagic fever can result in septic shock. As can Gram-Negative Bacteria, Ebola, Crimean-Congo, and Rift Valley Fever.
Leading cause of death of critically ill patients in the US.
Associated with response from Gram-negative bacteria LPS, often resulting from a systemic infection
TNF-alpha and other cytokines induce a “cross-talk” cascade
Originally thought to be a random cascade of uncontrolled signalling.
Led to failed treatments trying to down regulate individual aspects (increased mortality).
Now thought of as a highly regulated response that In VHF, may be induced by lymphocyte apoptosis which may fill the body with signals for more signalling
Also results in anergy of T-cells (over stimulation), commonly seen in VHF…leads to immunosuppression.

14. Mammalian TLRs Different Toll-like receptors bind to various PAMPs.
There are ten total TLR genes expressed on human macrophages.
Why a TLR for double stranded RNA?
Even single-stranded RNA viruses are double stranded when they transcribe their genetic material.
Unmethylated CpG motifs are 20-fold more common in bacterial DNA than in vertebrate DNA.

15. Interferon Cytokine that induces an anti-viral state in other cells
Stimulated by TLR-3, which binds to dsRNA
Also degrades intracellular RNA and increase protease activity
Not induced directly by viral infection.
Interferron does very little to save an infected cell, rather it signals neighboring cells to protect themselves
A very complicated pathway that can involve the upregulation ~100 genes
Can cause 5’ cap snatching, which prevents some RNA viruses from replicating.
Can increase MHC-II expression on surrounding APCs.

16. lipoteichoic acid is another example of a PAMP
As is LPS and peptidoglycan

17. TLR Toll TNFR Imd
They are called Toll-like receptors in humans because they are like Toll Receptors in Drosophila.
Toll induces antifungal and antibacterial responses.
Imd stands for “immune deficiency” because knockouts are highly susceptible to gram-negative bacterial infections.
TNFR = TNF-alpha receptor (same cytokine that can lead to sepsis)
Imd induces transcription of genes encoding antimicrobial peptides.

19. Toll and TLR
Toll is stimulated by a host-protein that is cleaved after encountering a pathogen
TLRs are stimulated by direct pathogen-receptor interaction
Homology = common ancestor/descent
Spaetzle is cleaved and this product initiates the toll cascade.
Toll acts on gram-positive bacteria and fungal pathogens

20. Imd and TNFR Homologous proteins exist in both pathways.
Both can result in apoptosis
Imd pathway deals with Gram negative bacteria

21. Natural Killer Cells
Some viruses downregulate MHC-1 expression on infected cells
NK cells induce apoptosis in cells missing MHC-1
Derived from same lineage as B and T cells and are techinically lymphocytes (although not involved in adaptive immunity).
They can also recognize other changes in cell-surface proteins, such as glycoproteins.
Why is it advantageous for viruses to downregulate MHC-1 expression?
Some cancer cell lines also downregulate MHC expression, which results in their killing by NK cells.

22. Complement System
Secreted as inactive enzymes known as zymogens (enzymes that must be modified in order to be active)
Plasma proteins that attack extra-cellular pathogens
Being coated in Complement can result in:
Phagocytosis
MAC (Membrane-Attack Complex)
Found in blood and lymph
Synthesized in the liver
Loss of phagocytosis stimulating complement leads to increased infection by bacteria
Loss of MAC associated complement leads to increased Neisseria infection (gonorrhoea/meningitis).
This is a highly regulated system with many “fail-safe” mechanisms to prevent killing of host cells, but these will not be discussed in detail here.
But trust me, they exist.

23. Complement Pathways
Three different pathways that all lead to the same result.
Classical was discovered first, but we will discuss it last because it is not strictly part of the innate immune system and it is the most complicated.
Please don’t memorize all of the zymogens, enzymes, and protein factors involved.
All you need is the geshtalt.

24. The Alternative Pathway
Complement proteins bind to pathogen surfaces, which are unable to repel the attack.
This coating with C3b signals macrophages and neutrophils to phagocytize the pathogen.
Host cells have regulatory proteins to prevent this cascade.
Named alternative, but is probably the most ancient pathway
Give brief overview of pathway:
Spontaneous binding of C3 to form C3 convertase, deposits C3 on bacterial/viral surfaces.
Leads to phagocytosis or destruction of pathogen membrane.

25. C3b binds to pathogen surfaces
Spontaneous formation of C3 Convertase, which converts C3 into C3a and C3b
C3b binds to pathogen surfaces
C3a is cleaved and mediates inflammation
C3a is an anaphylatoxin.
Leads to recruitment of complement, antibodies (if they have been produced), and other immune system cells.
Why is inflammation good?

26. Results from release of histamines and other vasoactive substances that increase membrane permeability.

27. On Pathogens, C3 forms a stable C3 Convertase
On Host Cells, a whole slew of other enzymes join the party

28. Host Pathogen Host: Innactivation of completment cascade
Pathogen: C3b all over which allows of phagocytes to eat em.
C3b binds to a specific receptor on macrophages.

29. Membrane-Attack Complex
Although C3b can act to signal phagocytes, it can also initiate another cascade that causes the MAC to form on bacterial cell walls by stimulating the binding and cleavage of other complement proteins.
Can also be effective against eukaryotes and enveloped viruses.
MAC leads to the loss of membrane integrity and destruction of the proton gradient.
The details of the cascade are not important, but its outcome is.
However, the MAC is not as impoprtant as phagocytosis as its loss only results in increased Neisseria infection

30. Mannose-Binding Lectin Pathway
Mannose on bacterial cells stimulates MB-lectin to deposit C3b on pathogen which forms a C3 Covertase.
Mannose is commonly found on bacterial cell surfaces.
Not found on host cells
MASP-1 = mannan-binding lectin serine peptidase 1

31. Classical Complement Pathway
Two different forms of antibody are both good at stimulating complement binding. IgG is the Ig you are most familiar with.
This is NOT part of the innate immune system, but it involves similar pathways and many of the same mechanisms.

32. Once complement (C1s) binds to antibodies, it stimulates a cascade to build C3 Convertase which coats the pathogen in C3b.
This results in phagocytosis and/or MAC formation

33. Also found in Sea Urchins.
Which complement systems are likely the most ancient? Why?
Drosophila also has a complement-like cascade

34. Evolved Viral Responses to the Complement System
Mechanisms to block and degrade C3 Convertase formation and depositing of C3b.
Also mechainisms to downregulated the anaphylatoxins.
In addition, viruses have methods of mimicing complement regulators and preventing the binding of complement to their surfaces.
Retroviruses and Togaviruses (+RNA) also have resistance. HIV encorporates complement regulators in its membrane upon cell release.
Not as involved a mechanism, and doesn’t require much additional genetic material.
Favoreel et al. (2003) J. Gen. Virol
Why are most of the viruses that have evolved resistance in the Poxviridae, Herpesviridae, and Coronaviridae familes?

35. And now for something completely different…

36. Hypermutation as a Defense Against Retroviruses
Hypermutation in B-cells is caused mainly by AID (Activation-Induced Cytidine Deaminase)
Causes all sorts of mutations during Affinity Maturation, mainly C to T
HIV genomes have been found with increases in G to A mutations
Walk through Affinity Maturation.
Assuming a common mechanism, why C to T in mammals and G to A in HIV?

37. APOlipoprotein B mRNA-editing Enzyme-Catalytic polypeptide-like 3G
A gene coding for a protein closely related to AID, APOBEC3G, has the ability to hypermutate retroviruses.
Prevents initiation of infection.
Why could this be a very good thing?
Natural Selection = an already existing function that is co-opted for a novel use
(somatic hypermutation to antiretroviral funtion)
The protein responsible for the hypermutation is APOBEC3G.
Expressed in many cells in the human body.
Retroviruses include HIV/SIV/Hepatitis B.
Drug design possibilities.
Unlike in B-cells, hypermutation is bad because it essentially increases the mutation rate (drastically), which increases the chance that each genome produced will be less fit than its parent. Multiple changes in each protein mean that virus will likely not survive a round of replication in the presence of APOBEC.
Therefore prevents infection from initiating.

38. APOBEC3G Action in HIV
If vif is not present, or not effective, APOBEC3G will be incorporated into the budding virus.
When the virus infects a new cell and undergoes reverse-transcription, APOBEC3G will deaminate the new DNA strand.
Just to clarify: HIV is a positive-stranded RNA virus. For those who got a free point on the exam, you’re welcome.
Vif is an accessory protein found in human and simian immunodeficiceny viruses.
Figure 1. Innate immunity mediated by APOBEC3G.Expression of APOBEC3G in the virus-producing cell (left) can lead to abortive retroviral infection of the target cell (right). APOBEC3G is incorporated into progeny virions as they assemble and bud5. After entry of the viral core into the target cell, the viral RNA genome (red) is reverse transcribed into minus-strand cDNA (light blue). APOBEC3G triggers dCdU deamination of the minus-strand cDNA (probably some time after degradation of the plus strand RNA by RNase H; not shown). The dU residues in the first-strand cDNA may then lead to excessive hypermutation capable of inactivating viral functions (left); degradation mediated by base-excision-repair enzymes (middle); or misreplication of the second cDNA strand. The assignment of target cell events to the cytoplasm or nucleus has been intentionally avoided, as this does not impinge on the basic aspects of this innate defense strategy. HIV Vif can counteract APOBEC3G-mediated immunity, but how this occurs is not yet known.

39. Cytidine Deamination of a Retrovirus
A C G U A C G U A C G U
T G C A T G C A T G C A
A C G T A C G T A C G T
RNA (+)
cDNA (-)
cDNA (+)
A C G U A C G U A C G U
T G T A T G C A T G T A
A C A T A C G T A C A T
The exact antiretroviral mechanism is unknown. Clearly hypermutation can result in defective virus particles.
However, APOBEC still seems capable of disabling retroviruses even in the absence of cytidine deamination.
Possibly two distinct mechanisms exist to combat retroviruses.
Normal Reverse Transcription
APOBEC3G Hypermutation

40. Evolution of AID Gene Family
Genes are very ancient (found in Xenopus & Fugu)
Massive expansion of gene family in primates, especially human lineage
Even though the phylogeny shows an increase in human genes, other closely related species aren’t included that are now known to possess homologs of most all of these genes.
Many other genes closely related to APOBEC3G have antiretroviral functions.
FIG. 2.— Phylogenetic relationships within the AID/APOBEC family. Neighbor-joining tree generated from a DNA alignment of the exon encoding the presumed zinc-coordinating motif. The different clusters in the AID/APOBEC family are identifed with the APOBEC3 cluster, further divided into Z1a, Z1b, and Z2 domains. The N-terminal and C-terminal domains in the double-domained APOBEC3 sequences are labeled [N] and [C], respectively. The labels at the end of each branch indicate the organism from which the sequence derives (Bos taurus, Bt; Danio rerio, Dr; Equus caballus, Ec; Gallus gallus, Gg; Homo sapiens, Hs; Monodelphis domestica, Md; Mus musculus, Mm; Oryctolagus cuniculus, Oc; Oryzias latipes, Ol ; Rattus norvegicus, Rn; Sus scrofa, Ss; Takifugu rubripes, Tr; and Xenopus laevis, Xl). Bootstrap values greater than 50 are shown. Phylogenetic trees with a similar topology were generated using sequences from other exons.
Conticello, S. G. et al. Mol Biol Evol :

41. Evolution of AID Gene Family
Gene duplication
Genomic movement
3A, 3F, and 3G all have documented antiretroviral function
This is just to give you an idea of what the gene family looks like.
Now, even 3H has been identified.
Figure 1. The Primate APOBEC Family(A) The human genome contains nine known members of the APOBEC family. AID and APOBEC1 are located approximately 900 kb apart on human Chromosome 12. The primate-specific APOBEC3 cluster of six genes resides on human Chromosome 22, and likely arose through a series of gene duplication events (Jarmuz et al. 2002; Wedekind et al. 2003). The single APOBEC3-like gene found in mouse resides on Chromosome 15 (not shown), which is syntenic to human Chromosome 22 (Sheehy et al. 2002). There is EST evidence for both APOBEC3D and APOBEC3DE (see Materials and Methods), and we treat these as three separate transcripts in our analysis because currently there is no evidence for the relevant protein products.(B) All members of the APOBEC family contain an active site that encodes a zinc-dependent cytidine deaminase domain with the HXE, PCXXC signature (Mian et al. 1998), a linker peptide, and a pseudoactive domain (Navaratnam et al. 1998; Jarmuz et al. 2002). The active and pseudoactive domains are related by structure only, and likely originated from a gene duplication event followed by degeneration of the catalytic activity of the pseudoactive domain. Several members of the human APOBEC3 gene cluster (APOBEC3B, 3DE, 3F, and3G) have undergone an additional duplication/recombination event and now contain two each of the active and pseudoactive sites (Jarmuz et al. 2002; Wedekind et al. 2003), as does the single APOBEC3-like gene found in mouse.
Sawyer et al. (2004) PLOS Biol e275

42. Controlling lentiviruses: Single amino acid changes can determine specificity
Species-specific APOBEC3G blocks infection with virus from other species
Not even have the chance to evolve in the new host
Fig. 1. Host-specific interactions of Apobec3G with viral Vifs.
Human Apobec3G encodes aspartate (D) at amino acid 128 and functionally interacts with HIV-1 Vif but not with SIVagm Vif, whereas AGM Apobec3G encodes a lysine (K) at amino acid 128 and functionally interacts with SIVagm Vif but not with HIV-1 Vif.
The functional interactions can be reversed by mutating human Apobec3G to K at amino acid 128 and by mutating AGM Apobec3G to D at amino acid 128. Interactions are denoted by + or -.
A positive interaction indicates that Apobec3G will be sensitive to Vif, and a negative interaction indicates that it will be resistant.
Kaiser, Shari M. and Emerman, Michael (2004) Proc. Natl. Acad. Sci. USA 101,

43. Phylogeny of HIV and SIV
Many SIVs, although humans have only acquired it from two species (albeit on multiple occasions from each).
Lots of cross-species transmission among monkeys (but not always).
Also, we are interested in the factors that influence the spread of SIV from monkey to monkey, as well as from monkey to human.
Gordon et al. (2005) LANL HIV Database Review Articles

44. Positive Selection on APOBEC3G
Natural selection favors rapid change in protein sequence
Leads to rapidly divergent genes between species
Happened in almost every OWM and Ape lineage
Brief description of positive selection.
What could account for this positive-selection for amino-acid change in APOBEC3G and for the diversification of the AID family as a whole?
“Our own genomes reveal the remarkable effects of retrotransposition, as about 45% of our genomic DNA results directly from this process (Lander et al. 2001).”
Sawyer et al. (2004) PLOS Biol e275