HIV and DCs + Autophagy

Question 1

HIV prevalence

Answer 1

HIV was first seen in Africa probably in 1920s, but it took 1980s to see the cases of HIV for real, and people hoped to have a vaccine in couple of years, we still don’t have a vaccine. There’s a cured patient –completely HIV free- seen with bone marrow transplant from CCR5 mutated donor in 2011, he died a while ago because he had a cancer relapse.
HIV is a huge problem in South Africa especially, and new cases keep increasing in world.  
Antiretroviral therapy (ART) stopped a lot of deaths. But a lot of people still dies.

Question 2

How does HIV transmit?

Answer 2

Sexual intercourse is main route of infection, involves mucosal tissues in genital tract and it transmits through mucosa.
The chances of getting HIV infection while having sexual contact with an infected person is at best %1.
Enhanced when there’s inflammation or genital coinfections.

If the main route of infection is sexual contact, there are no T cells in mucosal tissues! How does T cells get infected after all if they aren’t on the primary site of infection? = DCs might be the answer, since there’s a lot of DCs in mucosal tissue.
So when there’s DCs on mucosa, they capture the pathogen and bring it to draining LN, and there’s T cells there. So it might be same mechanism for HIV.

Question 3

How DCs induce adaptive immunity?

Answer 3

At immature stage, DCs can sense pathogens very good and capture them, they reside in almost all tissues including mucosal tissues. Upon interaction you get danger signal with PRRs. Then DCs get a genetic reprogramming (maturation) and change shape, and now instead of sensing, they can activate naïve T cells. So they migrate to LNs, and in LNs they instruct naïve T cells to become CD4 or CD8 T cells.

Question 4

HIV structure

Answer 4

EM picture on left. Has a nuclear capsid, (protein p24) in middle there’s RNA. And it has RT to translate into DNA and integrate. Envelope glycoprotein is critical, gp120 + gp41. = heavily glycosylated.

Gp120 is like the spike protein on SARS, main recognition molecule, and it binds to CD4 on T cell, and with co-receptor that can be CCR5 or CXCR4 or even both in different types of HIV. R5 TROPIC / X4 TROPIC / DUAL TROPIC on some cells
. T cell recognizes the HIV with CD4-CCR5/CXCR4 always.
In DCs, gp120 binds a completely different receptor.

gp120 is covered with carbohydrates called mannoses: so its covered with mannosylated glycans. This is also why immune system has a trouble with making Abs against it, almost all protein is covered with sugars, protein is inaccessible.

Question 5

Study theory of DCs bringing HIV to T cells

1) Binding of HIV to DCs

Answer 5

Gp120 binds to DCs, it might also bind to CD4 on DCs, but when you block it, there’s no change, so it doesn’t; and it doesn’t bind to CCR5 either.
But when you block DC-SIGN, interaction of HIV with DCs are blocked.
So HIV binds DC-SIGN on DCs.
DC-SIGN binds to carbohydrates. Gp120 is covered with carbohydrates too

Question 6

DC-SIGN

Answer 6

DC SIGN is found as tetramer on the surface, it has neck region (transmembrane) and a cytoplasmic domain for signaling. On top, there’s carbohydrate recognition domain that binds glycans.
Lewis X etc. is recognised, mannose.
DC-SIGN binds to other pathogens too.

Question 7

Study theory of DCs bringing HIV to T cells

2) What happens when HIV binds to DC-SIGN? + infectious synapse

Answer 7

DC-SIGN can internalize HIV. If you give DC SIGN any carbohydrate ligand, it’s targeted directly to the lysosomes. = So any carbohydrate is leaded to degradation.

HIV is in between T cells and DC. Exactly in the place of synapse = infectious synapse. Virus accumulates in synapse, then transferred to the T cells!

Question 8

What happens when HepC binds to DC sign? Random info

Answer 8

HepC isn’t taken to lysosomes? Where does it go? It goes to early endosomes. =then it escapes.

Question 9

Study theory of DCs bringing HIV to T cells

3) Study DC-SIGN + HIV + T cell interaction synapses: + transinfection / cisinfection

Answer 9

1) Give DC-SIGN very low dose HIV. If you give same dose to T cells, no T cell infection would occur. So the dose is only enough for presentation. Even no need to wash since u can’t infect T cells.
2) After a certain time, give the T cells.
3) Check the presentation: Whether DC sign hands over the HIV to T cells. Note: it doesn’t process hiv or break it down, gives it directly as a whole.
4) Once T cells get infected by DC, they produce HIV on their own. Virus has p24 protein, and if you do an ELISA for it, you can see whether HIV proliferates or not. (p24 count is correlated with HIV count from T cells – since DC doesn’t produce any virus.)

After a couple of days, virus production increases. So it means T cells are infected, and HIV is proliferating inside T cells. If CD4 or CCR5 is blocked, no inhibition is observed (T cells still get infected) but if you block DC-SIGN, infection is 2.5 folds reduced.
So it doesn’t bind via CD4 or CCR5 to DCs, via DC-SIGN.

So DC captures the virus, somehow keeps it inside intact, and hands it over to T cells. In this way, DC-SIGN brings HIV from mucosa to the LNs!
If you get infected via blood, you don’t need this DC carrying, T cells will get infected anyway with high concentration of HIV.
For the mucosal route, DCs are critical.
Handing over pathogen from one cell to another = Trans infection. If DCs would hand it over to another DC, then it would be a cis infection.

In some cells, DC-sign can also mediate cis-infection, then it really acts as an attachment receptor. And binding of the HIV is enough to infect the DC itself. DC-SIGN is also a signaling molecule, and it might lead to immune modulation, eg. Signaling via DC-SIGN can inhibit Type I IFN production by DCs, which is critical for antiviral immunity

Question 10

Study theory of DCs bringing HIV to T cells

3) How long HIV remains protected inside DCs?

Answer 10

Even at Day 6, DCs are preserving HIV to be handed over to T cells.
So DC-SIGN picks up the virus at mucosa, internalizes it, and HIV stays inside the DC in a protected way. Staining shows that indeed HIV is preserved in an internal compartment.
Upon encounter with a T cell, virus is handed over again with DC SIGN to the CD4 + coreceptor. = Synapse formation is observed.

Question 11

Thick mucosal layer + LCs

Answer 11

DC-SIGN (+) cells are in the submucosal layer. So to get infected, virus has to first transfer through the thick mucosal layer. Can explain why you get easily infected when there’s wounds/inflammation.

mucosal layer has other DCs that doesn’t have DC-SIGN: Langerhans cells. Langerhans cells reside more in the upper layer. DC-SIGN positive cells are found usually in deeper layers, both in mucosa and skin.

Question 12

What happens when HIV interacts with LC? 1) Get LCs to study - same method for getting DCs

Answer 12

How to get LCs for studying: from skin, 1) Take skin from plastic surgery/tummy tuck lol.

2) Cut the upper layer with dermatome (knife), so you get epidermis and dermis.
3) Incubate it with the enzyme Dispase, and get the epidermis only.
4) Add trypsin, degrade, using magnetic beads, isolate pure immature LC from skin.
5) You can also separate epithelia and subepithelia = get DCs

Question 13

What happens when HIV interacts with LC?
2) DC -Sign - Langerin - Mannan study
What’s Langerin?

Answer 13

DCs = infect T cells
DCs + mannan = can’t infect T cells, mannan binds DC-SIGN
LCs = can’t infect, no DC-SIGN
LCs + mannan = can infect? LANGERIN IS BLOCKED.

Langerin is specific receptor for LCs. So if Langerin captures the HIV, you don’t get transmission! Opposite to DC-SIGN. When Langerin is blocked, transmission occurs.

Very similar to DC-SIGN structure, has also carbohydrate recognition domain with similar specificity, they both bind to mannose structures on HIV.
If a cell is Langerin (+) it makes tennis like structures called Birbeck Granules, it’s part of the endolysosomal route

Question 14

What happens when HIV interacts with LC?
2) Langerin - HIV binding study
What happens after binding?

Answer 14

Anti-DC-SIGN Ab doesn’t prevent the binding, well since LCs don’t have DC-SIGN, mannan and Anti-langerin Ab stops the HIV binding. So Langerin is the primary receptor on LC that HIV binds.

Electron microscopy proof:
Bigger particles = HIV, LC is smaller particles, and they colocalize.

Langerin + HIV binding causes HIV to be internalized and they go to Birbeck granules. So Langerin captures the virus, but instead of handing it over to T cells, it degrades them inside the Birbeck granules.

Question 15

HIV - normal mucosa vs injured mucosa

Answer 15

1) Normal mucosa, HIV is captured by LC = degraded, no infection.
2) Injured mucosa, then they reach DC-SIGN (+) cells in submucosa –lower layers-, and they bring the virus to LN, and you get T cell infection.

Question 16

Risk factors for increased susceptibility to HIV infection

Answer 16

Langerin inhibition
High viral load / different viral subtypes
T/F= transmission founder strains, isolated from the patients upon the first infection. Very virulent strains. Lab strain really can’t infect any LCs, but founder strains can (a small percentage)! They can bypass a bit of the Langerin protection.
Inflammation / TNFalpha
If we incubate skin/vagina with pathogens or TLR ligands = we get high TNF-alpha. = more infection.
When you give TNF alpha or Pam3cysK4 (TLR ligand) = a lot of infection.
Now check T cell transmission: (Percentage of T cells getting infected – GFP positive) - with ex vivo human explant model
Same pattern is observed; cells get more transmission to T cells with TNF alpha or PAML

Coinfection - fungi, bacteria, HSV

Question 17

p24 ELISA alternative - see whether LCs hand over HIV to T cells

Answer 17

Ex vivo human explant infection model

Day 0 = epithelia + HIV GFP
If LCs get infected, they will also have GFP with them. Once LCs get activated, they migrate out of the epidermis, they go to the medium under the epidermis. From that medium, you can check the LC infection (expression of GFP).
Then add T cells, to see whether or not LCs can hand over the HIV to them. (T cells will also get GFP positive) – Alternative for p24 ELISA.

Question 18

Langerin and infection relation

Answer 18

Normally: No infection since HIV gets degraded inside LCs.
Langerin gets saturated/or decreased less: happens when there’s inflammation = Infection & transmission occurs.
Give TLRL: Infection and transmission observed. This can mimic like an inflammation, and Langerin expression gets downregulated, therefore protection is removed.
Surrounding cells, eg. Epithelia might produce TNF-alpha, and it can also activate LCs, downregulate Langerin, then infection/transmission occurs.

Question 19

Glycan levels on pathogens

Answer 19

HIV contains a lot, flu viruses have it, LAS Virus, some of the coronaviruses have significant amount of glycans, not as much as HIV but they have.

Question 20

Does DC-SIGN transmit SARS-Cov-2? - Ebolavirus

Answer 20

1) Check cis-infection, whether the DCs themselves get infected or not: Ebolavirus: DCs get infected, block DC-SIGN, they don’t get infected = DC-SIGN is the main receptor, same in macrophage.

Do it with SARS-Cov-2, DCs don’t get infected no matter what. Sars-Cov-2 doesn’t infect neither DCs nor macrophage.

2) Check trans-infection of SARS-Cov-2:
They can hand over the virus to an epithelia cell line (original target of Sars-Cov-2) though. And it can be blocked with anti-DC-SIGN. So DC-SIGN can transmit, but doesn’t get infected itself.

Question 21

Autophagy

Answer 21

2 ways of degradation inside the cells: Proteosomal degradation and Autophagy = lysosomal degradation pathway. Autophagy can be induced after stress, starvation, infection.

1) Isolation (autophagosome) membrane is formed, and it starts engulfing the pathogen/proteins.
2) The membrane expands and covers the bacteria, and autophagosome is formed.
3) Autophagosome fuses with lysosome (proteases, acidic) + cargo degraded.

Atg5/16 is very critical for elongation of phagosome and formation of it. It allows LCD3 to bind to autophagosomes. Autophagosomes strictly need this molecule on their surface for function.

Question 22

History of Autophagy

Answer 22

Mid 1950s, lysosomes found. 1990s Japanese scientist found autophagy pathway.

Question 23

HIV infection of T cells

Answer 23

CD4 positive cells get infected by HIV. T cells are main target, but also DCs, macrophages etc. have it too.

1) HIV binds to CD4 + coreceptors CCR5/CXCL4 on T cells
2) After binding it fuses to the cell membrane
3) And capsid is delivered to cytosol, viral RNA is RTed
4) DNA integrated into host genome, so virus uses host machinery for transcription.
5) It produces new viruses to infect new hosts.

Question 24

Current HIV therapies

Answer 24

Antiretroviral therapies increased the life expectancy of HIV patients a lot. %60 of current patients have it, and they are trying to make sure everyone will get it.
Problem: After 20-30 years of inducing ART to patients, now large cohort studies show that patients have increased chronic inflammation, not so clear it’s because of the therapy itself or due to living with HIV for so long, but also residual HIV replication is increased, not the one that circulates in blood, they can be transmitted. But some HIV goes to tissues, like mucosa, where DCs are, and they form reservoirs there. ART can’t reach those reservoirs. So if you stop the treatment, your HIV will replicate back from the reservoirs anyway.

Question 25

Current HIV therapies + problems

Answer 25

Antiretroviral therapies increased the life expectancy of HIV patients a lot. %60 of current patients have it, and they are trying to make sure everyone will get it.
Problem: After 20-30 years of inducing ART to patients, now large cohort studies show that patients have increased chronic inflammation, not so clear it’s because of the therapy itself or due to living with HIV for so long, but also residual HIV replication is increased, not the one that circulates in blood, they can be transmitted. But some HIV goes to tissues, like mucosa, where DCs are, and they form reservoirs there. ART can’t reach those reservoirs. So if you stop the treatment, your HIV will replicate back from the reservoirs anyway.

So these chronic inflammation/reservoir problem drives more problems in patients: Increased dementia risk, cardiovascular diseases etc.
Therapies target the host should be found, rather than the virus itself. ART stops viral cell cycle.
Which can be useful in stopping inflammation and even curing HIV completely.

Question 26

What happens when HIV interacts with LC?
3) Langerin - HIV binding study
What happens after birbeck granules - Autophagy - TRIM5 alpha

Answer 26

Is HIV targeted for autophagy after interacting with Birbeck granules? Confocal microscopy
Yes, they colocalize.

Steps:

1) LC uptakes HIV with Langerin
2) Targets it to Birbeck granules
3) Birbeck granules go to autophagy

We know that HIV membrane fuses with LCs, and capsid is released inside. But this is it, further steps can’t be measured in LCs. So something happens here.

It’s known that there’s a E3 ligase that can bind to HIV capsid!

1) We know that HIV binds TRIM5 alpha
2) It’s known that it binds to HIV and drives them for autophagy in macaques.

Human study shows: When you silence TRIM5 alpha with RNAi, you get infected / transmitted.

Question 27

What happens when HIV interacts with LC?
3) TRIM5 alpha autophagy direct relation - pulldown studies = mechanism
Langerin + TRIM5 alpha binding

Full mechanism

Answer 27

TRIM5alpha and HIV capsid pulls down together. TRIM5 alpha recruits Atg5/16 and drives autophagosome formation for degradation.

TRIM5alpha was already bound to Langerin, and it stayed bound during HIV infection.
TRIM5alpha is bound to Langerin so that it can ubiquitinate whatever comes and targets it for degradation!

Summary:

1) HIV comes and binds to Langerin, on steady state Langerin is already bound to TRIM5alpha E3 ligase and ATG for autophagy
2) HIV is internalized into Birbeck granules
3) After internalization, carried HIV virus now dissociates from its membrane and capsid binds to TRIM/Atg that’s carried near the autophogosomal membrane
4) Then via Atg5 recruitment, whole complex binds to LC3 and targeted for degradation when autolysosome is formed.