Hygiene Hypothesis: Old Friends

Question 1

What’s the trend of diseases amongst the years? (increase/decrease)

Answer 1

-Immune regulatory disorders increase (especiallly after 1995, in Westernized countries): MS, Crohn’s, Type I diabetes, asthma
-Infectious diseases decrease: Measles, mumps, TB, HepA, parasite infections
Before WW2: %30-40 of kids had helminth infections, now its very rare.
Due to vaccination + better healthcare

Question 2

Where’s the asthma/allergy increase observed?

Answer 2

Northern-Southern USA, Australia, Europe: so much asthma
Asia, Russia, Africa: some places hardly any asthma/allergy
Especially increases in urban centres/cities, but less in poor countries/rural areas

Question 3

Hygiene hypothesis

Answer 3

Found by David Strachan, UK, epidemiologist
He found: first kids have more chance to develop hay fever compared to others
Others have less because they are exposed to germs from older siblings (obv doesn’t count for all germs)
Since westernized countries have less kids: they get more inflammatory diseases

Question 4

What’s the differences in lifestyle that might effect our prevalence of getting inflammatory diseases?

Answer 4

After WW2 in Europe, now in developing countries:

• People move to cities/urban areas from rural areas
• Food system depends on fast food
• Sitting professions are more common rather than physical labor now
• We are always indoors rather than outdoor
• We have better housing: isolation, central heating, less humidity depending on condition
• We have better healthcare, we can prevent infectious diseases pretty good with antibiotics/penicillin has an important effect + also there’s vaccination
• Sanitation has improved, its often too much, hospital cleanliness for the immunocompromised is the new normal = very high personal hygiene, very clean homes, soaps w antibiotics lmao xD

Question 5

How do we perceive microbes now?

Answer 5

-We see them as disease source: and think absence of them is key to health
But= microbes can be beneficial!!

Question 6

Old Friends Hypothesis

Answer 6

Graham Rook, UK, improved hygiene hypothesis
Said that: there was microorganisms back then that were abundant in our system to train our immunity.
They were tolerated, and our immune system evolved with them instead in evolution.
ex: Lactobacilli, mycobacterium from the soil, organic material

Helminths/parasitic worms in gut or blood: Lost: used to provide Tregs/Bregs/antiinflammatory cytokines
Ectoparasites (fleas, lice, mites, ticks): Mostly lost
Carrier states (only): Salmonella, HepA, H.pylori, TB, toxoplasma positivity: Mostly lost
Microbiota of skin, gut, airway, genitalia: Changed/less diverse (due to going indoor, no contact with environmental microbes/food)
Microbiota of the natural environment (soil, animals, air, plants): Less diverse
= They both provided Treg, TLR-2, TGFbeta, SCFA

-50 years ago everyone was positive for HepA IgG, so everyone was infected at some point, no one has it now. Also the same with lice & helminths.
Probably they caused a lot of problems: but they had useful insights to teach our immune system.

Question 7

How does the immune system change between rural and urban residents?

Answer 7

Mass cytometry study:
(Mass cyt. is like flow cyt. but with metal labeled Abs, causes less spillover so you can use more markers)
-A subset of CD8 T cells, ILCs and gamma delta T cells were lost
-B cell subsets switched to something different
Conclusion: Less exposure to urban life develops immune system differently
-Vaccines and drugs in two different groups might act differently
-In urban: more inflammatory diseases, rural: protected from those

Question 8

What is the hygiene hypothesis about?

Answer 8

Contact with microbes

Question 9

How many types of helminths exist?

Answer 9

2. Gut-intestinal helminths / blood-lymphatic system helminths

Question 10

Helminth infections: Observed where, how much, what do they cause?

Answer 10

• Almost 3 billion people are infected with chronic parasitic infections (1/3 of the whole world)
• It’s especially a huge problem in the tropics
• Helminth infection can persist for years, normally immune system is there to fight with them, but often it cant get rid of it
• How do they effect immune system/or whats their effect on allergies/asthma is studied
• In some areas, almost %100 of the kids are infected, they don’t kill, might cause some reaction in some cases: often small complaints so they ignore or don’t notice it at all
• But in some cases: prolonged bladder and liver inflammation can even cause cancer

Question 11

Association of helminths and allergy/asthma meta-analysis review

Answer 11

• Allergy results: Negative association: once you have helminths, you don’t have allergy! Clear association with hygiene hypothesis
• Asthma results: Not clear, only hookworm showed a negative association
• Ascaris even increased asthma: it has a lung stage that drives a significant immune response in lungs, other worms often have lung stage too but probably not that invasive
• Overall results were inconsistent, the clinical outcome of allergy/asthma varies (severity), IgE levels vary, pathology due to worm infection varies, helminth infection intensity varies etc.
• Also while doing such meta-analysis: there’s a significant difference between results from 2021 and 2006. The environment changes a lot. Even in Indonesia there was 1 plane a week, now there’s a lot even in rural areas, everyone uses cars instead of walking = conclusion is very hard

Question 12

Antihelminthic treatments results

Answer 12

• In pregnancy: if you use Albendazole/Praziquantel: kids have an increased risk of egzema and wheeze in 1-5 years, but not anymore in 9 years.
• In children: Albendazole/Praziquantel for 2 years: more IgE, more SPT conversion? in Gabon Vietnam / no effect on eczema, IgE reduced, SPT conversion was observed = So there’s environmental difference variability

Question 13

Why hygiene hypothesis is prevalent on kids and toddlers?

Answer 13

-Early priming: Immune system is immature in kids, susceptible to infections, both adaptive and regulatory arm is developing = so teaching them properly is critical!

Question 14

Schisto / H. polygyrus (Pig hookworm) animal study- airway responsiveness results

Answer 14

OVA model + once they give Schisto, allergic airway inflammation = BAL eosinophilia numbers decrease
Airway hyperresponsiveness: once you give methacholine inhaled, once you have asthma: you respond greatly in low doses, if you don’t: you respond in very high dose
OVA: hyperresponsive
OVA+ Schisto: acted like no asthma at all
= Both immunological and functional level = asthma reduced

Similar results in pig hookworm: decreased eosinophils w infection, and less immune cell invasion in tissue

Question 15

Schisto infection :Immune responses step by step

Answer 15

1) Cercariea step: Weak Th1
Schistosome - Adult worm
2) Eggs: strong type II response: macrophage, DCs, IL-4, IL-5, IL-13
3) Chronic stage: Treg, Breg, reg. DCs, IL-10 and TGFbeta increase

Question 16

Regulatory network cells/and what they secrete?

Answer 16

Tregs= Foxp3, IL-10
Bregs= TGFbeta, IL-10
Alternatively activated macrophages
regulatory DCs

Question 17

What does Tregs do? / on other immune cells for suppression

Answer 17

They block everything around them! Can use either of these mechanisms depending on circumstances:

• Inhibit antigen presentation to T cells/they can prevent access to DCs by surrounding them
• Modulate B cells
• Tissue remodeling
• Inhibit innate cells
• Downregulate Teffs (Th1/Th2) or kill them w granzyme perforin
• They secrete IL-10, TGFbeta, IL-35
• They can starve Teffs w cytokine deprivation: eg. they express IL-2R, so they collect all IL-2 around and it needed for T cell growth
• They can deprive adenosine
• they can change nutrient balance around (glycolysis, oxphos substrates)
• they can suppress DCs directly with checkpoint inhibitors eg. CTLA4, LAG3

Question 18

How do T regs suppress the overall environment? /benefits

Answer 18

• They protect from autoimmunity: If T cell recognizes autoantigen, they prevent its development to Teff = helps differentiating self/nonself
• Critical in mucosa response, there’s a lot of microbes in microbiota, T-regs avoid building response to every microbe = also avoid allergies
• They drive tissue repair: Secrete amphiregulin, induce epithelial/fibroblast repair

Question 19

Schisto /H.polygyrus study - Treg results

Answer 19

H.polygyrus secretes HES, and increases Foxp3+ Tregs

Schisto also show increased Foxp3+ Tregs

Question 20

Deworming then malaria infection study

Answer 20

After 9 months of deworming (compared to worm control): TNF-alpha response to plasmodium increased significantly

• CTLA4 on CD4 T cells decreased/brake released
• So helminth makes the response much more suppressed: cancer cells also use a similar strategy (especially w checkpoint inhibitors).

Question 21

What’s observed as a bystander effect in kids infected w helminths? -T cells only-

Answer 21

Increased Tregs/enhanced activity

Suppressed Th1, they drive Th2 response tho

Question 22

Good things/bad things in helminth infection?

Answer 22

The positive effect of helminth infections:
-Reduced hyperresponsiveness in allergy/asthma
-Reduced autoimmunity
-Reduced IBD
Negative: 
-overall suppression to other pathogens
-Reduced vaccine response
-Reduced anti-tumor immunity

Question 23

Treatment of IBD with Trichuris suis trials

Answer 23

Joel Weinstock-GI doctor from the USA, thought there’s a lot of pig farms in Texas, and pig farmers don’t really have IBD, or it’s very mild.
So he thought the pigs on the farms are infected with helminths, especially Trichuris (whipworm), and farmers kept continuously getting exposed to its eggs = so they are immune for IBD.
Pro: This can be tested easily in humans because this Trichuris cant infect humans, very controlled infection, you eat eggs, they don’t hatch, they just wash away = temporary exposure

So he gave his patients eggs: every 2 weeks for 12 weeks, 2500 mature eggs. He saw significant improvement in colitis index after week 6

Then they did all those trials for the same eggs on Crohn’s, MS, UC, Rhinitis etc. mostly didn’t work. Only very small-scale ones w selected patients worked, and once it passes to Phase II/III= bummed.
Hookworm larvae trials was the same only worked for limited ppl
Other larvae studies: also didn’t work

Question 24

Why Trichuris whipworm trials didnt work/criticisms?

Answer 24

1) Maybe it only works for kids:
- The hygiene hypothesis does not say that helminth infection helps adults.
- In low-middle-income countries: the biggest effect is seen on kids.
- Also, the hypothesis says that it’s useful in early priming once their immune system still develops.
- Trying this on adults, also adults with full-blown disease: the effect might not be enough to reduce the whole inflammation
- Also you cant try this on kids with no disease, another half of the world is trying to eliminate these worms completely xDDD But that’s why rather this pig worm trial was good: no one gets infected but gets exposed.

-Egg administration (like in the case of pig worm study) with no infection might not be enough to provide protection at all.

2) Treatment period is short
-Might be, but pharma companies don’t fund that long (more than 5-10 years)
-Also trial participants won’t want that: they want 6m max treatment period.
Irl: those kids have those helminths either all their life/or prolonged exposure at least 2-3 years

3) Insufficient dose
- They cant know that, they just try to make it similar to original infections
- Also ethical committees make you use the lowest dose possible/also no side effects

4) Wrong worm species
- Often ppl in endemic areas are infected w multiple worms instead of one
- Do you need a specific one for each disease for eg.?

5) Ppl immune responses differ

Conclusion: We should rather learn how helminths suppress/teach immunity. What type of molecules they secrete and use those instead, harder to learn but better.

Question 25

Overall helminth response? both suppressive and inflammatory

Answer 25

Inflammatory: helminths also cause Th2 response: ILC2, M2, eosinophils, IgE increase
Antiinflammatory: regulatory network: Treg (IL-10, Foxp3), Breg (IL-10), M2, tol. DCs
Two happen at the same time.
Too little Type II immunity: no clearance, Too much: fibrosis, tissue damage, but also clears the infection
Same balance in Tregs
To help helminth treatment: study proinflammatory path
To help protection against asthma allergy: understand antiinflammatory path

A worm is not a single entity: it drives different response w different structures of itself, already identified - molecules that effect B or T cells can be an interesting candidate to study

Question 26

Helminths and their effect on immune system: mice model only

Answer 26

H. polygyrus (pig hookworm) = excretes HES, it mimics TGFbeta, acts on Tregs, gives protectivity in autoimmunity
releases ARI (allergic response inhibitor), blocks IL33 on epithelia, protects for allergy
Schisto eggs= produce omega1, acts on Th2 & foxp3 T cells: good for allergy response
Other worms produce cystatins: act on macrophages protects for colitis

All on mice: but can be applied for humans in correct doses/times for different studies

Question 27

Risk/good factors of asthma /allergy - immune response +

Answer 27

Good:
-Breast milk feeding (probably better than formula feeding)
-Having older siblings/bigger household
-Going daycare
-Farming
-Having helminth infection
-Having pets
-Microbial exposure
Early years of life is important, that’s what regulates the immune response
Induces Treg/Breg/Th1 response = less allergy asthma

Bad:
-being a single child
-Live in city
-Too sterile environment
-Use antibiotics on very early life: this kills both infection and gut microbiome
Induces Th2 response: asthma/type II diabetes

Not sure but definitely alters the microbiome:
-Vaginal vs Caesarean section birth: Vaginal canal normally gives kids first microbiome: good Lactobacillus, Caesarean birth gives skin microbiome to kids, which is often random so might not be good
They now wipe the babies w vaginal microbiome to reach the same composition
No study showed that it actually increases susceptibility

Question 28

Why intestine balance is important?

Answer 28

• It’s the largest organ w the most microbiome exposure
• Helps us digest food
• Calibrates immune response against microbiome
• Even can influence our mood
• Gut Lung axis/gut brain axis etc.

Question 29

The effect of antibiotics on microbiome in early life study / effect of antibiotics in general

Answer 29

The odds ratio for asthma if exposed to antibiotics in the first year of life: very high
Other diseases: diabetes, MS, eczema can also occur
But often antibiotics at 1 year of age are only given once there’s a very serious infection, nevertheless, people should be careful w antibiotic usage even as adults
Doctors tend to give antibiotics for the diseases they cant diagnose: but its bad bcs 1) antibiotic resistance 2) it changes the microbiome and you can get something nasty like C.diff, you can’t remove C.diff easily= as a last resort you need fecal transplant lmao to reverse everything

Which bacteria it will reduce: probably depends on the antibiotic itself, not everything is wiped out in bacteria usage especially in adults but some decreases significantly

Question 30

Importance of commensal bacteria in microbiome

Answer 30

-They keep the space so that pathogens cannot reside. Commensals and symbiotes are more common and better at colonizing
-Germ free mice w no microbiota: More susceptible to inflammatory diseases
If you make only 1 specie the microbiota: Gnotobiotic mice: you can study the effect of a single bacteria as well

If pathogens increase: you get infection/inflammation, if symbiotes do: you get better regulation, better everything

Question 31

SCFAs?

Answer 31

Good microbes in microbiota produce short-chain fatty acids (SCFA) with their fermentation of dietary fibers, like those found in complex food
Those SCFAs: 1) regulate better immunity: Th1/Th17/neutrophil chemotaxis
Although Th17 is proinflammatory normally: in gut, it produces IL22 which induces repair/maintenance of epithelia
2) help better barriers: better mucus, tight junction response
3) help inducing more tolerance, IL-10, Foxp3, m1
= this helps both pathogen clearance/suppression of excessive inflammation

These SCFAs consists of acetate, butyrate and propionate
Butyrate is histone deacetylase inhibitor: so it changes gene expression

Question 32

Scfa in allergic inflammation HDM model

Answer 32

HDM: house dust mite allergy model
Low fiber diet: Most eosinophil in BAL: shows allergic inflammation
Normal: middle
High fiber: Least eosinophil in BAL
Conclusion: diet determines whether you get an allergy or not too!!!

Question 33

Gut lung axis?

Answer 33

• SCFA move to blood= act on bone marrow for forming new immune cells (DC, Tregs, macrophage) = they migrate to lung/they are much more regulatory now = they inhibit Th2 asthma, or excessive influenza response (ARDS)
• Whole cells can migrate to the lungs directly from the gut
• microbial metabolites can also travel and inhibit Type I IFN response, also too excessive on influenza sometimes

Question 34

H.polygyrus - SCFA- Asthma model

Answer 34

Antibiotic treatment model
-Hookworm infection reduces eosinophils, but once you destroy microbiome with antibiotics, helminth cannot help the increase

Cohousing model: Once you put mice together, they eat each other’s feces, so get each other’s microbiota
-So even if your microbiota is destroyed, if you can transfer that from someone else with helminth infection, you can reduce your eosinophils
This effect is seen due to the effect helminth caused on the microbiome!

SCFA-R CKO model:
if you CKO SCFA receptor, no protection of helminths: no reduction in eusinophils
Helminth protection is due to SCFAs

Conclusion: Helminths change the microbiota to a more protective phenotype: they secrete SCFA together
they also secrete acetate/other molecules, they both derive Foxp3 Tregs, they migrate to lungs= reduce type II inflammation = protect from asthma