Immunity and microbiome

Question 1

What is the intestinal microbiome?

Answer 1

The intestinal microbiome is a signalling hub that integrates environmental inputs, such as diet, with genetic and immune signals to affect the host’s metabolism, immunity and response to infection.

Question 2

Discuss microorganisms and the human host

Answer 2

• Microbial cells outnumber cells 2:1
• Microbial genes outnumber genes 100:1
• 1.5kg of microorganisms –> more than the average human brain
  Not all cause disease
• Pathogen –> do cause disease
• Commensal bacteria –> don’t

Question 3

Where does the human microbiome come from?

Answer 3

Some evidence some microbiome development occurs in utero - may not be as a sterile environment as expected

Within 20 minutes of being born there is a colonisation of the microbiome that will harbour and be used for life
- depends on type of birth –> vaginal birth tend to be colonised with a microbiome that resembles the mother’s vagina –> caesarean section tend to be colonised with a microbiome that resembles the skin, could be mother or healthcare professionals
- studies indicates that the difference in birth types can lead to differences in susceptibility to autoimmune disease, allergies etc.

From birth to 1 year old, there is an increase in the number of microbes and an increase in the diversity. There is a lost of colonisation which is thought to come from maternal factors such as breastfeeding, skin contact, environmental exposures, diet, antimicrobial therapies can all influence the balance of species of bacteria that are colonising the infant during their first year –> where most of the microbiome is being formed

After 1 year, the residual microbiome stabilises and stay in similar numbers, unless there is infection etc.
The composition continuously evolves.
Have a set standard, might be times in life where there are more or less of a certain type due to various factors.

Question 4

Where is the human microbiome established?
What bacteria?

Answer 4

Comprised of different species depending on the area of the body and the environmental niche that that body site provides e.g.
- Nutrients
- Respiratory requirements –> aerobic or anaerobic
- Space and binding sites to colonise
- Other bugs providing binding sites and nutrients
Is it due to what the host can provide or what the breakdown of other microbes in that area can provide.

Different sites colonised by microbes:
- Skin
- Scalp
- Nose
- Teeth –> Streptoccocus mutans, Bacteriodes, Fusobacterioum, Streptococci
- Mouth –> Types of Strepotococci
- Throat
- Lung
- Intestine
- Urethra and vagina
- Groin and perineum
- Feet

Question 5

What are the differences between bacteria populations in the GI tract?

Answer 5

Species change as migration through GI tract which reflects the environment.
e.g. stomach has very few bacteria as it is a very hostile environment designed to kill bacteria and prevent illness from ingestion such as food poisoning.
As the GI progresses down there is an increase in diversity.
The beginning of the GI tract has a higher oxygen concentration so promotes more aerobic respiration so suits bacteria of that type. Also has a low pH of around 2.
Travelling through the tract, the oxygen concentration decreases and the environment becomes more anaerobic so suits bacteria that use that respiration. It also has a higher pH of around 7.
The diversity reflects the conditions and environment.

Oesophagus and stomach
- Lactobilli
Duodenum
- Lactobilli
- Streptococci
Jejunum and Ileum
- Enterobacteria
- Bacteroides spp.
Large bowel
- Many different types such as Bifidobacteria, Pseudomas, Lactobillus, Clostridium
- Most diversity
Faecal material
- Bacteroides
- Bifidobacteria

Some species translocate from other parts of the body such as Fusobacterium spp that is found in the large bowel, it has originated from the oral tissues and moved down

Question 6

How does the microbiota composition of the intestinal epithelium lumen change down the GI tracts?

Answer 6

At the epithelial surface of the intestine next to the epithelial cells there are a few species such as
- Clostridium
- Lactobaciilus
- Enterococcus
That stick to the epithelial lining cells

Moving towards the lumen of the intestine there is large increase in species diversity. Entering the more mucus dense area there are a wider variety of types of bacteria. Especially moving towards the faeces.
The species will be encouraged into these areas by nutrients from the host and by other bacteria.
Microbes that have direct contact with the epithelial cells will be able to help regulate the immune system by contact.
Further away microbes can interact through soluble factors such as things they secrete that can interact with the host tissue.

Question 7

How do microbes and hosts co-exist?

Answer 7

Commensalism
- One species benefits and the other is unaffected
- e.g. in the large intestines, hosts provides the environment, the bacteria ferments the digested food. The bacteria is usually harmless but can cause harm if tissues are damaged, gut flora changes or immunity if reduced

Mutualism
- Both species benefit
- Bacteroides benefit in the gut by being provided an environment and the host benefits by the breakdown of metabolites that the bacteria produces from dietary fibre into SCFA which can be used as an energy source

Parasitism
- Once species benefits at the expense of another
- E.g Entamoeba Histolytica, host provides the environment, Protozoa feed on mucosa causing ulcers and dysentery.

Question 8

What are the functions of a norma microbiome?

Answer 8

• Outcompetes pathogenic species for colonisation, space and food –> so having either commensal or mutualistic bacteria present takes up space away from the pathogenic species so they have no where to stick to or eat
• Synthesises vital nutrient –> provides things we cannot generate without the bacteria
• Produces anti-microbial substances affective against pathogens
• Activate the host immune system (maintaining a tolerance of ‘good’ bacteria)
• Emerging roles for appetite and weight loss

Question 9

How do commensals outcompete pathogens for colonisation, space and food?

Answer 9

By the commensal microbes colonising e.g. the whole mucosal epithelium and taking up all the binding sites and using all the nutrients. When there is an entry of pathogenic system that is introduced to the system, it has no where to go as there are no cells to bind to and all the nutrients have been used up

Question 10

How does the gut microflora breakdown dietary components and synthesise new ones?

Answer 10

Intestinal microbes can breakdown dietary components and turn them into something bioactive e.g.
- Histidine –> Histamine –> related to hypersensitivities and allergy type reactions. Can modulate the immune system
- Glutamate –> GABA –> a neurotransmitter important in the perception of pain
- Dietary fibre –> SCFA –> nutrition impact

Question 11

How can the breakdown of dietary fibre impact appetite and regulation of calorie intake?

Answer 11

By microbes breaking down dietary fibre into SCFA.
Animal studies have found that a SCFA, Acetate, stimulates the parasympathetic nervous system by the vagus nerve. Vagus nerve stimulation would stimulate pancreatic beta cells to produce insulin so there would be an increase in insulin production.
The vagus nerve stimulation also stimulates the stomach to produce ghrelin (hunger hormone) and increase the intake and contribute to weight gain
??

Question 12

How do commensal microbes have anti-microbial affect against pathogens?

Answer 12

Commensal bacteria can produces substances which have anti-microbial affect against pathogens
e.g. Mucin is produced by commensal bacteria that live on the intestine, which is broken down by Bacteroides species into Fucose. Fucose is helpful in binding up pathogenic species such as E. Coli. E. Coli get trapped in fucose which is a sticky substance which prevents it from binding to epithelial cells.

Question 13

What is tolerance?

Answer 13

The active ignoring of certain bacteria
Has mechanisms to stop itself from responding

Question 14

Why does the body not respond to the microbes all the time?

Answer 14

The immune system has immune tolerance, which is an active decision whether to respond or ignore different microbes. Has mechanisms to stop itself from responding.
A healthy system requires a balance between an immune response towards pathogenic microbes and tolerance.
The presence of microbes in the immune system helps to trains the microbes whether to tolerate or respond

Question 15

What are the key concepts in gut mucosal immunity?

Answer 15

• Epithelial barrier and anti-microbial peptides –> need a strong epithelial barrier as gaps between are potential reach points for bacteria to get into the system
• Microbiome and tolerance–> balance
• Gut-associated lymphoid tissue

The gut mucosal immune system is part of an extensive immunologically distinct network called Mucosa-Associated Lymphoid Tissue (MALT)
The mucosal immune system is highly specialised which is important as the mucosa is such a potential breach point for microbe entry.

Question 16

What is the anatomy of gut-associated lymphoid tissue and its antimicrobial defecnes

Answer 16

Active constructive complex network of communication between bacteria and immune cells.
Villi lined with epithelial cells and some other specialised cells.

In small intestine:

• Goblet cells –> produce mucins, and make the mucosal layer which helps to trap the microbes to prevent entry
• M cells –> allow the transport of immune stimulating molecules or antigens (stimulates an immune response) from the lumen of the gut to the underlying immune system. Moves the antigen into the Peyer’s patch which contains a large proportion of immune cells such as antibody producing B cells and dendritic cells (antigen presenting cells)
• Paneth cells –> produce antimicrobial peptides which helps to regulate and keep in check the growth of microbes. Are commensal but can be harmful if they cause overgrowth, become opportunistic pathogens and cause infection
• Specialised immune cells –> anti-inflammatory and prevent the immune system reacting to all the microbes present. Involved in the mechanism of tolerance. Function by surveillance and produce anti-inflammatory peptides to reduce immune response to microbes in a certain area. They produce cytokines such as Il-22 which stimulates the tightening the epithelial cell barrier to prevent microbe entry. A defect in IL-22 production can then lead to a compromised barrier function and a risk of illness from microbe entry
• IgA plasma cells –> activated B cells that produce IgA antibodies. IgA functions by trapping and ‘mopping up’ bacteria to prevent them invading into our system. Acts as a barrier

Large intestine

Similar structure

• Isolated Lymphoid Follicle instead of Peyer’s Patch –> hot spot of immune cells, especially antigen presenting cells and lymphocytes

Question 17

How is the response balanced with tolerating commensal bacteria?

Answer 17

Commensal bacteria are sampled by the immune system and determined to be harmless
Immune responses to gut bacteria damped down to avoid overreaction and tissue damage
Errors in the mechanism are when microbes can begin to cause harm
Active constructive complex network of communication between bacteria and immune cells to ensure tolerance is maintained.

Question 18

What are some major influences on microbial composition?

Answer 18

• Host genetics –> tells you body how to respond to microbes
• Age
• Immune system –> how it responds, whether it tolerates or initiates a response
• BMI
• Diet/ Lifestyle
• Environmental factors –> where you go
• Evolutionary history (gut architecture)
• Antibiotics –> not always specific so can destroy some healthy bacteria causing microbiome disruption (broad spectrum)

Question 19

How does atherosclerosis link to the microbiome?

Answer 19

Atherosclerosis is an inflammatory condition involving macrophage harvesting of LDL (foam cells) –> bacteria within plaques can come from the mouth, affect macrophage ability
Microbes isolated from plaques originate from GI (Porphyromonas gingivalis) –> alive, often come from the mouth, transiently travel from mouth.
Trimethylamine in red meat, metabolised by intestinal microbiota, increases risk of atherosclerosis and CVD.

Studies have linked the microbiome with the inflammatory response by increasing it and macrophages harvesting more cholesterol from the bloodstream and contributing to atherosclerosis.
Evidence has show treating mice with antibiotics reduces risk of CVD

Question 20

Why do mouth microbes matter?

Answer 20

Mouth is gateway to body
Often neglected - the GI system doesn’t start at the oesophagus
Unique environment for microbes to grow in (lots of niches)
Biofilms form –> rarely forms anywhere else
Diseases elsewhere in the body can manifest in the mouth and vice versa
More than 700 species of bacteria in oral cavity, plus fungi and viruses

Question 21

What are some major change is microbial composition in disease?

Answer 21

T2D –> higher proportion in bacteroidetes, less firmicutes
IBD –> less firmicutes + bacteroidetes, influx of proteobacteria, actinobacteria
Necorotising enterocolitis –> more proteobacteria, less firmicutes

Alteration in species ratio
Microbiome composition correlates with disease

Question 22

What is dysbiosis?

Answer 22

An imbalance in bacterial composition, changes in bacterial metabolic activities, or changes in bacterial distribution within the gut.
Symptoms include digestive disturbance which may cause gas, bloating, diarrhoea, constipation, mucus in stool.

Treatment
- Antibiotics e.g Ciproflaxin which treats gut infections resulting from dysbiosis, Rifaximin which treats symptoms of IBS

Cause
- Genetic background
- Health status –> infections, inflammation
- Lifestyle habits –> high sugar, low fibre diet
- Environmental factors
- Antibiotics
- Some medications
- Food additive
- Hygiene

Question 23

How can obesity affect the microbiome and immune function?

Answer 23

Obesity can be associated with dysbiosis

Chronic, low grade inflammation leads to increased PRR (pattern recognition receptors) expression (recognise antigens) in antibodies, muscle and circulating monocytes, which can alter the composition of microbe
- can see microbes as a threat
- inflammation may be triggered by dysbiosis

Over expressed PRR in chronic low grade inflammation which can alter the composition of the microbiome further due to initiation of immune system.

Question 24

What are the main mechanisms that link oral microbes with systemic health/disease?

Answer 24

Translocation of bacteria from one site to another
- e.g. infective endocarditis, transient bacteraemia, portal of energy to microbe via dental treatments and tooth brushing –> inoculate bacteria into the bloodstream
- e.g. Scar tissue on heart from pacemaker can causes microbes to settle and stick to the scar tissue/ grow out and cause a vegetation, particular streptoccocus species grow out in a biofilm on heart valves –> cause loss of function and heart tissue infection

Chronic inflammation
- Cytokines and inflammatory markers elevated, collateral tissue damage, cancer
- e.g. periodontitis and diabetes, leucoplakia and OSCC
- Chronic inflammation rarely remains localised

Molecular mimicry
- e.g. HSP 60 (fold proteins) and Porphyromonas gingivalis has a similar protein called GroEL. So an gum infection which causes an immune response on GroEl, can mean HSP 60 can be targeted (autoreactivity). Immune system cannot differentiate
- Structural similar to normal molecules in tissues, so bacteria mimic molecules of own cells —> usually not a problem unless there is an immune response

Bacteria has the capability to become pathogenic if they move to an area of the body they are not supposed to be

Question 25

Why target the microbiome for novel therapies?

Answer 25

• New research suggests the microbiome plays a significant role in health and disease
• Advances in technology mean better analysis of microbiome, and invention of new diagnostic techniques
• Lean towards personalised medicine for the future
• Move away from antibiotics

Looking for ways to disrupt or promote particular species in the microbiome
Easily accessible so treatment can be delivered

Question 26

How can the microbiome play a role in cancer

Answer 26

Direct toxin production by bacteria
- Bacteroides Fragilis –> Activates c-MYC oncogene (cancer causing gene –> promotes progression of cells into cancer)

Chronic inflammation
- Prolonged stimulation of DNA-damaging molecules (NO, ROS) by innate immune cells –> collaterally damage tissues instead of original aim of bacteria
- Fusobacterium Nucleatum – From mouth –> colorectal cancer
- Helicobacter pylori –> Stomach cancer –> first ever microbe to be classed as carcinogenic

Inflammation cause dysbiosis
- Translocation of microbes to transformed tissue, driving proliferation (F. Nucleatum –> colorectal cancer)
- Affects chemotherapy efficacy

Question 27

What is Fusobacterium Nucleatum?

Answer 27

An gram positive anaerobic oral commensal and a periodontal pathogen associated with a wide spectrum of human diseases.
- Long and sticky
- Allows many bacteria species to stick to it
- Translocate to colon and uses specific receptors to bind to colorectal cells and drive cellular proliferation (cell cancer risk is uncontrolled cell proliferation) –> override cell checkpoints causing, increase in inflammation and cell growth

Oral disease
- Bridging species in dental plaque biofilm
- Can lead to gingivitis/ peridontitis

Systemic disease
- Colorectal cancer –> moves to gut, cause opportunistic infections –> increases CRC cell proliferation, promotes chronic inflammation

Treated with antibiotics

Question 28

What current microbial therapeutic mechanisms of action are involved in IBD?
How can we use the microbiome to our advantage?

Answer 28

Ulcerative Colitis:
- Probiotics –> Alter bacterial diversity
- Antibiotics –> Reduce bacterial diversity –> Pouchitis
- Faecal Microbial Transplantation –> Dramatically alters structure and function of gut microbial community

Crohn’s Disease –> attempting to reduce pathogenic affect
- Exclusion diets –> Reduce bacterial substrates –> Microbial pathogenic potential –> Reduce bacterial diversity –> Emulsifiers
- Antibiotics –> Microbial pathogenic potential
- Faecal Microbial Transplantation –> Case reports only, need randomised control trial evidence

The imbalance between missing or depleted key species associated with disease and excess of pathogenic species –> exacerbation of IBD

Question 29

What diseases can the microbiome influence?

Answer 29

• Atherosclerosis
• Cancer
• Obesity
• NAFLD
• Pulmonary disease and atopy
• Rheumatoid arthritis
• Type 1 diabetes

Question 30

How does the microbiome link to IBD?

Answer 30

Related to the homeostasis (tolerance) and inflammation disrupted.

In a healthy individual homeostasis is maintained by
- Symbiont bacteria (mutualisitically, commensally), some a very beneficial and help to train the immune system to be tolerant –> increase amount of regulatory cells to stop the immune system responding when it doesn’t need to
- Increase production of anti-inflammatory molecules and transcription factors that will drive immune cells into an anti-inflammatory profile
- Bacteria that will help train and develop gastro-associated lymphoid tissue by generating the right kind of immune cells needed to protect it, increasing barrier function
- By these working together can help protect against pathogens

Dysbiosis
- Can be due to genetic factors, such as mutations in certain genes that code for parts of the immune system that are important in tolerance. Without those mechanisms means tolerant cells can’t be made
- Environmental factors –> diet, stress, infection

Leads to
- Destruction in the health beneficial bacteria, by either antimicrobials or pathogens –> losing benefits and immune training
- All anti-inflammatory tolerance mechanisms broken down and the immune system will switch from protective to immunogenic –> tissue inflammation

This tissue inflammation can then lead to IBD

Question 31

Describe the intraoral space and microbes

Answer 31

Only part of the body where there is exposure to soft tissues, hard tissue and the external environment.
Due to this it provides lots of new and unique microbial niches which means that lots of the types of bugs in the mouth can only really live in the mouth.
Many anaerobic species live in the picks of the tongue –> can have some impact on some physiological mechanisms like blood pressure regulation
Location depends on whether the species will be aerobic or anaerobic

Teeth
- Hard surfaces perfect for biofilms to form

Space between teeth and gum –> Gingival crevice
- Provides binding sites for bacteria which is sheltered from tooth brushing

Question 32

How do oral microbes contribute to systemic health?

Answer 32

General mechanisms
- More diversity = better health
- Different microbiome between individuals may explain why some individuals are more prone to oral disease than others despite good oral hygiene

Specific mechanisms
- Anaerobes on dorsum of tongue reduce nitrate to nitrite which is a vasodilator and can help regulate blood pressure

Porphoromonas Gingivalis and Fusobacterium Nucleatum produce short-chain fatty acids from dietary fibre
- SCFA can regulate BG and lipid metabolism, epigenetic, regulation of cell proliferation, immune function and hormone release?

Question 33

What functions can Porphoromonas Gingivalis and Fusobacterium Nucleatum do?

Answer 33

Porphoromonas Gingivalis and Fusobacterium Nucleatum produce short-chain fatty acids from dietary fibre
- SCFA can regulate BG and lipid metabolism, epigenetic, regulation of cell proliferation, immune function and hormone release?

Question 34

What is an example of oral bacteria and systemic disease link?

Answer 34

Porphyromonas Gingivalis and Atherosclerosis

Scientists have isolated PS from plaques from atherosclerosis, its is found to be
- elevated levels of inflammation –> more foam cell production
- increasing numbers of cytokines
- up-regulate vascular cell adhesion molecules so they accumulate even more immune cells –> lipid accumulation
- platelet aggregation –> affect clotting

PS is associated with the picking up of more cholesterol