GM, Immunity & Barrier Function

Describe prevailing hypotheses on the early gut microbiota and incidence of immune mediated diseases

Explain ways to measure the gut permeability

Provide examples of microbial regulation of mucosal immune cell
subsets and gut barrier function

Discuss potential pathways of how gut immunity and barrier
function may contribute to extra-intestinal disease

Ussing chamber:


The Ussing chamber provides a physiological system to measure the transport of ions, nutrients, and drugs across various epithelial tissues


How it works:
Measurement of short-circuit current as an indicator of active ion transport taking place across the intestinal epithelium. The active ion transport produces a potential difference across the epithelium (VEP). The voltage difference generated is measured using two voltage electrodes that are placed as near as possible to the tissue/epithelium.


Limitations:
It is invasive due to the need for fresh intestinal tissue, and the lack of correlations between Ussing chamber data and other permeability assays raising the question of which aspects of the intestinal barrier can be assessed by this approach.

The Hygiene Hypothesis:


Early childhood exposure to particular microorganisms (such as the gut flora and helminth parasites) protects against allergic diseases by contributing to the development of the immune system

The farm effect:
Same lifestyle and genetic background but two different farming practices


Early-life environmental exposure has a stronger effect on asthma pathogenesis

The biodiversity hypothesis:


Lack of microbiological diversity in the everyday living environment is a core reason for the dysregulation of immune tolerance and – eventually – the epidemic of immune-mediated diseases


Lack of diversity = lack of antigens for correct immune stimulation and immune maturation

The Old Friends Hypothesis - Friend or foe?


This hypothesis states that humans must be exposed to symbiotic microbes during childhood in order for adaptive immunity to properly develop. Children primarily encounter these bacteria through contact with others and the outdoors.

Invariant Natural Killer T cells (iNKT cells)


Recognise and be activated by foreign lipids found din pathogenic bacteria or commensals such as B/ fragilis


A germ-free postal period increases iNKT cell accumulation and correlates with increased sensitivity to induced colitis


The effect of the microbiota on the iNKT cell population seems to be particularly important early in life

B cells:


Essential to immune system as a source of the five different isotopes of functionally distinct antibodies: IgA, IgE, IgG, IgD and IgM.


  1. Critical that the correct GM is introduced early in life


  2. GF: Skew towards type-2 immunity with increased IgE:IgA ratio in serum


  3. High diversity GM early in life protects against 2-type phenotype



Lack of antigens can result in an improper developed immune system with less regulatory cells resulting in increased risk of developing auto-immune diseases

Innate lymphoid cells
Lack antigen-specific receptors


Main function is to produce cytokines, inducing or maintaining immune responses

ILC3s are programmed in off-spring during pregnancy


Reversible microbial colonization of germ-free mice (transiently colonized with E. coli). Pregnant dams were GF at birth again.
Germ-free pups born to transiently colonized dams harbour have increased small intestinal ILC3 populations

Biomarkers of inflammation/immunity:


Faecal calprotectin test has a relatively high specificity and sensitivity (approximately 90%) for distinguishing between non-inflammatory bowel disorders (e.g. irritable bowel syndrome) and inflammatory bowel disease


There is also IgA and defensins. Whereas secretory IgA has been examined in patients with celiac disease, defensins have been analyzed mostly in patients with IBD

In vivo FiTC-dextran permeability


This assay is an indirect measure of total intestinal permeability.


Permeability assays usually use oligosaccharides of large size. The large size molecule is thought to cross the paracellular intestinal pathway only if the intestinal barrier function is compromised.


In case of barrier function loss such probes cross the intestinal barrier, appear into the circulation and can be detected in urine after renal excretion. The small size molecule is thought to traverse the intestinal barrier freely, independent of barrier function loss, and is affected in the same way as the large molecular probe by the pre- and postmucosal confounders like as gastric dilution, gastrointestinal motility, bacterial degradation, and renal function.


Bacteria-related markers:


LPS measurement
Several studies have successfully used LPS assays to show endotoxemia, mostly in patients with sepsis. Enhanced levels of LPS were found also in patients with obesity and metabolic syndrome, which might indicate bacterial translocation from the gut lumen to the circulation as a consequence of intesTIinal barrier function failure.


Limitation:
While LPS can be quite easily measured in portal vein blood in animals, it remains a challenge to measure LPS in peripheral blood in humans and it requires careful standardisation of the measurement

Biomarkers of epithelial cell integrity:


There are several: glutathione s-transferases, zonulin, diamine oxidases, citruline etc.


Citruline:
Plasma levels of citrulline, an amino acid not incorporated into proteins, but produced by small intestinal enterocytes from glutamine have been proposed as a marker of functional enterocyte mass.


Loss of small bowel epithelial cell mass results in impaired intestinal permeability and in declined circulating levels of citrulline, as is shown in haemopoietic stem cell transplant recipients suffering from severe oral and gastrointestinal mucositis following intensive myeloablative therapy.

Bacteroides fragilis influences T cell differentiation:


B fragilis naturally present in human GM composition


Polysaccharide A (PSA) from B fragilis can reverse the type-2 dominated phenotype observed in germ-free mice

ILC3s and diet:


HFD enriched with fermentable fibre increases production of ILC3s leading to expression of IL-22, enterocyte proliferation and mucosal defence

Invariant natural killer T (iNKT) cells express an invariant TCR-a chain and a diverse array of TCR-b chains and recognize endogenous and exogenous (bacterial) lipid antigens when pre- sented by CD1d, a MHC class I–like molecule

Treg cells in lung and skin


Exposure to house dust mite (HDM) antigen— which possesses CD1d-restricted antigens for iNKT cells, during the first 2 weeks of life, but not thereafter—induces a helios-negative (presumably induced) subset of Treg cells from conventional CD4+ T cells in the lung


Consequently, initial HDM exposure during adult life results in increased AHR relative to that observed when HDM exposure first occurs in early life.


Mechanistically, the allergen-induced development of the helios-negative Treg cells in early life is dependent on microbiota and involves the expression of the immune checkpoint molecule programmed cell death protein 1 (PD1) on the T cell and PD-L1 on dendritic cells so that early-life blockade of PD-L1 abrogates the expansion of the helios-negative Treg cell and increased asthma

Importance of gut barrier: It prevents against loss of water and electrolytes and entry of antigens and microorganisms into the body while allowing exchange of molecules between host and environment and absorption of nutrients in the diet.

TLR5 and obesity development:

Gut dysbiosis affect ILC3s in metabolic syndrome.