Gut Dysbiosis and Immunosenescence

My obsession with ethologist John Calhoun’s Universe 25 experiment this past spring/summer (see the five-part Instagram series) inspired me to vacate the CLRLY study and venture into the real world in order to survey current social conditions. One must live to learn, so what better destination to do so than Miami? The densely populated shark tank in which elites, nouveau-rich and service class alike tear each other to shreds in the hyper-individualistic pursuit of shiny objects, social status and oceanfront views— modern man’s equivalent of rat chow. Was Vice City truly the Mecca e-comm bros and TikTok thots had made it out to be? Or was Calhoun’s warning to humanity becoming a reality almost 60 years after his first experiments began? My first-hand discoveries came at the temporary cost of my own immune function but provided brand new insights into Calhoun’s research and the current state of society. Here’s part one of the CLRLY blog series examining the pathology of infectious disease and its damning social implications.

The Universe 25 experiment

In 1968, Calhoun constructed a “mouse utopia”; an enclosed environment in which he introduced several pairs of breeding mice with the intent of observing what would happen to the population given unrestricted expansion. The basic survival needs of the mice were provided for absent all predation, however, space was limited. As the population grew exponentially sans any form of selective pressure, overcrowding became prevalent and led to social stress and the breakdown of social roles. Calhoun believed that over-socialization forced the mice into simpler, autistic behavior in order to handle the sheer quantity of social contact points experienced on a daily basis. The majority of the mice regressed into degeneracy, became violent and exhibited dysfunctional sexual behavior that culminated in the inability to raise young. These mice had succumbed to what Calhoun deemed “the behavioral sink”.  A small minority self-isolated in their living compartments, only venturing out to feed at odd hours so as to avoid the masses. They exclusively focused on sleep and self-care, losing all reproductive drive in the process. Although these “beautiful ones” lived significantly longer than the others, both groups ultimately failed to reproduce successfully and the population of Universe 25 collapsed two years after the experiment began.

Calhoun’s theory: social stress alters behavior

Calhoun was partially correct in his theory that attributed the regression in behavior to social stress, but failed to take into account the underlying biological processes at play. Psychological stress activates sympathetic nervous system activity, or the “fight or flight” response. The excessive social contact amongst the mice in Universe 25 induced a nearly constant sympathetic state in which stress-related hormones like adrenaline and cortisol remained chronically elevated. Over time, these stress hormones can erode immune system function, specifically by triggering apoptosis in thymocytes and causing the thymus gland to gradually lose mass. This is referred to as thymic involution; a natural process associated with aging that is expedited by stressors, whether physical, psychological or oxidative in nature. Since the thymus is responsible for producing immune-related T-cells, as its mass and function decline, so does T-cell output which increases susceptibility to pathogens and leads to immunosenescence.

Calhoun missed something huge…

While immune resilience amongst the mice declined, the population continued to expand. This entailed more social contact amongst a growing number of immunocompromised mice, and also more contact with the pathogens they carried and left behind in their wake. But what were these pathogens and where did they come from if these were sterile lab mice and it was a closed environment? The study’s major flaw, and something Calhoun failed to take into account: the pen was only cleaned every 6-8 weeks! Universe 25 wasn’t a utopia after all, it was no better than the slums of New Delhi…

Biome = microbiome

Since the immune system is responsible for parsing good bacteria from bad, constantly differentiating what is “self” from what is “not self”, when the mice came into contact with more bacteria from peers and the environment, their immune systems had to work harder and more often— especially so as harmful bacteria became more prolific. When the immune system detects antigens like bacteria, viruses and fungus it flags them for elimination, thus activating an immune response which recruits immune components like dendrites, antibodies, T-cells and macrophages to deal with the invaders. This immune response hinges upon sympathetic nervous system activation, and just like social stress, if sustained, it promotes thymic involution. Think of the thymus gland as a candle— there is only so much wax to burn before the flame flickers then finally goes out.

Gram-negative bacteria and dysbiosis

As antigenic pressure in Universe 25 snowballed out of control, the mice became colonized by the pathogenic bacteria ingurgitating their would-be paradise. In an immunocompromised state, “bad” gram-negative bacteria like proteobacteria and bacteroides can proliferate within the microbiome unchecked while “good” gram-positive strains like bifidobacteria or lactobacillus become outnumbered in the competition for limited nutrients. This bacterial imbalance is referred to as dysbiosis (note: bacterial strains aren’t always inherently good or bad, proper ratios are what’s important). As gram-negative bacteria are killed off in their struggle for dominance, they release endotoxins called lipopolysaccharides (LPS) that bind to various immune cells and endothelial cells and activate an immune response. If this becomes chronic, it leads to immune dysfunction and intestinal permeability whereby endotoxins enter into circulation.

Why are SCFAs important for immune function?

Gram-positive bacteria are essential in maintaining immunity and gut barrier function as they ferment soluble fiber and resistant starches from the diet— fruits and vegetables— to produce the short-chain fatty acids (SCFAs) acetate, butyrate and propionate in the process. These SCFAs serve as the energy substrate for endothelial cells and immune cells to use more oxygen ie. run mitochondrial oxidative phosphorylation (OXPHOS). As we’ve discussed in previous blogs, OXPHOS shifts the cell away from glycolysis and allows the cell to produce more ATP. Increased ATP availability drives a higher metabolic rate which shifts epigenetic expression in these cells to favor anti-inflammatory states— think AMPK vs. MTOR activation and a higher NAD+/NADH ratio. As a result, gut barrier function is maintained and immune cells remain in a vigilant “surveillance” state, primed and ready to deal with any invaders. 

Antibodies and mucosal immune surveillance

The gut lining consists of an outer mucosal layer which serves as the first line of defense against pathogens and a single layer of epithelial cells which lies beneath it. The integrity of the outer mucosal layer is largely dependent on the presence of antibodies— the first responders that bind to pathogens in the gut and neutralize them. The primary antibody found in the mucosal lining, secretory IgA (SIgA), is produced by bone-marrow-derived B-cells that are located below the epithelium in a space called the “lamina propria”. This is where most of the other immune cells are hanging out, including dendrites.

Why is acetate important for antibody production?

Dendrites are cells that can extend from the lamina propria up into the mucosal layer to detect antigens. They are the immune watchdogs; if something is amiss topside, the immune response gets activated. SCFAs are essential for dendrites to function properly. Specifically, acetate allows dendrites to express a greater quantity of aldehyde dehydrogenase (ALDH) enzymes which convert vitamin A to its active metabolite, retinoic acid (RA). Retinoic acid acts as a transcriptional factor for B-cells to initiate the production of SIgA which supports mucosal immune surveillance and maintains the integrity of the mucosal lining.

Vitamin A toxicity impairs immunity

Not to digress too far, but vitamin A has been a highly debated (highly played out) topic in the space and deserves a brief encore. In later iterations of the experiment, Calhoun determined that elevated levels of vitamin A in the chow expedited the formation of the behavioral sink, which we now understand correlated to a decline in immune function. Although he failed to identify the link, it’s related to the above mechanism; just like low levels of acetate reduce dendritic ALDH expression, high levels of vitamin A inhibit ALDH expression through feedback saturation and oxidative stress thus decreasing IgA production and lowering mucosal immune surveillance. Calhoun determined that the threshold for vitamin A toxicity in humans was in excess of 5,000 IUs per day— I usually recommend a max of around 2,500 IUs. Moral of the story: not enough vitamin A and you’ll become the poster child for this year's Christmas food drive, too much and you’ll catch the ‘tism like the beautiful ones.

How does butyrate influence T-cell differentiation?

An environment rich in SCFAs encourages dendrites to promote a “tolerogenic”, or tolerant, state conducive to healthy immune function. Under these conditions, they produce RA along with anti-inflammatory cytokines like interleukin-10 (IL-10) and transforming growth factor-beta (TGF-ß) which work together to tell thymus-derived CD4+ T-cells within the lamina propria to differentiate into protective T-regulatory cells (Tregs). Tregs are anti-inflammatory and help suppress inappropriate immune activation which also helps maintain the mucosal lining. Butyrate is especially important to this process, not only as an energy substrate for dendrites, but because it inhibits histone deacetylases (HDACs) within their nuclei which promotes overall tolerogenic gene expression. If gram-positive bacteria decline along with the acetate and butyrate they produce, mucosal immune surveillance falters and the mucosal lining degrades allowing endotoxin to come into direct contact with the epithelium.

How does butyrate protect gut barrier function?

Underneath the mucosal layer, butyrate is metabolized by intestinal endothelial cells via OXPHOS, a process which uses up oxygen. This creates an hypoxic (also think acidic) environment necessary in maintaining the integrity of the endothelial lining via stabilization of the regulatory protein, hypoxia inducible factor-1a (HIF-1a). As SCFAs decline, local oxygen levels increase and HIF-1a becomes degraded causing “leaky gut”— the separation of the tight junctions between the endothelial cells of the gut lining.

As within, so without

As a result, LPS are able to leak across the gut barrier and access the lamina propria where they activate immune components. When dendrites sense endotoxin, they signal T-cells to differentiate into their pro-inflammatory phenotypes and release cytokines like tumor necrosis factor-alpha (TNF-⍺) and interleukin-6 (IL-6) that perpetuate the immune response. This “cytokine storm”, along with endotoxins and waste from the digestive tract, enters the bloodstream and initiates a positive feedback loop of “auto-intoxication” which, among other things, induces acute thymic atrophy. If the immune system becomes overwhelmed, as in the case of the ill-fated residents of Universe 25, these toxins reach the brain and begin to alter behavior…

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I stepped out of the Uber into the stagnant humidity of South Beach that carried with it a hint of something rank. The complex loomed 30 stories above, casting a shadow over the lush, well manicured maze that led me past trending juice shops and fitness studios before delivering me to the lobby of the luxury residences. “Fingerprint ID required”… not a chance. I waited for one of the Alo types to enter, sliding in behind and quickly skirting by the front desk like I knew what I was doing. Biometric checkpoint number two. Same thing. I had gained access to the central “green space”; lawns and gardens with palm trees interspersed amongst them, overgrown jungle plants of unknown varieties formed canopies over walkways pungent with the smell of dog excrement. I advanced towards the North Tower, passing a handful of Residents led on leashes by their real-life Tomagotchis, eyes disinterestedly directed at blue-lit devices. The ocean breeze coming off of Biscayne hit me as the Miami skyline came into view. Jan Hammer played in my head. Checkpoint number three. I confidently strode by another concierge desk and took the elevator to the 11th floor, located the unit, dialed in the six digit code and entered Universe 2025…