Redox and the gut microbiome

Host redox biology shapes the gut microbiome and vice versa; relationships which may be important in oxidative stress-associated disease and ageing. Here’s an overview…

Gut oxygen

Reduction-oxidation (redox) processes play fundamental roles in biology, while shifting redox environments have shaped the evolution of life on this planet. The primordial earth was virtually anoxic when life appeared ~3.8 billion years ago, with the advent of photosystem II (i.e. early photosynthesis) and geochemical changes eventually increasing atmospheric oxygen (O₂) (i.e. Great Oxidation Event). This exposed life to a double-edged sword: a toxic oxidant and an energetically favourable respiratory acceptor. Consequently, while some committed anaerobes became confined to anoxic zones, others went aerobic, creating the present dichotomy ^1,2. Moreover, aerobic metabolism facilitated the evolution of complex multicellular metazoa ^3,4, and novel biogeographical redox environments therein. In particular, the human gut is populated by trillions of microbes, predominantly anaerobes, which have co-diversified with us acquiring traits such as O₂ intolerance ⁵.

Gut O₂ is dynamic and follows gradients ⁶; levels decline radially from epithelium to lumen and distally from small to large intestine, culminating in the largely anoxic and reducing environment of the colonic lumen—reminiscent of primordial earth. The aerotolerance of gut microbes broadly parallels their distribution, with enrichment of aerobic and facultative anaerobes in the mucosa and obligate anaerobes in the lumen ^6,7. Further, it also relates to their initial colonisation by succession; infancy being characterised by initial abundance of facultative anaerobes, with obligate anaerobes increasing over 4 months ⁸ and 1 year ⁹. And notably, in children and adults, acute secretory diarrhea (i.e. V. cholerae or E. coli) depletes the gut microbiome, while repopulation also proceeded with facultative then obligate anaerobes over 30 days ¹⁰. As such, it has often been presumed that initial consumption of gut O₂ by aerobes underlies the successive growth of obligate anaerobes. However, luminal anoxia was largely unaffected in germ-free mice, while in vitro experiments implicated chemical oxidation and microbial biomass as major O₂ sinks ¹¹. Also, initial colonisation by aerobic or anaerobic bacteria supported that of dominant anaerobe Bacteroides thetaiotaomicron; suggesting early colonisation by aerotolerant bacteria may simply reflect better survival ex vivo and vectorization in vivo ¹². Several human studies are also informative. In 88 African-American newborns, E. coli (family Enterobacteriaceae) was the most common early coloniser and evidenced to perform anaerobic fermentation of amino acids (e.g. serine/threonine to succinate/acetate), while at 1 month bacterial diversity and carbohydrate fermentation genes increased ¹³. Going further, in 153 East Asian newborns followed for 4 months, Enterobacteriaceae and other facultative/aerobic bacteria were increasingly displaced by Bifidobacteria and strict anaerobes, correlating with faecal acetic acid; all of which was delayed with C-section and normalised by a Bifido-based synbiotic ⁸. And in 98 Swedish newborns, the microbiome was initially enriched in TCA cycle functionality (vs. pyruvate at 4 months), suggesting a more oxidative environment; while at 12 months cessation of breastfeeding marked the transition toward an adult/maternal microbiome, which included a decline in Bifidobacteria and emergence of Clostridia spp. ⁹. Thus Bifidobacteria and breastfeeding may shape early anaerobiosis.

Gut O₂ principally arises from bloodstream delivery to the submucosa for cellular respiration, where diffusion from the epithelium ¹⁴ establishes a dynamic radial gradient ^6,7; and perhaps in relation to surface area (decreases from small–large intestine) ¹¹. Modulating intestinal oxygenation (e.g. via hyperbaric oxygen, intermittent hypoxia or mechanical ventilation) correspondingly alters the gut microbiome ¹⁴. Conversely, microbial metabolism also modulates epithelial metabolism. In particular, unabsorbed carbohydrates (i.e. fibres and resistant starches) are normally the major carbon source for microbes and (anaerobically) fermented to short-chain fatty acids (SCFAs), mainly acetate (C2:0), propionate (C3:0) and butyrate (C4:0); the latter being the preferred carbon source for colonocytes and their (aerobic) respiration. Here colonocytes exist in a state of ‘physiological hypoxia’, where butyrate supports O₂ consumption and stabilises hypoxia-inducible factor (HIF), a transcription factor coordinating barrier function and antimicrobial defence ^14,15 (incl. β-defensin-1 ¹⁶). Further, butyrate also induces PPARg-dependant β-oxidation and inhibition of iNOS, thereby limiting O₂ and nitrate (NO₃^–), respectively, which can serve as respiratory acceptors for growth of facultative Enterobacteriaceae ^17–19. Consequently, antibiotic suppression of the gut microbiome can increase host-derived O₂/nitrate ^15,17,18,20. Notably, to a lesser extent than butyrate, acetate also supported epithelial O₂ consumption (but not HIF) ¹⁵. This may be particularly relevant during infancy, where facultative Bifidobacteria (supported by breastfeeding) may be an early driver of anaerobiosis, and perhaps via metabolism of carbohydrates to acetate in the ‘bifid shunt’ ⁸. Whereas cessation of breastfeeding and increased Clostridia spp. possessing the acetyl-CoA pathway (e.g. E. rectale and F. prausnitzii) have correlated faecal butyrate over 12 ²¹ and 24 months ²².

Gut redox

O₂ can directly oxidise a subset of biomolecules (e.g. flavins, quinones and metal centres), where its reduction can generate reactive oxygen species (ROS) with capacity to oxidise many others (e.g. lipids, proteins and DNA). This can occur incidentally or intentionally, while elaborate antioxidant systems serve to eliminate ROS and buffer redox changes. In particular, cellular redox poise is determined by the thiol/disulfide redox couples, whose redox potentials (E_h in mV) are held low (favouring reduction), with dynamic changes regulating cell physiology via redox signalling ²³. For instance, the intestinal epitheliums rapid turnover and cell transitions (i.e. proliferation–differentiation–apoptosis) are regulated by progressive oxidation of glutathione (GSH) and thioredoxin (Trx) ^24,25. The epithelium further secretes mucus and antimicrobial peptides, which co-associate to form a frontline physiochemical barrier to microbes ²⁶. Mucus is composed of mucin oligomers assembled via disulfide bridges (S–S bonds) which stabilise its structure and confer redox-dependent fluidity, which may be modulated by luminal thiols ^24,27. Moreover, reduction of α-defensin-6 (small intestine) and β-defensin-1 (esp. colon) unmasks their antimicrobial activity against commensal bacteria and opportunistic fungi, which may be mediated by epithelial Trx and the reducing luminal milieu ^28–30. Reciprocally, commensal microbes stimulate epithelial receptors (e.g. TLRs and FPRs) and ROS-dependent pathways regulating cell turnover, gut barrier and immune functions ^24,31,32. In particular, oxidative/xenobiotic signals are sensed by Nrf2, a master transcription factor mediating redox and epithelial homeostasis ³³. Accordingly, eubiotic Lactobacilli (e.g. LGG) can induce cytoprotection via NOX1/ROS-dependant activation of Nrf2 ³⁴. And of microbial metabolites, butyrate induces Nrf2 ^35,36 (which may also support PPARg/β-oxidation ^37–39), while urolithin A induced AhR/Nrf2-dependent enhancement of gut barrier integrity and anti-inflammatory activity ⁴⁰.

In a study on 70 diverse individuals, gut microbiome aerotolerance correlated the faecal redox potential (r = 0.41) ⁴¹; a relationship which may involve various factors. Foremost, microbes depend on antioxidant activity for aerotolerance, while anaerobic metabolism may be particularly susceptible to poisoning by O₂/ROS due to its dependence on radical chemistry and low-potential metal centres ^1,2. Episodic disruptions can reversibly inhibit growth, whereas excessive aeration and internal Fenton chemistry can be lethal ¹; i.e. electron transfer from ferrous iron (Fe(II)) to hydrogen peroxide (H₂O₂) forms hydroxyl radicals (^•HO) with capacity to irreparably damage DNA. Among antioxidants, bacteria express various catalases, a family of heme/iron or manganese-containing enzymes which decompose H₂O₂ to H₂O and O₂ ⁴²; and often used to identify aerobic/facultative bacteria (i.e. catalase test). In humans and mice, microbial catalase expression was greater in rectal mucosa vs. stool, paralleling radial O₂ gradients ⁷. Moreover, one of the major butyrate-producing bacteria in humans, Faecalibacterium prausnitzii (i.e. ~5–10% of adult microbiome), may utilise an extracellular flavin–thiol electron shuttle (i.e. Fig. 3; resembling GSH reductase) to survive close to the epithelium; and at the expense of electron disposal via SCFA production ⁴³. Notably, antioxidants (e.g. GSH/cysteine, ascorbic acid and uric acid) can maintain the viability of such highly sensitive anaerobic bacteria in ambient air ^44,45 and even enhance Clostridia butyrate production ⁴⁶.

The gut microbiome may also directly affect luminal reducing activity. Accordingly, in mice on broad antibiotics for 5 days, an increased faecal redox potential occurred within hours, which was not associated with luminal O₂ and even occurred ex vivo ²⁰. Subsequent microbiome recovery followed ecological succession (and required dispersal via cohousing), where an early increase in Enterobacteriaceae associated with a lowering of the faecal redox potential; suggesting they may act as pioneer taxa directly affecting luminal redox and driving their own displacement by secondary colonisers ²⁰. Mechanistically, antibiotics led to a sustained increase in nitrate, a potent respiratory acceptor after O₂ ²⁰ (e.g. standard redox potentials), which can particularly be exploited by Enterobacteriaceae with their high prevalence of nitrate reductase ^47,48. In ruminants ⁴⁹ and pigs ⁵⁰, luminal redox also correlates negatively with pH and acetate, and positively with propionate. Faecal organic acids themselves may have some very mild antioxidant activity (e.g. lactic and acetic acids ⁵¹), although are tied to microbial hydrogen (H₂) economy ⁴⁹. H₂ is a diffusible gas which selectively scavenges hydroxyl radicals ⁵², while also representing a primordial electron donor with potential for broad interspecies transfer and metabolism (i.e. 70% of human colonic microbes contain hydrogenases) ⁵³; generally being released during carbohydrate fermentation and oxidised in sulfate reduction, methanogenesis and reductive acetogenesis ⁵⁴. Of SCFAs, production of acetate and butyrate may favour net H₂ release (e.g. via NADH–ferredoxin–hydrogenase; Fig. 1 ⁵⁵), whereas propionate its consumption ^49,54. Notably, in 32 healthy newborns followed for 6 months, luminal H₂ increased and stabilised after 1 month, while faecal SCFAs kept increasing, but with a lower proportion acetate (i.e. 99–82%) and higher propionate (i.e. 0–15%) ⁵⁶. Finally, some bacteria can metabolise various sulfur-containing molecules (e.g. sulfate, cysteine and taurine) to hydrogen sulfide (H₂S) gas ⁵⁷, a highly reducing thiol which can modulate mucus integrity ²⁷ and be oxidised by colonocyte mitochondria (via SQR) to support bioenergetics ⁵⁸.

Microbial redox is a direct target of host immunity. In particular, the oxidative/destructive power of ROS/RNS is weaponised by innate immune cells, including phagocytes and epithelia, as a conserved form of defence. In response to microbes, mammalian epithelial barriers can produce NOX-dependent superoxide (O₂^•–), DUOX-dependant hydrogen peroxide (H₂O₂) ^59–61 and iNOS-dependent nitric oxide (^•NO) ^62–64, all of which may mediate antimicrobial effects. For instance, in mice, stomach infection with Helicobacter felis (a model for H. pylori) induced epithelial DUOX2 at the apical surface and release of H₂O₂ into the mucus layer, causing bacterial oxidative stress and limiting infection/inflammation ⁶⁵; and similarly, intestinal DUOX2 regulated microbial redox and access to lymphatic tissues, thereby limiting immune responses ⁶⁶. In humans, DUOX2 was upregulated in IBD/IBS ⁶⁶, while rare loss-of-function variants associated with elevated blood IL-17C and mucosal expansion of Gram-negative Enterobacteriaceae ⁶⁷. Notably, IL-17 cytokines support type-3 immunity against extracellular pathogens ⁶⁸; including recruitment of neutrophils expressing myeloperoxidase (MPO) for production of hypochlorous acid (HOCl) ⁶¹, a chlorine-based oxidant and potent antimicrobial (also used commercially as a disinfectant). In animal models, microbes also induce co-expression of NOX and iNOS ⁶², and formation of peroxynitrite (ONOO^–; i.e. O₂^– + NO) ⁶⁴, another antimicrobial ^62–64. Moreover, epithelial NOX deficiency increased H₂O₂-producing Lactobacilli which mediated compensatory colonisation resistance ⁶⁹ and ameliorated colitis ⁷⁰. Notably, all these oxidants have potential to engage host redox signalling, where antioxidant responses may even support immunity. For instance, sulforaphane-rich broccoli sprouts induced a Nrf2-depentant reduction in gastric H. pylori colonisation and inflammation in mice and humans ⁷¹. Also, in a urinary tract infection (UTI) model, urothelial cell infection with E.coli (typically thought to originate from faeces ⁷²) induced ROS and Nrf2, which subsequently suppressed oxidative stress and inflammation, and promoted bacterial expulsion via regulation of the RAB27B GTPase ⁷³.

Gut dysbiosis

A dysfunctional gut–microbiome redox dialog may underlie disease. For instance, in human gastritis, tissue oxidative stress correlated microbiome redox traits (i.e. O₂ respiration and antioxidant genes) used to define a ‘microbial-redox index’, as a putative indicator of mucosal health ⁷⁴. Moreover, many conditions may feature general shifts from obligate to facultative anaerobes (e.g. C-section ⁸, malnutrition ^41,75, HIV ^76,77, IBD ^78,79, CRC ⁸⁰, anxiety/depression ⁸¹, ME/CFS ^82,83, post-stroke ^84–86, Parkinson’s ^87,88 and MCI–Alzheimer’s ^89–91), marking an increase in aerotolerant bacteria and implicating O₂ as a common driver of colonic dysbiosis ^78,92. Accordingly, mitochondrial dysfunction is a common feature of disease phenotypes, while mutations conferring increased mtROS decreased microbiome species diversity and major families of SCFA-producing obligate anaerobes (i.e. Lachnospiraceae and Ruminococcaceae/Oscillospira) ⁹³. Further, in IBD, a pro-inflammatory response to β-fructan fibres (i.e. FOS/inulin) was accompanied by low faecal SCFA-producing bacteria and riboflavin (i.e. vitamin B₂) ⁹⁴, an antioxidant cofactor for F. prausnitzii ⁴³; while impaired mucosal butyrate oxidation was associated with markedly decreased mitochondrial thiolase activity, which can result from increased mtROS/H₂O₂ ⁹⁵. On the other hand, oxidising conditions can favour the growth of facultative anaerobes and opportunistic pathogens (e.g. Salmonella ^17,96, E. coli ⁷⁸, C. rodentium ⁹⁷ and V. cholerae ⁹⁸) by conferring bioenergetic advantage; a situation promoted by epithelial inflammation and metabolic dysfunction ¹⁹. For instance, gut inflammation generates various novel carbon sources and respiratory acceptors ⁹⁹; in particular, the iNOS–NO pathway can yield nitrate (NO₃^–). As above, oxidising conditions may initially favour formation of peroxynitrite, which can rapidly isomerise to nitrate ^48,86, a reaction also catalysed by haemoglobin (i.e. ferrous heme) ^100,101, itself a faecal correlate of intestinal inflammation and cancer ¹⁰². Nitrate may induce blooms of Enterobacteriaceae—a common feature of inflammatory dysbiosis ^{47,78,103,104}. Similarly, antibiotics can disrupt redox dynamics in the gut and promote outgrowth of Enterobacteriaceae ²⁰; here depletion of SCFA-producing bacteria ¹⁵ was reported to increase epithelial O₂/nitrate, allowing aerobic expansion of Salmonella and E. coli ^17,18.

A primary function of the gut is to digest food and assimilate water and nutrients, which themselves have inherent redox activity. Even common liquids (e.g. spring, tap or soda water) may have markedly different redox potentials of relevance ⁴¹; in a colitis model, H₂-enriched/reduced water restored microbial SCFA production, colonocyte metabolism (i.e. ↑ PPARg and ↓ iNOS), gut barrier and anaerobiosis, while suppressing E. coli expansion ¹⁰⁵. Reducing activity may also be conferred by dietary antioxidants (e.g. ascorbate ^106,107, polyphenols ^75,108 and fibres ^109,110). In particular, since many phyto-antioxidants are poorly absorbed, the gut was suggested to be a major site of action ¹¹¹. An early 2-week trial with an antioxidant-rich diet (with matched macros and fibre) increased stool bulk and antioxidant content, but not Lactobacillus or Bifidobacteria (selective measurement only) ¹⁰⁸; while in porcine models, antioxidant supplementation (as NAC ¹¹² or antiox-blend ¹¹³) did increase intestinal levels of Lactobacillus and Bifidobacteria, at the expense of E. coli. Moreover, more recent trials with high-dose or delayed-release (colon-targeted) ascorbate have modulated the gut microbiome ¹⁰⁶ and increased SCFA/butyrate production ¹⁰⁷, respectively. Another important nutrient is iron, a redox-active transition metal which is incorporated into metalloproteins employed throughout metabolism ¹¹⁴; hence deficiency may disrupt many pathways mentioned herein (e.g. ↓ RBC/O₂, NOX/MPO and catalase; ↑ mtROS) ^114–116. However, free/labile iron can catalyse the Fenton reaction forming hydroxyl radicals, while high-dose iron supplementation has increased faecal ROS ¹¹⁷ and Enterobacteriaceae ^118,119, in association with inflammation ¹²⁰. Accordingly, in vitro, iron can favour the growth and virulence of enteric pathogens ¹²¹; and similarly, chronic exposure of titanium dioxide (a white pigment—E171) nanoparticles to E. coli induced adaptive morphogenesis and commensal–pathogen transition via hydroxyl radicals ¹²².

Children with severe malnutrition have positive/oxidised faecal redox potentials and corresponding dysbiosis ^41,75. On the other hand, high fat/lard diets can also induce intestinal oxidative stress which highly correlates bacterial dysbiosis ¹²³ and its amelioration by tea polyphenols ¹²⁴. Moreover, pre-IBD was associated with a history of antibiotics and high fat intake (combined RR = 8.6; higher saturated fat p = 0.03), which in mice cooperatively impaired colonocyte mitochondrial O₂ consumption resulting in overgrowth of Enterobacteriaceae, ameliorated by the PPARg agonist 5-ASA (i.e. Mesalamine) ¹²⁵. Diets high in saturated fat specifically can also induce taurine-conjugated bile acids and growth of H₂S-producing bacteria ¹²⁶ with disruption of the gut barrier ^127–129. Taurine provides organic sulfonate (SO₃^–) for sulfite respiration by Bilophila ^57,130; and notably, sodium sulfite (a preservative—E221) also induced Bilophila and E.coli in vitro ¹³¹. Excess H₂S can reduce disulfide bonds in the mucus network (e.g. 2x > NAC) ²⁷, increasing microbe/toxin exposure to the epithelium and inflammatory sequelae ¹³², and also impair colonocyte respiration (i.e. SCFA metabolism) ^{57,58,130,133}. High intake of sulfur-rich proteins (e.g. meat) may also increase colon delivery of sulfur amino acids (i.e. Met/Cys) for sulfide generation ^58,134. Moreover, diets high in red meat specifically can increase colonic nitrite in humans, perhaps via heme iron-dependent delivery of nitrosating agents ¹³⁵; while in mice, heme (red pigment) can also suppress Gram-positive Firmicutes and induce nitrate-reducing bacteria ¹³⁶ and faecal sulfide ²⁷.

As above, host–microbe interactions regulate epithelial redox and physiology ²⁴. Crucially, a healthy gut microbiome produces metabolites with growth-repressive effects on tumorigenesis ¹³⁷, which is attenuated in people with colorectal cancer (CRC) ¹³⁸. In particular, butyrate prevents genotoxicity via induction of phase 2 detox pathways (i.e. GSTs ¹³⁹ and Nrf2 ^35,36), and has anti-proliferative and pro-apoptotic effects on colon cancer cells via induction of ROS and depletion of GSH ^140–142; similarly to phytochemicals (e.g. glucosinolates and polyphenols) ^{36,137,139,140}. Note, ROS-inducers may have differential effects on cancer cells due to their higher basal ROS and antioxidant dependence ¹⁴³. Moreover, proliferating/cancer cells exhibit aerobic glycolysis (aka. Warburg effect) due to increased glucose processing saturating and outpacing mitochondrial metabolism ¹⁴⁴. Consequently, in colon cancer cells, inefficient butyrate oxidation promoted nuclear accumulation and function as a HDAC inhibitor ¹⁴⁵, which further decreased its oxidation ¹⁴⁶; although others report that butyrate can suppress glucose metabolism ¹⁴⁷ and promote its own oxidation with differential glutamine metabolism, while inhibiting cell proliferation ¹⁴⁸. Recently, CRC was also associated with downregulation of Lactobacillus reuteri and its metabolite reuterin, which can induce protein oxidation in colon cancer cells and suppresses tumour growth ¹³⁸. Conversely, chronic colonic inflammation promotes dysplasia and cancer ¹⁴⁹. For instance, epithelial TLR4 activity was upregulated in IBD and CRC, and in mice induced tumorigenesis dependent on both DUOX2 (produces H₂O₂) and dysbiosis, which further increased ROS, potentially establishing a feedforward loop ¹⁴⁹. Notably, in vitro this pathway was induced by adherent-invasive E. coli (contains LPS), but not F. prausnitzii ¹⁴⁹. Moreover, inflammation shifts colonocyte metabolism toward glycolysis ¹⁹, while epithelial oxygenation can promote aerobic expansion of colibactin-producing E. coli, a pro-oncogenic driver species ^150,151. Also, nitrate/nitrite-reducing bacteria may promote formation of carcinogenic N-nitroso compounds (NOCs; e.g. nitrosamines) ¹³⁵; while sulfide-producing bacteria may open the mucus barrier to dietary heme, facilitating epithelial damage-induced hyperproliferation ²⁷.

Systemic pathways

Beyond local effects, the gut microbiome may influence systemic redox and health via pathways affecting nutrient metabolism and redox signalling. Indeed germ-free mice reveal a broad effect of the gut microbiome on blood metabolites ^152,153 and induction of phase 2 metabolism ^154,155. For instance, blood indole 3-propionic acid (IPA) results entirely from bacterial deamination of tryptophan ¹⁵⁴ and is associated with microbiome diversity (and F. prausnitzii) ¹⁵⁶ and cardiometabolic health ^157–159. IPA is a potent antioxidant in vitro (i.e. > melatonin) ¹⁶⁰, which may underlie some beneficial effects in cell/animal models ^157,158. On the other hand, in women newly diagnosed with breast cancer, gut microbiome indole biosynthesis was suppressed and correlated lymphocyte infiltration to tumours, while serum levels of IPA induced AhR/PXR-dependent oxidative/nitrosative stress (via ↓ Nrf2; ↑ iNOS and mtROS) and cytostasis in breast cancer cells ¹⁶¹. Beyond IPA, the gut microbiome was also linked to induction of the hepatic Nrf2-antioxidant pathway and protection against xenobiotics via Lactobacilli (e.g. LGG) and production of 5-methoxyindoleacetic acid (5-MIAA) ¹⁵⁵. And beyond indoles, gut microbes can directly regulate systemic metabolism of the major thiol-antioxidant GSH by consuming serine/glycine precursors and limiting host availability, which may be of particular importance in age-related metabolic disease (e.g. NAFLD) ^153,162. Reciprocally, biliary secretions also deliver significant GSH to the intestine ²⁵, while liver diseases can involve GSH deficiency ¹⁶³.

Age-related oxidative stress and organ dysfunction has been linked to the meta-organismal trimethylamine oxide (TMAO) pathway ^164–168. Here dietary precursors (esp. carnitine and choline) fuel microbial production of TMA, which is converted by liver FMO3 to TMAO, which circulates blood before being excreted by kidneys or reduced/detoxified by the ubiquitous enzyme mARC1 ¹⁶⁹. In animals and humans, blood TMAO increases with age ^164–168, and in relation to oxidative stress and vascular dysfunction, where ex vivo experiments implicate excess superoxide and impaired NO bioavailability ^165,167,168. In animals this occurs alongside gut dysbiosis, including overgrowth of Proteobacteria and Desulfovibrio (a sulfate-reducing and TMA-producing genus), and increased FMO3 expression ¹⁶⁸, while suppression of microbial TMA lyase (with DMB) normalises vascular dysfunction ^165,167. Similarly, western diet-induced TMAO, vascular dysfunction, exercise intolerance and frailty were ameliorated by DMB ¹⁷⁰. Mechanistically, a high fat diet was reported to induce colonocyte mitochondrial dysfunction, thereby increasing luminal O₂/nitrate, E. coli respiration-dependent choline catabolism and blood TMAO ¹⁷¹. In the vasculature, TMAO may induce inflammation/oxidation via gradual accumulation of advanced glycation end products (AGEs) ¹⁷² and suppression of SIRT1/3 ^164,173 and Nrf2 ¹⁷⁴. Several conditions are also associated with small intestinal bacterial overgrowth (SIBO) or dysbiosis, which can be associated with vitamin B₁₂ deficiency—thought to result from increased nutrient competition with the host and/or production of inactive cobalamin analogues ^175–178. B₁₂ is a cofactor for the remethylation of homocysteine, a source of cysteine for thiol-based redox molecules. On the other hand, gut microbes are also a potential source of several vitamins for the host ¹⁷⁹, including folate ^180–182 (another cofactor for homocysteine remethylation) and the NAD-precursor niacinamide (NAM) ¹⁸³; both linked to redox biology. Moreover, an RCT with B-vitamins lowered homocysteine and TMAO proportionally ¹⁸⁴, which might involve modulation of gut microbiome or host metabolism.

As above, SCFAs are the major end products of carbohydrate/fibre fermentation which support colonocyte metabolism; and this extends to systemic metabolism ^185,186. For instance, in an RCT on 12 overweight men, acute colonic infusion of physiological SCFA mixtures increased fasting fat oxidation and resting energy expenditure ¹⁸⁷. Accordingly, SCFAs are absorbed into peripheral circulation and can regulate extraintestinal cells via G-protein coupled receptors (GPRs) and histone deacetylases (HDACs), while butyrate in particular can induce Nrf2-dependent effects in disease models ^188,189. For instance, susceptibility to sepsis-associated encephalopathy was associated with lower faecal/plasma/hippocampal butyrate, while sodium butyrate (NaB) administration normalised plasma/hippocampal butyrate, improved survival and neuroinflammation, and suppressed microglial activity in vitro via a GPR109A/Nrf2/HO-1 pathway ¹⁹⁰. In a uveitis model, NaB suppressed the ocular inflammatory response and shifted IL-6R-dependent Th17 differentiation to Treg via a Nrf2/HO-1 pathway ¹⁹¹. And regarding Alzheimer’s, in neuronal cells under high cholesterol, NaB suppressed BACE1-dependent amyloidogenesis via an SMCT1/Nrf2/SOD1 pathway ¹⁹². More generally, the ability of SCFAs to support the (epithelial) gut barrier extends to the (endothelial) blood–brain barrier (BBB) ¹⁹³, where propionate at least was shown to suppress mtROS and induce Nrf2 ¹⁹⁴. Of further interest, in a 2-week trial on 20 adults, switching from a western to low fat/high fibre diet not only increased colonic butyrate, but also folate ¹⁸²; a required nutrient for various butyrate-producing Clostridiales (e.g. Fig. 2) ¹⁹⁵ and the survival of colonic Tregs ¹⁹⁶. Thus folate may support the production and protective effects of SCFAs in inflammatory/autoimmune disease (incl. uveitis) ¹⁹⁷. Conversely, in a 2-week trial on 10 obese adults, an isocaloric low carb diet (i.e. C4%/F72%/P24%) rapidly lowered liver fat and VLDL-TGs, alongside carbohydrate-fermenting bacteria and SCFAs, while increasing folate-producing Streptococcus/Lactococcus and faecal/serum folate in association with β-hydroxybutyrate and 1C metabolism ¹⁸¹. Notably, SCFAs and ketones are similar molecules (esp. butyrate and β-hydroxybutyrate, respectively), but with differential immuno-metabolic effects ^198–201, as may ketogenic diets long-term ^202–204.

In addition to dietary fibre, plant polyphenols also have limited absorption and accumulate in the colon; here microbial metabolism generates smaller secondary metabolites with high bioavailability and bioactivity (e.g. antioxidant and anti-inflammatory), which can persist in circulation for days ^205,206 and target distant organs such as the brain ²⁰⁷. For instance, the major green tea polyphenol, EGCG (C₂₂H₁₈O₁₁), is poorly absorbed but degraded by gut microbes to pyrogallol (C₆H₆O₃), a major metabolite excreted in human urine and more potent activator of Nrf2 in vitro ²⁰⁸. And dietary ellagitannins/ellagic acid (i.e. pomegranate, berries, nuts, tea, etc.) are metabolised to urolithins, which in rodents induce Nrf2-dependent health effects in the intestine ⁴⁰ and elsewhere (e.g. liver ²⁰⁹, heart ²¹⁰, brain ²¹¹, retina ²¹², etc.). Notably, polyphenols may also support SCFA production. For instance, in an 8-week RCT on older adults (n=51, ≥60yrs; MaPLE trial), a polyphenol-rich diet lowered intestinal permeability (i.e. zonulin) and blood pressure, while increasing butyrate-producing bacteria (e.g. Ruminococcaceae and Faecalibacterium) ²¹³ in association with serum polyphenol metabolites and methylxanthines ²¹⁴. In animal models, polyphenols can also restore SCFA production during colitis ^215,216, post-antibiotic dysbiosis ²¹⁷ and TMJ inflammation ²¹⁸. Furthermore, diets high in carbs/fibre ^156,219,220 and polyphenols ^221,222 may increase blood IPA. For instance, in a MaPLE trial post-hoc analysis, the polyphenol-rich diet increased blood IPA, but only in those with normal kidney function, and in association with changes to CRP and gut microbiome (esp. + Clostridiales and – Enterobacteriales) ²²¹.

Commensal microbes may also modulate metabolism of systemic gaseous mediators. In particular, endogenous nitric oxide (^•NO) is eventually oxidised to nitrate (NO₃^–), some of which is taken up by the salivary glands and secreted into the oral cavity where it can be reduced by (nitrate-respiring) bacteria to nitrite (NO₂^–), which can be absorbed and support systemic production of vasoactive NO ²²³. This pathway may be induced by exercise; for instance, in an RCT on 23 healthy adults, acute administration of antibacterial mouthwash (0.2% chlorohexidine) attenuated post-exercise (at 1–2hrs) hypotension, tissue oxygenation and salivary/blood nitrite ²²⁴. Moreover, this pathway may be supplemented by diet; for instance, in a 10-day RCT on 18 healthy young and older people, nitrate-rich beetroot juice altered the oral microbiome (e.g. ↑ Proteobacteria/Neisseria and Rothia), increased plasma nitrite and lowered blood pressure (old group only), which were correlated (e.g. ∆NO₂^–/∆SBP r = –0.73) ²²⁵. Notably, a prior meta-analysis of such RCTs suggests that even nitrate-depleted beetroot juice (as a placebo vs. others) may slightly lower blood pressure, implicating other components ²²⁶. Importantly, the fate of gastric nitrite also depends on the local redox environment. In particular, ingestion of antioxidant polyphenols may support gastric and systemic NO production, via direct reduction of nitrite and generation of relatively stable NO-donors (e.g. S-nitrosothiols), respectively, while supressing formation of carcinogenic NOCs ¹³⁵, which may underlie their positive health effects ²²⁷ (contrasting those of heme iron ¹³⁵). As above, intestinal microbes are also a source of H₂, which can diffuse into circulation and be exhaled in breath, with interim potential to support systemic redox homeostasis and health ^228,229. In particular, H₂ can rapidly penetrate biomembranes and diffuse into organelles, where it may function as a selective antioxidant, removing cytotoxic radicals without disrupting normal ROS/RNS signalling (unlike vitamin C) ^52,230. Furthermore, the murine gut microbiota was also shown to support systemic transsulfuration activity and endogenous H₂S ²³¹, which generally has antioxidant and anti-inflammatory activity ²³².

The gut microbiome also shapes systemic immunity and consequently immunometabolism. In particular, ageing and disease are associated with impaired gut barrier function and increased translocation of LPS into the body ^233–235 and brain ²³⁶; while blood LPS correlated NOX2 (r = 0.441) and oxidative stress in elderly people with neurodegenerative diseases ²³⁷. Accordingly, LPS/endotoxin is recognised by TLR4 and stimulates innate immunity and ROS/RNS, particularly via the phagocyte oxidative/respiratory burst ^61,238. In mice cognitive ageing was accompanied by depletion of butyrate-producing bacteria (i.e. ↓ Roseburia and F. prausnitzii), gut/brain barrier dysfunction, elevated blood/brain LPS and systemic/cerebral inflammation ²³⁹; while age-related microglial activation was ameliorated by NaB or soluble fibre ²⁴⁰. Moreover, to gut–brain reciprocity, increased faecal Enterobacteriaceae has been identified as an independent risk factor for poor outcome in stroke; while in mice brain ischaemia induced rapid intestinal ischemia and oxidant production (i.e. ↑ NOX1, DUOX2 and iNOS), with ROS/RNS-dependent nitrate, Enterobacteriaceae expansion and barrier dysfunction (i.e. ↑ LPS, LBP and D-lac), which reciprocally induced systemic inflammation and worsened brain infarction ⁸⁶. Note, the initial brain-to-gut pathway was suggested to involve sympathetic/β-adrenergic activation ⁸⁶. A follow-up study further showed that post-stroke cognitive impairment was associated with low butyrate/Lachnospiraceae at baseline and increased Enterobacteriaceae at 3 months, when they negatively and positively correlated blood LPS, respectively; and was transmissible to mice via the gut microbiome (i.e. FMT), where it was associated with BBB disruption, hippocampal microglial activation and degeneration, and thalamic amyloid-β deposition, all of which could be replicated by injection of LPS (from E. coli) and rescued by NaB ²⁴¹. Finally, in people with HIV, an increased abundance of aerotolerant bacteria was associated with worse emotional states (incl. perceived stress) and a history of depression ⁷⁷; while in mice psychosocial stress induced a gut inflammatory­/oxidative response (i.e. ↑ DUOX2 and iNOS) associated with dysbiosis (incl. ↓ Lachnospiraceae) and increased microbial catalase expression ²⁴².

Perspective

Redox processes play a defining role in metazoan and microbial biology and consequently shape the symbiosis between us and our microbiome; a relationship which may be important in oxidative stress-associated disease and ageing. From an evolutionary perspective, the huge diversity of the gut metagenome may serve to greatly augment host (or ‘holobiont’) digestive and biosynthetic capacity, supporting survival and reproductive fitness; while reciprocally, gut microbes have co-diversified with humans, acquiring traits such as reduced genomes and O₂ intolerance, implying host dependency ⁵. In particular, maintenance of an anoxic and reducing colonic microenvironment may support a mutual redox metabolism, wherein commensal microbes (esp. obligate anaerobes) partially oxidise indigestible carbohydrates/fibres via anaerobic fermentation to SCFA end products which can be salvaged by host cells and fully oxidised via aerobic respiration, meanwhile engaging signalling pathways coordinating epithelial and immuno-metabolic homeostasis. On the contrary, increasingly aerobic and oxidising conditions may generally favour shifts from obligate to facultative anaerobes, resulting in heterogeneous and dysbiotic relationships of competition and parasitism, typified by barrier breakdown and immuno-metabolic dysregulation, and potentially representing a common pathway underlying gastrointestinal ¹⁹ and systemic disease ⁹⁹. In essence, 2 key molecules may be particularly representative of this dichotomy: butyrate is a product of redox symbiosis which transduces redox homeostasis, while LPS is a product of oxidative dysbiosis which transduces oxidative stress. Many environmental factors discussed above may modulate this balance and which could be employed to support a mutual redox symbiosis and systemic health.

In memory of HC: ‘Keep smiling’.

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