Toll-like receptors are a family of pattern recognition receptors expressed on the surface of immune and other cells. Toll-like receptors are the principal inducers of innate immunity, activating transcription factors such as NF-kB that increase the expression of inflammatory cytokines. Among toll-like receptors, TLR4 is notable for recognizing lipopolysaccharide (LPS), a structure found on the outer membranes of Gram-negative bacteria. This overview will predominantly focus on TLR4 and its involvement in human health, disease, and behavior.
Toll-like receptors are a gateway to understanding how innate immunity influences aging.
While the innate inflammatory response is necessary for immunity against bacterial infection, chronic activation of the TLR4 pathway can accelerate aspects of aging, a phenomenon termed inflammaging. One source of TLR4-mediated innate immune activation is thought to be chronic exposure to lipopolysaccharide by certain conditions, such as intestinal permeability.
In many ways, the immune system's response to lipopolysaccharide via TLR4 is a canonical example of innate immune activation in response to common pathogens. This effect is so robust and reproducible across species, that researchers routinely use "LPS challenge" to measure inflammation under varying environmental and physiological circumstances. Through extensive investigation of the TLR4 system, scientists have illuminated a variety of relevant relationships between LPS exposure, aging, and disease, for example: 
Unlike the adaptive immune system, which uses very specific antibodies to fight pathogens, the innate immune system is limited in the specificity of its response and uses highly inflammatory cells to fight infection. Pattern recognition receptors allow the innate immune system to differentiate between different groups of pathogens (e.g., bacteria, fungi, parasites), but not specific strains or species. The toll-like family of pattern recognition receptors are a foundational component of the innate inflammatory response in animals as complex as humans and as simple as fruit flies.
By recognizing molecular patterns from Gram-positive and Gram-negative bacteria separately, toll-like receptors enhance the specificity of pathogen identification. Gram-positive bacteria have a thick shell around their cellular membrane, mainly composed of peptidoglycan, that protects the bacterium from harmful compounds in its environment. Gram-negative bacteria have a thin peptidoglycan layer encased by a second cellular membrane studded with many surface structures, such as LPS. Peptidoglycan, which is recognized by TLR2, and LPS, which is recognized by TLR4, are examples of the pathogen-associated molecular patterns (PAMPs), which bind to pattern recognition receptors.
In addition to PAMPs, TLR4 and other immune receptors recognize damage-associated molecular patterns (DAMPs), which are endogenous (i.e., produced by the body) compounds that indicate injury. DAMPs include DNA, adenosine triphosphate (ATP), and heat-shock proteins, which are normally bound inside cells and not accessible in the extracellular space where immune cells patrol; hence, their presence in the extracellular space may be as a consequence of cell lysis and thus signal the damaging activities of a pathogen. Cells that express PAMP and DAMP receptors, such as macrophages, are vital to amplifying the immune response to infection and stimulating repair pathways.
Endotoxemia is the condition of having above-average levels of LPS or other endotoxins in the bloodstream. The most extreme example of this condition is sepsis, which occurs when an infection overwhelms the immune system, causing excessive inflammation and tissue injury that can be fatal. Before the advent of antibiotics, the most common causes of sepsis were gram-positive bacteria such as Staphylococcus. Currently, gram-negative bacteria such as Escherichia coli are the most common causes of sepsis, increasing blood LPS levels 100 times their normal level, resulting in an overwhelming overproduction of proinflammatory cytokines effected by TLR4, leading to cell damage and consequent organ failure.
Metabolic endotoxemia, on the other hand, is the name researchers have given to the condition of having persistent, moderately increased endotoxin levels (two- to three-fold increase), which often occurs in people with metabolic disease. Research increasingly suggests that metabolic endotoxemia plays a role in chronic disease, potentially facilitated through the translocation of bacteria from the intestinal lumen as a result of intestinal permeability, (sometimes referred to as "leaky gut").
Increased intestinal permeability is a feature of metabolic diseases such as obesity,, non-alcoholic fatty liver disease, type 2 diabetes, and cardiovascular disease. Patients with metabolic diseases tend to have higher blood levels of endotoxin, leading to chronic activation of TLR4 and its downstream pro-inflammatory pathways. In addition to genetic susceptibilities, environmental exposures contribute to increased intestinal permeability; however, diets high in fiber-  and polyphenol-rich plant foods,  exercise, and probiotics have been shown to strengthen the gut barrier.
In healthy people, the largest reservoir of endotoxin in the body is the colon, where commensal gram-negative bacteria such as Bacteroides and Prevotella support a healthy gut microbiota and immune system. But under conditions of increased intestinal permeability, endotoxin produced by the gut microbiota is absorbed into the bloodstream. Once absorbed, endotoxin may be intercepted by TLR4 on circulating innate immune cells such as monocytes, facilitating systemic inflammation, or leak from the bloodstream into the extracellular space in tissues such as adipose tissue or liver tissue. Tissue-resident cells that express TLR4 include immune cells (e.g., macrophages, neutrophils, dendritic cells) and various non-immune cell types including liver cells, muscle cells, blood vessel (i.e., endothelial) cells, and adipocytes (i.e., fat cells). In tissue-resident cells, TLR4 activation initiates localized inflammation and the recruitment of pro-inflammatory immune cells. Previous research has demonstrated that the number of TLR4 receptors expressed by muscle  and endothelial cells increase with age, suggesting that TLR4 plays a functional role in inflammaging.
"In the setting of metabolic endotoxemia, chronic TLR4 activation can lead to excessive reactive oxygen species (ROS) formation in the liver, increasing the risk of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, a precursor of liver cancer." Click To Tweet
Endotoxins that seep through the intestinal barrier infiltrate the mesentery, a network of connective tissue, blood vessels, lymph vessels, nerves, and other cells that envelops and supports the intestines. As part of normal digestion, nutrient-rich blood is carried from the intestines to the liver, where compounds are transformed, repackaged, and exported to cells in the rest of the body. Because the liver has the first pass at metabolizing substances in the blood, it is necessary to detoxify harmful chemicals and PAMPs before they disperse throughout the body. In the setting of metabolic endotoxemia, chronic TLR4 activation can lead to excessive reactive oxygen species (ROS) formation in the liver, increasing the risk of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.
In addition to the liver, skeletal muscle is a vitally important site of glucose absorption from the bloodstream. Healthy skeletal muscle is sensitive to insulin and has the capacity to absorb and utilize carbohydrates efficiently. However, chronic TLR4 activation in muscle directly interferes with insulin sensing pathways. Loss of insulin sensitivity in muscle increases blood sugar levels, contributing to type 2 diabetes and cardiovascular disease development.
The endothelial cells that line blood vessels are also sensitive to the ROS produced by excessive TLR4 activation. Endothelial dysfunction is one of the first steps in the formation of atherosclerotic plaques, suggesting that metabolic endotoxemia may promote atherosclerosis and other cardiovascular diseases. Observational evidence demonstrates a relationship between cardiovascular disease and pathogens such as Chlamydia pneumoniae, Porphyromonas gingivalis, and _ Helicobacter pylori_ in the gut and oral microbiota. Animal studies demonstrate that these pathogens can be found within atherosclerotic plaques, suggesting that the gut microbiota plays an important role in cardiovascular disease progression. However, the precise mechanisms require further research.
Adipose tissue is another important site of glucose absorption that is dependent on insulin and impaired by reactive oxygen species. Adipose tissue grows by hypertrophy (i.e., increase in the size of cells) instead of hyperplasia (i.e., increase in the number of cells). Because of this, excessive weight gain causes adipocytes to swell and leak free fatty acids into the surrounding cellular environment. These fats bind to TLR4, acting similar to a DAMP, a molecule that should not normally be abundant in the intercellular space unless cells are damaged.
Excessive TLR4 activation by endotoxin absorbed by the digestive tract or free fatty acids secreted by dysfunctional adipocytes recruits macrophages to the signal site, potentiating inflammation. Inflammatory macrophages clear free fatty acids and cellular waste from dysfunctional adipocytes, but may themselves become dysfunctional due to oxidative damage. In this case, macrophages form crown-like structures, which are aggregates of adipose and immune cells that increase localized tissue inflammation. Adipose tissue dysfunction contributes to a further decline of insulin sensitivity and increase in baseline inflammation, a hallmark of chronic disease.
Chronic inflammation is instrumental in the pathophysiology of many diseases, including depression, and compelling evidence suggests this relationship is causal. Elevated biomarkers of inflammation, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), are commonly observed in people with depression. These pro-inflammatory cytokines coincide with the development of depressive symptoms and induce changes in the brain and neuroendocrine function.
As the principal inducer of innate immunity, toll-like receptors can be expected to play a profound role in mediating inflammation-associated depression. In a double-blind, placebo-controlled trial, the TLR4 pathway was activated by administering low-dose LPS (0.6 ng/kg) to healthy adults. This low-dose endotoxemia rapidly induced a 25-fold increase in plasma TNF-alpha and a 100-fold increase in plasma IL-6.
Alcohol use disorder has complex environmental and genetic causes and is associated neuropsychiatric diseases such as depression attention deficit hyperactivity disorder , psychotic disorders, and dementia, among others. These disorders involve disruption of the brain's normal functions, which may include hyperactivity of the immune system at the blood-brain barrier.
Alcohol exposure can disrupt the delicate lipid membranes that enclose cells, increasing the permeability of cellular barriers in the gut and brain. Similar to leaky gut, leaking of the blood-brain barrier increases endotoxin transport into brain tissue due to disruption of the tight-junction proteins that hold neighboring barrier cells together. Previous research in animals demonstrates that chronic excessive alcohol exposure increases intestinal permeability, endotoxemia, and liver inflammation.
Chronic mild endotoxemia increases inflammation in TLR4 sensing tissues, such as the blood vessels that comprise the blood-brain barrier. TLR4 activation locally or systemically increases production of inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, which have been shown to cross the blood-brain barrier and increased symptoms of depression and cognitive impairment. Therapies that strengthen the integrity of the blood-brain barrier have the potential to improve a wide range of neuropsychiatric disorders.
Drugs that modulate neurotransmitter (e.g., serotonin, dopamine, acetylcholine) concentrations are a common strategy for treating alcohol misuse and neuropsychiatric disorders; however, many patients don't adequately respond to these drugs.
Findings from one study utilizing gene therapy methods demonstrates efficacy in reducing alcohol use and highlights the role of TLR4 signaling in alcohol use disorders. The authors utilized gene therapy to silence gamma-butyric acid (GABA) receptors or TLR4 in a brain region called the central nucleus of the amygdala in a mouse model of binge drinking. Silencing either pathway reduced binge drinking behavior. TLR4 is activated downstream of GABA receptors in this region of the brain, which researchers believe may contribute to brain injury as a result of binge drinking.
"Previous research has demonstrated that environmental factors, such as trauma, can alter the epigenome of immune cells, increasing the expression of pro-inflammatory genes that increase the risk of post-traumatic stress disorder (PTSD) and other illnesses." Click To Tweet
TLR4 activation results in changes to gene expression that increase inflammatory mediators. The process of regulating gene expression involves epigenetic modification, the process of chemically modifying DNA structures to enhance or silence specific genes. These modifications include histone acetylation, DNA methylation, and non-coding RNAs. Previous research has demonstrated that environmental factors, such as trauma, [can alter the epigenome of immune cells, increasing the expression of pro-inflammatory genes that increase the risk of post-traumatic stress disorder (PTSD) and other illnesses. The omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have potent anti-inflammatory effects but aren't yet used to treat PTSD or similar illnesses.
One study measuring the effects of a five-gram daily EPA + DHA supplement for six months in women at high risk of breast cancer found that supplementation did not change average genome-wide methylation density. However, when the researchers focused on genes related to breast cancer progression specifically, omega-3 supplementation led to increased methylation in the promoter region of two genetic pathways – focal adhesion, a process of connective tissue remodeling that contributes to cancer metastasis, and toll-like receptor signaling, which involves a family of immune receptors that recognize molecular patterns from pathogens and promote inflammation. Hypermethylation in the promoter region of a gene reduces the expression of all genes downstream of that promoter, suggesting that omega-3s reduce inflammation by selectively turning off pro-inflammatory genes.
These results provide valuable insight into the precise mechanisms that underlie the health benefits of omega-3s. While the researchers initially predicted that omega-3 supplementation would reduce inflammation by removing methyl groups from the anti-inflammatory genes (increasing their expression), the opposite mechanism – adding methyl groups to pro-inflammatory genes – was discovered.
Intestinal permeability - Increased intestinal permeability causes leaking of endotoxin from the gut into the bloodstream, activating whole-body TLR4 signaling and contributing to chronic inflammaging.
Blood-brain barrier - The blood-brain barrier is regulated in part by toll-like receptors that respond to pathogen-associated molecular patterns to protect the brain from infection and diseases of aging.
Polyphenols - Bioactive plant compounds found in food that strengthen the gut barrier and reduce overactive toll-like receptor signaling.