THE INNATE IMMUNE SYSTEM
E. HARMFUL EFFECTS ASSOCIATED WITH MICROBIAL-INDUCED INFLAMMATION AND WITH ABNORMAL PATTERN-RECOGNITION RECEPTOR RESPONSES
The overall purpose of this Learning Object is:
1) to learn some of the harmful effects associated with microbial-induced inflammation, improper pattern-recognition receptor responses, and/or excessive cytokine production; and
2) to introduce potential therapeutic possibilities associated with improper pattern-recognition receptors responses.
A. The Ability of Pathogen-Associated Molecular Patterns or PAMPs to Trigger the Synthesis and Secretion of Excessive Levels of Inflammatory Cytokines and Chemokines
In order to protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns (PAMPs). (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-associated molecular patterns or MAMPs.)
Molecules unique to bacterial cell walls, such as peptidoglycan monomers, teichoic acids, LPS, porins, mycolic acid, and mannose, are PAMPs that bind to pattern-recognition receptors (PRRs) on a variety of defense cells of the body causing them to synthesize and secrete a variety of proteins called cytokines (def). These cytokines can, in turn promote innate immune defenses such as inflammation, fever, and phagocytosis. The binding of PAMPs to PRRs also leads to activation of the complement pathways (def) and activation of the coagulation pathway (def).
Cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interleukin-8 (IL-8) are known as inflammatory cytokines (def) because they promote inflammation. Some cytokines, such as IL-8, are also known as chemokines (def). Chemokines promote an inflammatory response by enabling white blood cells to leave the blood vessels and enter the surrounding tissue, by chemotactically attracting these white blood cells to the infection site, and by triggering neutrophils (def) to release killing agents for extracellular killing.
- Inflammation (def) is the first response to infection and injury and is critical to body defense. Basically, the inflammatory response is an attempt by the body to restore and maintain homeostasis (def) after injury. Most of the body defense elements are located in the blood, and inflammation is the means by which body defense cells and defense chemicals leave the blood and enter the tissue around an injured or infected site. The release of inflammatory cytokines eventually leads to vasodilation of blood vessels. Vasodilation (def) is a reversible opening of the junctional zones between endothelial cells (def) of the blood vessels and results in increased blood vessel permeability. This enables plasma, the liquid portion of the blood, to enter the surrounding tissue.The plasma (def) contains defense chemicals such as antibody molecules (def), complement proteins (def), lysozyme (def), and beta defensins (def). Increased capillary permeability also enables white blood cells to squeeze out of the blood vessels and enter the tissue. As can be seen, inflammation is necessary part of body defense. Excessive or prolonged inflammation can, however, cause harm as will be discussed below.
(Scanning electron micrographs of a cross section of a capillary showing an endothelial cell and a capillary with a red blood cell; courtesy of Dennis Kunkel's Microscopy.)
- Products of the complement pathways (def) lead to: 1) more inflammation; 2) opsonization of bacteria; 3) chemotaxis of phagocytes to the infected site; and 4) MAC lysis of gram-negative bacteria, viral envelopes, and human cells recognized as foreign.
- The products of the coagulation pathway (def) lead to the clotting of blood to stop bleeding, more inflammation, and localization of infection.
At moderate levels, inflammation, products of the complement pathways, and products of the coagulation pathway are essential to body defense. However, these same processes and products when excessive can cause considerable harm to the body.
During minor local infections with few bacteria present, low levels of cell wall PAMPs are released leading to moderate cytokine production by defense cells such as monocytes, macrophages (def), and dendritic cells (def) and, in general, promoting body defense by stimulating inflammation and moderate fever, breaking down energy reserves to supply energy for defense, activating the complement pathway (def) and the coagulation pathway (def), and generally stimulating immune responses (see Fig. 9). Also as a result of these cytokines, circulating phagocytic white blood cells such as neutrophils (def) and monocytes stick to the walls of capillaries, squeeze out and enter the tissue, a process termed diapedesis (def). The phagocytic white blood cells such as neutrophils then kill the invading microbes with their proteases and toxic oxygen radicals.
However, during severe systemic infections with large numbers of bacteria present, high levels of cell wall PAMPs are released resulting in excessive cytokine production by the defense cells and this can harm the body (see Fig. 10). In addition, neutrophils (def) start releasing their proteases and toxic oxygen radicals that kill not only the bacteria, but the surrounding tissue as well. Harmful effects include high fever, hypotension (def), tissue destruction, wasting, acute respiratory distress syndrome (ARDS) (def), disseminated intravascular coagulation (DIC) (def), and damage to the vascular endothelium. This can result in shock (def), multiple system organ failure (MOSF), and death.
3D animation illustrating innate and adaptive inflammatory response in Chron's disease.
Gardenia Gonzalez Gil, Living Pixels. This animation takes some time to load.
Keep in mind that a primary function of the circulatory system is perfusion, the delivery of nutrients and oxygen via arterial blood to a capillary bed in tissue. This, in turn, delivers nutrients for cellular metabolism and oxygen for energy production via aerobic respiration to all of the cells of the body.
Sepsis is an infection that leads to a systemic inflammatory response resulting in physiologic changes occurring at the capillary endothelial level. This systemic inflammatory response is referred to as Systemic Inflammatory Response Syndrome or SIRS.
Based on severity, there are three sepsis syndromes based on severity:
1. Sepsis. SIRS in the setting of an infection.
2. Severe sepsis. An infection with very low blood pressure and end-organ dysfunction as a result of hypoperfusion, the reduced delivery of nutrients and oxygen to tissues and organs via the blood.
3. Septic shock. Severe sepsis with persistent hypotension (def) and tissue hypoperfusion (def) despite fluid resuscitation.
We will now take a look at the underlying mechanism of SIRS that can result in septic shock.
Systemic Inflammatory Response Syndrome (SIRS) Resulting in Septic Shock
During a severe systemic infection, an excessive inflammatory response triggered by overproduction of inflammatory cytokines such as TNF-alpha, IL-1, IL-6, IL-8, and PAF in response to PAMPs often occurs.
The release of inflammatory cytokines eventually leads to vasodilation of blood vessels. Vasodilation (def) is a reversible opening of the junctional zones between endothelial cells (def) of the blood vessels and results in increased blood vessel permeability. Normally, this fights the infection by enabling plasma, the liquid portion of the blood, to enter the surrounding tissue.The plasma (def) contains defense chemicals such as antibody molecules (def), complement proteins (def), lysozyme (def), cathelicidins, and beta defensins (def). Increased capillary permeability also enables white blood cells to adhere to the inner capillary wall, squeeze out of the blood vessels, and enter the tissue to fight infection, a process called diapedesis (def) or extravasation.
Excessive productions of cytokines during a systemic infection results in the following events:
1. During diapedesis (def), phagocytic WBCs called neutrophils (def) adhere to capillary walls in massive amounts.
- Chemokines such as IL-8 activate extracellular killing by neutrophils, causing them to release proteases (def) and toxic oxygen radicals (def) while still in the capillaries. These are the same toxic chemicals neutrophils use to kill microbes, but now they are dumped onto the vascular endothelial cells to which the neutrophils have adhered.
- These events result in damage to the capillary walls and leakage of blood into surrounding tissue (see Fig. 3). This contributes to a decreased volume of circulating blood (hypovolemia) (def).
- Hypovolemia then contributes to hypoperfusion (def).
2. Prolonged vasodilation (def) and the resulting increased capillary permeability causes plasma (def) to leave the bloodstream and enter the tissue. Activation of the complement pathways and production of vasodilators such as C5a, C3a, prostaglandins, and leukotrienes further contributes to fluid loss.
- This contributes to a decreased volume of circulating blood (hypovolemia) (def).
- Hypovolemia then contributes to hypoperfusion (def).
Prolonged vasodilation also leads to decreased vascular resistance within blood vessels.
- This, in turn, contributes to a drop in blood pressure (hypotension).
- Hypotension then contributes to hypoperfusion (def).
3. At high levels of TNF, vascular smooth muscle tone and myocardial contractility are inhibited.
Cytokine-induced overproduction of nitric oxide (NO) by cardiac muscle cells and vascular smooth muscle cells can also lead to heart failure.
4. Activation of the blood coagulation pathway can cause clots called microthrombi to form within the blood vessels throughout the body. This is called disseminated intravascular coagulation (DIC) (def).
- These microthrombi block the capillaries and interfere with perfusion (def).
- Activation of neutrophils also leads to their accumulation and plugging of the vasculature.
- Depletion of clotting factors leads to hemorrhaging in many parts of the body following neutrophil-induced capillary damage.
5. In the lungs, the increased capillary permeability as a result of vasodilation in the lungs, as well as neutrophil-induced injury to capillaries in the alveoli (def) leads to acute inflammation, pulmonary edema (def), and loss of gas exchange in the lungs. This condition is called acute respiratory distress syndrome (ARDS) (def).
- As a result, the blood does not become oxygenated.
- Lack of oxygenation of the blood via the lungs then causes hypoperfusion (def).
6. In the liver, hypoperfusion (def) and capillary damage results in impaired liver function and a failure to maintain normal blood glucose levels.
- Overuse of glucose by muscles and a failure of the liver to replace glucose can lead to a drop in blood glucose level below what is needed to sustain life. (Glucose is needed to make ATP via aerobic respiration.)
7. Hypoperfusion can also leads to kidney and bowel injury.
8. The combination of hypotension (def), hypovolemia (def), DIC (def), ARDS (def), and the resulting hypoperfusion (def) leads to acidosis (def).
- Without oxygen, cells switch to fermentation and produce lactic acid that lowers the pH of the blood. A blood pH range between 6.8 and 7.8 is needed for normal cellular metabolic activities in humans.
- Changes in the pH of arterial blood extracellular fluid outside this range lead to irreversible cell damage.
In summary:
- Neutrophil-induced damage to the capillaries, as well as prolonged vasodilation, results in blood and plasma leaving the bloodstream and entering the surrounding tissue. This can lead to a decreased volume of circulating blood (hypovolemia).
- Prolonged vasodilation also leads to decreased vascular resistance within blood vessels while high levels of TNF, inhibit vascular smooth muscle tone and myocardial contractility. This results in a marked hypotension.
- Activation of the blood coagulation pathway can cause clots called microthrombi to form within the blood vessels throughout the body causing disseminated intravascular coagulation (DIC).
- Increased capillary permeability as a result of vasodilation in the lungs, as well as neutrophil-induced injury to capillaries in the alveolileads to acute inflammation, pulmonary edema, and loss of gas exchange in the lungs (acute respiratory distress syndrome or ARDS). As a result, the blood does not become oxygenated.
- Hypotension, hypovolemia, ARDS, and DIC result in marked hypoperfusion.
- Hypoperfusion in the liver can result in a drop in blood glucose level from liver dysfunction.
- Hypoperfusion leads to acidosis and the wrong pH for enzymes involved in cellular metabolism resulting in cell death.
- Hypoperfusion also can lead to cardiac failure.
Collectively, this can result in :
- end-organ ischemia (def). Ischemia is a restriction in blood supply that results in damage or dysfunction of tissues or organs.
- multiple system organ failure (MSOF) (def).
- death.
Septicemia (def) is a condition where bacteria enter the blood and cause harm. There are approximately 500,000 cases of septicemia per year in the U.S. and the mortality rate is between 20% and 50%. Over 125,000 people a year in the U.S. die from septic shock. Those that survive may have permanent damage to the lungs or other organs. Approximately 45% of the cases of septicemia are due to gram-positive bacteria, 45% are a result of gram-negative bacteria, and 10% are due to fungi (mainly the yeast Candida).
B. Harmful Effects Associated with either an Overactive or an Underactive Innate Immune Response
There are a number of harmful effects that are known to occur as a result of either an overactive or an underactive innate immune response. This occurs as a result of people possessing different polymorphisms in the various genes participating in PRR signaling.
These include:
a. People with an underactive form of TLR-4 (def), the toll-like receptor for bacterial LPS, have been found to be five times as likely to contract a severe bacterial infection over a five year period than those with normal TLR-4. People with overactive TLR-4 receptors may be more prone to developing SIRS from gram-negative bacteria.
b. Most people that die as a result of Legionnaire's disease have been found to have a mutation in the gene coding for TLR-5 (def) that enables the body to recognize the flagella of Legionella pneumophila.
c. B-lymphocytes (def), the cells responsible for recognizing foreign antigens (def) and producing antibodies (def) against those antigens, normally don't make antibodies against the body's own DNA and RNA. The reason is that any B-lymphocytes that bind the body's own antigens normally undergo apoptisis (def), a programmed cell suicide. People with the autoimmune disease systemic lupus erythematosis have a mutation in a gene that signals the cell to undergo apoptosis. As a result, these B-cells are able to bind and engulf the body's own DNA and RNA and place them in an endosome (def) or phagolysosome (def) where the the DNA can be recognized by TLR-9 (def) and the RNA by TLR-7(def). This, in turn, triggers those B-lymphocytes to make antibody molecules against the body's own DNA and RNA. Another gene error enables these B- cells to increase the expression of TLR-7.
d. TLR-4 (def), MyD88, TLR-1 and TLR-2 (def) have been implicated in the production of artherosclerosis in mice and some humans.
e. Mutations in the gene coding for NOD-2 (def) that prevents the NOD-2 from recognizing muramyl dipeptide make a person more susceptible to Crohn's disease, an inflammatory disease of the large intestines.
f. People with chronic sinusitis that does not respond well to treatment have decreased activity of TLR-9 (def) and produce reduced levels of human beta-defensin 2 (def), as well as mannan-binding lectin (def) needed to initiate the lectin complement pathway (def).
g. Pathogenic strains of Staphylococcus aureus producing leukocydin (def) and protein A (def), including MRSA (def), cause an increased inflammatory response. Protein A, a protein that blocks opsonization (def) and functions as an adhesin (def), binds to cytokine receptors for TNF-alpha (def). It mimics the cytokine and induces a strong inflammatory response. As the inflammatory response attracts neutrophils to the infected area, the leukocydin causes lysis of the neutrophils (def). As a result, tissue is damaged and the bacteria are not phagocytosed.
h. People with chronic mucocutaneous candidiasis disease have a mutation either in the gene coding for IL-17F or the gene encoding IL-17F receptor. Th17 cells secrete cytokines such as IL-17 that are important for innate immunity against organisms that infect mucous membranes.
i. A polymorphism in the gene for TLR-2 makes individuals less responsive to Treponema pallidum and Borrelia burgdorferi and possibly more susceptible to tuberculosis and staphylococcal infections.
j. Polymorphisms in a gene locus called A20, a gene that helps to control inflammation, are considered as risk alleles for rheumatoid arthritis, systemic lupus erythmatosus, psoriasis, type I diabetes, and Chron’s disease.
D. Therapeutic Possibilities
Researchers are now looking at various ways to either artificially activate TLRs in order to enhance immune responses or inactivate TLRs to lessen inflammatory disorders. Examples of agents being evaluated in clinical studies or animal studies include:
1. TLR activators to activate immune responses
a. Both TLR-4 (def) and TLR-9 (def) activators are being tried in early clinical trials as vaccine adjuvants to improve the immune response to vaccines. TLR-9 activators are being tried as an adjuvant for the hepatitis B and anthrax vaccines and a TLR-4 activator is being tried as an adjuvant for the vaccine against the human papillomaviruses that cause most cervical cancer.
b. Both TLR-7 (def) and TLR-9 activators are being tried in early clinical trials as an antiviral against hepatitis C. Activation of these TLRs triggers the synthesis and secretion of type I interferons that block viral replication within infected host cells.
c. TLR-9 activators are being tried in early clinical trials as an adjuvant for chemotherapy in the treatment of lung cancer.
d. TLR-9 activators are being tried in early clinical trials to help in the treatment and prevention of allergies and asthma. Activation of TLR-9 in macrophages and other cells stimulates these cells to kill Th2 cells, the subclass of T-helper lymphocytes responsible for most allergies and asthma.2. TLR inhibitors to suppress immune responses
a. General TLR inhibitors might onday be used to treat autoimmune disorders.
b. A TLR-4 (def) inhibitor, a mimic of the endotoxin from the gram-negative cell wall, is being tried in early clinical trials to block or reduce the death rate from gram-negative sepsis and SIRS (def).
c. TLR-4, TLR-2, and MyD88 inhibitors might possibly one day lessen atherosclerotic plaques and the risk of heart disease.
Of course using TLR activators or TLR inhibitors to turn up or turn down immune responses also carries risks. Trying to suppress harmful inflammatory responses may also result in increased susceptibility to infections; trying to activate immune responses could lead to SIRS or autoimmune disease.
A number of human cytokines produced by recombinant DNA technologies are now being used to treat various infections or immune disorders. These include:
1. recombinant interferon alfa-2a (Roferon-A): a cytokine used to treat Kaposi's sarcoma, chronic myelogenous leukemia, and hairy cell leukemia.
2. peginterferon alfa-2a (Pegasys) : used to treat hepatitis C (HCV).
3. recombinant interferon-alpha 2b (Intron A): a cytokine produced by recombinant DNA technology and used to treat Hepatitis B; malignant melanoma, Kaposi's sarcoma, follicular lymphoma, hairy cell leukemia, warts, and Hepatitis C.
4. peginterferon alfa-2b (PEG-Intron; PEG-Intron Redipen): used to treat hepatitis C (HCV).
5. recombinant Interferon alfa-2b plus the antiviral drug ribavirin (Rebetron): used to treat hepatitis C (HCV).
6. recombinant interferon-alpha n3 (Alferon N): used to treat warts.
7. recombinant iInterferon alfacon-1 (Infergen) : used to treat hepatitis C (HCV).
8. G-CSF (granulocyte colony stimulating factor): for reduction of infection in people after myelotoxic anticancer therapy for solid tumors.
9. GM-CSF (granulocyte-macrophage colony stimulating factor): for hematopoietic reconstruction after bone marrow transplant in people with lymphoid cancers..
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