Psoriasis, IL-17, Cathelicidin, TLRs, NFkB, Inflammation and Heparin Therapy

Host DNA Released by Keratinocyte Apoptosis Binds LL-37 and Activates Dendrocytes

Psoriasis is an inflammation of the skin that leads to overproduction of keratinocytes resulting in a thick crust.  Skin inflammation, in this case, is considered a result of autoimmunity, but an autoantigen has not been identified.  It is more likely that psoriasis results from an autoinflammatory condition, in which inflammation produces a complex of self molecules that mimic bacterial DNA and trigger TLR/NFkB inflammation signaling.  And of course, if this is going to be interesting, it has to involve heparin.

Vitamin D Binds to a Transcription Factor Receptor that Controls Antimicrobial Peptides
A significant component of the innate immune system is a group of antimicrobial peptide  (defensins, cathelicidins, e.g. LL-37).  These short polypeptides owe their natural antibiotic activity to numerous basic (positively charged, arginine and lysine) amino acids.  The transcription factor that controls the expression of these peptides is the vitamin D receptor.  Thus, various forms of vitamin D influence the amount of antimicrobial peptides produced in the mouth, skin and crypts of the intestinal villi.  Oral vitamin D3 would be expected to directly improve defensin production in the gut and LL-37 production in the skin.

IL-17 Stimulates Skin Inflammation and LL-37 Production
A specific group of lymphocytes, called T helper 17 cells, produce IL-17.  These Th17 cells accumulate in some sites of inflammation, such as psoriasis and their secretion of IL-17 is associated with ongoing inflammation and may contribute to LL-37 production, as well as apoptosis of keratinocytes in the thickening skin of psoriasis plaques.
http://www.ncbi.nlm.nih.gov/pubmed/19623255?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_SingleItemSupl.Pubmed_Discovery_PMC&linkpos=2&log$=citedinpmcarticles&logdbfrom=pubmed

Th17 Cells Are Produced in the Gut in Response to Segmented Bacteria
One of my readers brought to my attention an article that shows that one of the hundreds of species of gut bacteria, segmented filamentous baceria, stimulates the gut to develop T helper 17 cells that subsequently migrate to sites of inflammation.
http://www.medpagetoday.com/Gastroenterology/InflammatoryBowelDisease/16472
This emphasizes the link between the gut and inflammatory diseases and parallels other examples of gut influence on disease, such as the ability of Helicobacter pylori to affect asthma or parasitic worms to tame Crohn’s disease, allergies and asthma.

Inflammation Lowers Heparan Sulfate Production and Spreads LL-37
One of my students induced inflammation in cells in vitro and showed by quantitative PCR that genes involved in heparan sulfate proteoglycan production are selectively silenced.  This observation explains in part the loss of heparan sulfate in kidneys and intestines that contributes to the leakiness of these organs in response to inflammation and the partial repair of these organs by heparin treatment.  Decrease of heparan sulfate that normally coats cells and binds antimicrobial peptides, such as LL-37, would explain the enhanced movement of LL-37 in psoriatic skin.

LL-37 Binds to Host DNA and Triggers Toll-Like Receptors
DNA is released from keratinocytes in psoriatic skin and this host DNA binds the antimicrobial peptide cathelicidin LL-37.  The LL-37/DNA complex mimics bacterial DNA and triggers the Toll-like receptors (TLR) on the surface of immune cells, dendrocytes, to activate NFkB, the transcription factor controlling inflammation.
http://www.ncbi.nlm.nih.gov/pubmed/19050268?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_SingleItemSupl.Pubmed_Discovery_RA&linkpos=1&log$=relatedarticles&logdbfrom=pubmed

Heparin Treats Psoriasis
It seemed obvious to me that the heparin binding domains (Look at all the basic amino acids in blue in the illustration of LL-37.) of LL-37 were involved in DNA binding and the reason the LL-37 was binding to host DNA, was that heparan sulfate had been depleted as a result of local inflammation.  It also seemed obvious that topical heparin should eliminate psoriasis plaques.  So I did a Google search of psoriasis + topical heparin and got a hit on a 1991 patent application that claims a broad applicability for heparin use in curing symptoms of a wide variety of diseases, including psoriasis.
http://www.patentstorm.us/patents/5037810/description.html
The only topical form of heparin that I know of is Lipactin (available in Canada and Europe?), a treatment for coldsores, which makes sense because herpes viruses use heparan sulfate to infect cells.

Antimicrobial Heparin-binding Domains

Antimicrobial Digestion

Stomach enzymes produce antimicrobial peptides and intestinal enzymes inactivate heparin-binding domains of pathogens

The human digestive systems exhibits some amazing adaptations -- it can excise heparin-binding domains from ingested proteins and use them as antimicrobial peptides to inactivate ingested bacteria. Then in subsequent enzymatic steps in the intestines, the heparin-binding domains that otherwise could be used to adhere bacteria to the heparan sulfate proteoglycans of the intestinal lining, are chopped into inactive peptide fragments.

I was trying to find a cheap heparin-binding protein to use for a variety of research projects and finally found it in the form of whey lactoferrin. This protein is available in a relatively pure form for about $1 per milligram. Alternatively, I could buy it as a nutriceutical in 250 mg capsules for $0.50 per capsule or 1/500th the cost. My next step was to purify the cheap lactoferrin by binding it to chromatography beads with attached heparin. The lactoferrin stuck to the beads and other contaminating material was washed away. Finally, pure lactoferrin was released by increasing the salt concentration of the wash solution. My goal was to use proteolytic enzymes to hydrolyze the lactoferrin and produce peptide fragments containing heparin-binding domains. I naively pasted the known amino acid sequence of bovine lactoferrin into a website that would predict the cleavage locations of numerous proteases along the lactoferrin molecule. Amazingly, pepsin, the stomach protease, released a couple of peptides with heparin-binding domains, e.g. KCRRWQWRMKK, whereas trypsin, the intestinal protease, degraded all of the heparin-binding domains. I had a simple procedure for producing the peptides I wanted, but I also learned something about the beauty of the digestive system.

It took me a while to realize the utility of the alternative proteases. Production of heparin-binding peptides by pepsin enhances the sterilization of meals, because the heparin-binding domains are also generally antimicrobial. In fact, most antimicrobial peptides secreted by the skin or venom of a wide variety of organisms from primates to poison dart frogs have heparin-binding sequences. Degradation of heparin-binding domains by trypsin in the intestines is also advantageous, because numerous bacteria (e.g. E. coli O157;H7) and viruses (e.g. HIV and avian flu) use heparan sulfate regions of gut proteoglycans as receptors to immobilize these pathogens on the surface and initiate infections. Some toxins that rely on heparin-binding are also inactived in the intestines.

Rosacea: Alzheimer’s of the Face

Is Rosacea Caused by Amyloid LL-37, as Alzheimer’s Is Caused by Anti-microbial Abeta?
A recent article in PLoS One (Thanks Daniel!) suggests that the amyloid beta (Abeta) proteins that aggregate to form fibrous plaques in the brain tissue of Alzheimer victims, function as typical defensive anti-microbial peptides (AMPs), similar to the LL-37 cathelicidin implicated in facial tissue in rosacea.  The structural and functional similarities of Abeta and LL-37 suggest to me that Alzheimer’s and rosacea may also be similar in initiation and treatment.  Let’s compare amyloids and AMPs.

[The figure shows a model protein (from ref.) used to examine stain binding to amyloids.  The stains appear to bind to aromatic amino acids spaced evenly between adjacent proteins, but adjacent basic amino acids (blue) are spaced the same way and provide sites for heparin binding.]

Amyloids:

• Amyloid proteins/peptides align into stacks and fibers
• Stacked beta sheets bind amyloid stains: Congo Red, Thioflavin-T
• Fibers form on anionic polymers: heparin, DNA
• Short amyloid stacks are toxic to cells
• Proteases produce multiple sizes of amyloid peptides

Anti-microbial Peptides:

• AMPS typically contain heparin-binding domains -- basic peptides/ plus charge
• Some AMPs, e.g. LL-37, form fibers on DNA, heparin (stain with amyloid stains)
• Toxic to cell membranes
• Kallikrein stimulated by gut flora migrates to face and clips LL-37 to a smaller peptide that binds to host DNA and stimulates the TLR receptor to produce inflammation
• Stomach pepsin hydrolyzes dietary proteins into anti-microbial peptides (heparin is secreted by mast cells onto to the intestinal surface to protect from any amyloid-like effects)
• Defensins, cathelicidins and other AMPs are under transcriptional control of vitamin D receptor

Abeta Is Anti-microbial Like LL-37

Amyloid beta is the well-known source of the fibrous plaques forming brain lesions in Alzheimer’s disease.  The normal function of Abeta has not been firmly established.  The recent article shows data to support Abeta as an anti-microbial peptide comparable to LL-37 against several pathogenic bacteria and yeast.  Knock-out mice deprived of a gene corresponding to Abeta are susceptible to bacterial infections.  The anti-microbial activity present in extracts from Alzheimer’s disease brains was inactivated by anti-Abeta antibodies.

Implications of Abeta as an AMP Like LL-37

The similarities between AMPs and amyloid peptides suggest some implications for both Alzheimer’s disease and rosacea.  Vitamin D is a hormone that binds to a cytoplasmic receptor and the vitD/receptor complex then acts as a transcription factor that controls the expression of defensins in the intestines, LL-37 in facial skin and perhaps Abeta in brains.

Amyloids form fibers on a scaffolding of heparan sulfate (HS).  There is usually an excess of HS on the surface of cells and the HS is rapidly recycled back into cells.  During inflammation, mast cells release heparin, short fragments of HS, that should also inhibit amyloid fiber formation on HS.   Chronic inflammation, however, reduces HS production and may set the stage for amyloid fiber formation.  HS metabolism of the brain may be vitally important to the development of Alzheimer’s disease, especially since the increasing chronic inflammation of aging people should deplete brain HS.

LL-37 forms complexes with DNA from damaged host cells in rosacea skin.  The LL-37/DNA complexes trigger TLRs and inflammation.  LL-37 may normally bind to cell surface HS and chronic inflammation of the skin may cause the shift to pathogenic autoinflammation.  Topical application or perhaps low dose IV heparin may be effective in disrupting the autoinflammation due to LL-37.  Part of the toxicity of LL-37 in the skin may be due to amyloid like structures that could form with inadequate HS and overabundant LL-37 production.  Vitamin D metabolism should also be very important, since LL-37 synthesis is controlled by vitamin D.  This is consistent with the benefits that some rosaceans observe with high doses of vitamin D3 supplements.

references:
Soscia SJ, Kirby JE, Washicosky KJ, Tucker SM, Ingelsson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD.  The Alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide.  PLoS One. 2010 Mar 3;5(3):e9505.

Abedini A, Tracz SM, Cho JH, Raleigh DP.  Characterization of the heparin binding site in the N-terminus of human pro-islet amyloid polypeptide: implications for amyloid formation.  Biochemistry. 2006 Aug 1;45(30):9228-37.

Biancalana M, Makabe K, Koide A, Koide S. Molecular mechanism of thioflavin-T binding to the surface of beta-rich peptide self-assemblies.  J Mol Biol. 2009 Jan 30;385(4):1052-63. Epub 2008 Nov 14.

Crohn’s Disease and Cryptidins

The intestines produce enzymes to digest food, antimicrobial peptides to kill pathogens and have lots of surface area to absorb nutrient molecules released from the food macromolecules (protein, polysaccharides, fats). The epithelial cells that line the intestines, enterocytes, must communicate with bacteria in the gut, the gut flora, to maintain bacteria helpful in food digestion, i.e. probiotic bacteria, and trigger an immune response to eliminate pathogens. Probiotic bacteria are tolerated and pathogens are identified and attacked.

Enterocytes are produced by division of stem cells at the bottom of the crypts that are in the valleys between the villi that project into the lumen where the digesting food is. New enterocytes are added at the base of the villi and old enterocytes are sloughed off at the top of the villi. As the new enterocytes move up the villi, they differentiate to produce the dramatic surface of microvilli, the furry brush border that further expands their surface area. The mature enterocytes produce transport proteins on their microvilli to take up sugars, amino acids and fats. The small nutrient molecules pass through the base of the enterocytes and bath the cells below, the lamina propia. The nutrients enter the capillaries of the villi and travel to the liver. Fats are transported through the lymphatic system.

Bacteria that slip through the enterocyte layer encounter macrophages and other types of white blood cells of the lamina propia. Among these cells are the Paneth cells. Fragments of the cell walls of bacteria bind to the NOD proteins of the Paneth cells and trigger the secretion of antimicrobial peptides, the cryptidins. Cryptidins are antimicrobial because of their array of basic amino acids surrounded by hydrophobic amino acids. These short proteins are able to disrupt the membrane function of most bacteria. I think they work on bacteria the same way that amyloid proteins, e.g. amyloid plaque proteins of Alzheimer’s disease, kill human cells. In fact, amyloid fibers bind to heparin and so do antibiotic peptides.

Here is an example of an antibiotic peptide, cryptidin 4,

GLLCYCRKGHCKRGERVRGTCGIRFLYCCPRR

Note the pairs of basic amino acids (blue). These amino acids are necessary for toxicity to bacteria. Heparin binding domains from proteins are produced naturally as proteins are digested to peptides in the stomach by pepsin. Pepsin hydrolyzes proteins next to the basic amino acids and leaves antimicrobial peptides that sterilize incoming food. I have illustrated the cryptidin protein to show how the basic amino acids (blue) are displayed on its surface.

With each meal, the fat content normally stimulates the production of a hormone, cholecystokinin, that binds to a receptor and causes an anti-inflammatory release of cytokines from the vagus nerves that reach the villi. Thus, food normally makes the intestines more tolerant of food antigens.

If the intestines become chronically inflamed, then exposure to normal probiotic bacteria can lead to cycles of inflammation that damage the integrity of the intestines. The intestines lose the ability to discriminate between probiotic and pathogen.

Crohn’s disease is an inflammatory, autoimmune disease of the bowel. The chronic inflammation of the lamina propia eliminate the ability of the Paneth cells to produce cryptidins and bacteria set up residence in the crypts and cause continual inflammation. This disease is typically treated by suppressing inflammation and treating with antibiotics.

Other treatment approaches that have been found effective are omega-3 oils to stimulate production of anti-inflammatory prostaglandins, pre- and probiotics, heparin and helminth eggs, e.g. wireworm.

Crohn’s disease would seem to benefit from the standard recommendation of an anti-inflammatory diet and lifestyle.

Lactoferrin: Natural Anti-Microbial Milk Protein

Nosocomial infections happen when the immune system is compromised through a medical procedure and common bacteria, such as Staphlococcus aureus, get introduced. From my perspective, that means that the walking bacterial reservoirs, i.e. gut flora of healthcare practitioners, provide an inoculum directly to the damaged tissue, or indirectly by contaminating the patients gut flora and then spreading the pathogens from the patient’s digestive tract to the damage site. This is what happens with ventilator-associated pneumonia and sepsis in critically ill patients.

The trick is to keep the patient’s gut flora healthy -- healthy as a breastfed baby’s. The typical medical approach is to kill off lurking pathogens with a dose of antibiotics. The problem with this approach is that it is both indiscriminant and selective, i.e. it kills both pathogens and beneficial bacteria, but it also provides a selective advantage for the antibiotic resistant hospital strains of opportunistic pathogens.

Humor break: Why do babies spit up half-digested breastmilk and then smile? Answer: Pepsin produces antimicrobial peptides from milk proteins. The baby smugly acknowledges that she knows that she has just protected her upper respiratory and digestive tracts against bacterial pathogens.

Pepsin hydrolyzes proteins next to aromatic amino acids and away from the basic amino acids, arginine and lysine. That means that heparin-binding domains, which consist of groups of basic amino acids in a hydrophobic environment, are clipped out intact from proteins by pepsin. Thus, babies sucking down milk make their own isolated peptides with heparin-binding domains.

Lactoferricin with basic amino acids in blue.

Many organisms, from fruit flies to frogs to humans, produce anti-microbial peptides. They also produce proteins with nucleic acid-binding domains and nuclear localization signals and heparin-binding domains and IP3-binding domains. If all of those binding domains are clipped out by pepsin and the peptides are compared to the anti-microbial defensive peptides, amazingly they are all the same. All have groups of basic amino acids among hydrophobic neighbors, and all are toxic to bacteria.

Lactoferrin is a major component of milk whey. It binds iron and heparin. It can be digested by pepsin into an an anti-microbial peptide, lactoferricin. Baby’s smile and spit-up on your shoes when you say lactoferrin.

Transgenic mice that produce porcine lactoferrin in their milk, transfer extra lactoferrin their little suckling mouse pups and that extra lactoferrin gives extra protection against bacterial and yeast pathogens. That is the experimental justification to suggest that treating patients at risk of nosocomial infections (I guess that would mean every patient in contact with a nurse or doctor) with oral lactoferrin should selectively eliminate the pathogens. Lactoferrin is prebiotic and supports the growth of probiotic gut flora.

ref:
Yen CC, Lin CY, Chong KY, Tsai TC, Shen CJ, Lin MF, Su CY, Chen HL, Chen CM. Lactoferrin as a natural regimen for selective decontamination of the digestive tract: recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonates from pathogenic challenge in the gastrointestinal tract .J Infect Dis. 2009 Feb 15;199(4):590-8.