Innate Recognition

1. Innate Recognition Dr Kathy Triantafilou University of Sussex School of Life Sciences Falmer, Brighton

2. Recommended books • Immunobiology by Janeway and Travers

3. “Immunity”Ability to fight off infection • Innate (non-specific) immunity • basic resistance to disease • anatomic barriers • physiologic barriers • endocytic • phagocytic • inflammation • Acquired (specific) immunity • specificity • diversity • memory • self/non-self discrimination

4. Anatomic barriers • Skin • several layers of tightly packed epithelial cells • hair follicles • sweat glands • Mucous membranes (respiratory and urogenital tracks) • saliva • tears • mucous secretions

5. Physiologic barriers • Temperature • many species are not susceptible to certain diseases because of their body temperature (i.e. chickens and anthrax) • pH • gastric acidity • Various soluble factors • lysozyme (hydrolytic enzyme found in mucous) • interferons (produced by virus-infected cells) • complement (serum proteins that cause membrane-damaging reactions)

6. Endocytic barriers • Endocytosis • the macromolecules contained within the extracellular tissue fluid are internalised by the cells • internalisation occurs as small regions of the plasma membrane invaginate, or fold inward forming small emdocytic vesicles • endocytosis occurs either through pinocytosis or receptor mediated endocytosis

8. Inflammatory response • Inflammatory response: tissue damage caused by a wound or by invasion of a pathogenic microorganism induces a complex sequence of events • Celsus (First century A.D. Roman physician) described four cardinal signs of inflammation: • rubor (redness) • tumor (swelling) • calor (heat) • dolor (pain)

9. Signs of inflammation • Vasolidation • increase in the diameter of blood vessels • engorgement of the capillary network • tissue redness and increased temperature • Increased capillary permeability • influx of fluid into the tissue • Influx of phagocytic cells • migration of white blood cells and phagocytosis

10. Signs of inflammation • Influx of phagocytes from the capillaries into the tissue: • Margination • attachment of cells to the endothelial wall of the blood vessel • diapedesis or extravasation • emigration between the capillary-endothelial cells • chemotaxis • migration through the tissue at the site of infection

13. Pattern recognition receptors • Pattern recognition receptors are employed by the innate immune system in order to recognise pathogens • Pattern recognition receptors (PRRs) • receptors on lymphocytes able to recognise antigens • Antigenic arrays or patterns called pathogen-associated molecular patterns (PAMPs)

15. PAMPs • Pathogen-associated molecular patterns (PAMPs) are: • bacterial lipopolysaccharide (LPS) • bacterial lipoteichoic acid (LTA) • peptidoglycan • bacterial flagellin • bacterial DNA

16. Negative vs Positive

17. Bacterial recognition • Recognition of bacterial components by the innate immune system can sometimes provoke a strong pro-inflammatory response • The production and release of potent pro-inflammatory mediators (such as TNF-a,IL-6, IL-1 etc) is also triggered • Uncontrollable cytokine production can result in cardiovascular collapse, hemodynamic instability and eventually cause sepsis syndrome

18. Septic Shock • Causes approximately 175,000 deaths annually in the U.S. • Afflicts about 500,000 Americans annually • Gram negative sepsis alone accounts for up to 1/2 of the cases (70,000-300,000 cases per year) • Annual cost for treatment is an estimated $5-10 billion

19. Reasons why there is a high incidence of septic shock • Increased use of cytotoxic and immunosuppressive drug therapies • Increasing frequency of invasive devices, such as intravascular catheters in clinical medicine • Increased longevity of patients prone to develop sepsis • Increase in infections caused by antibiotic-resistant organisms

20. Clinical evidence of infection

21. Development of septicemia • Once in the bloodstream the bacteria multiply uncontrollably releasing toxins in the blood • Our immune system “senses” the presence of bacteria and attempts to eliminate them • In its attempt the immune system “over-reacts” leading to the over production of mediators • This causes damage to the walls of the blood vessels allowing blood to leak out under the skin, the person develops multi-organ failures

22. Types of microorganisms isolated from patients with septic shock • Gram negative bacteria, the incidence of gram negative organisms vary between 50%-80% of the cases • Gram positive bacteria, vary between 6%-24% of the cases • Fungi

23. Major gram negative bacteria producing infections in the U.S. • Enterobacteriaceae • Escherichia Coli (38%) • Klebsiella-Enterobacter-Serratia • Proteus-Morganella-Providencia • Citrobacter • Salmonella-Shigella • Pseudomonadaceae • Pseudomonas aeruginosa (10-15%) • Other species, Pseudomonas cepacia, Pseudomonas maltophilia • Other nonfermentative bacilli • Acinetobacter calcoaceticus • Alcaligenes faecalis

24. What is meningitis? • Meningitis is a disease characterised by the infection of the meninges (the membranes covering the brain and spinal cord) • This inflammation is caused by a bacterial or viral infection

25. Bacterial meningitis • In the United Kingdom alone an estimated 6,000 children between 1 month and 23 months will get meningitis in a given year • The mortality rates are high, with 1 in 10 cases being fatal • 15-30% of the children surviving meningitis will be left with severe handicap, such as deafness or brain injury

26. Common pathogens • Neisseria meningitidis (most common in the UK, accounting for more than half of the cases) • Streptococcus pneumoniae • Haemophilus influenzae

27. Development of meningitis • The initial step is the bacterial colonization of the throat • From the throat the bacteria enter the bloodstream • Once in the bloodstream the bacteria multiply uncontrollably resulting in septicemia before they can infect the meninges • In some cases infection in the bloodstream and the meninges develops at the same time

28. Septicemia • Bacteria that cause meningitis can also cause septicemia (blood poisoning) as well as meningitis • Of the 80% of cases that have meningococcal meningitis, approximately 55% have both meningitis and septicemia • Septicemia is a more serious infection with a higher mortality rate (80%)

31. Signs and symptoms • Patients suffering from septicemia often develop a rash, called haemorrhagic rash • It starts off as a cluster of tiny blood spots, which look like pin-pricks on the skin • These become bigger and become multiple areas of obvious bleeding under the skin, like fresh “bruises” • These “bruises” then join together to form large areas of purple skin damage

32. Signs and symptoms • Septicemia can develop very quickly • The patient rapidly becomes unwell: • loses interest in food and surroundings • feverish and cold, with cool feet and hands • followed by coma and sometimes death

33. Haemorragic rash • Early stage • Advanced stage

34. Meningococcal Septicemia

35. Development of septicemia • Once in the bloodstream the bacteria multiply uncontrollably releasing toxins in the blood • Our immune system “senses” the presence of bacteria and attempts to eliminate them • In its attempt the immune system “over-reacts” leading to the over production of mediators • This causes damage to the walls of the blood vessels allowing blood to leak out under the skin, the person develops multi-organ failures

36. Vaccines • Neisseria has five main Groups - A,B,C, W135 and Y • Most UK meningococcal disease is caused by groups B and C • There are combined vaccines for group A and C, that can give some protection • Effective vaccines for Group B are still some years away (which accounts for 65-70% of the cases)

37. Current Treatment • Antibiotics (such as Rifampin, ampicillin, penicillin, gentamycin sulfate, vancomycin) • Measures to decrease pressure within the brain • Measures to decrease the fever • Oxygen therapy if the person has problems breathing • …all measures are treating the effect and not the cause

39. Lipopolysaccharide (LPS) • LPS is a structural component of the outer membrane of all gram-negative bacteria • pmolar concentrations are sufficient to stimulate cells of the immune, inflammatory, vascular system.

40. Structure of LPS • LPS consists of two chemically dissimilar domains: • The hydrophilic, polysaccharide core and O-antigen structures • A hydrophobic region known as lipid A • Virtually all LPS-induced biologic responses are lipid A dependent

41. (A) Schematic structure of E. Coli LPS. (B) Chemical structure of lipid A.

42. Molecules initially identified to be involved in the innate recognition of LPS • Lipopolysaccharide Binding Protein (LBP) (Tobias et al. 1986) • LBP, a serum protein was shown to bind LPS and transfer it to a cellular receptor • CD14 (Wright et al. 1990) • receptor for LBP-LPS complexes

43. LBP LPS CD14 Activation of NF-kB, MAPK, SAPK, p38

44. LPS Binding Protein (LBP) • A 60 kD glycoprotein (single chain polypeptide) • An acute phase reactant • Functions as an opsonin that recognises the lipid A region of LPS and forms complexes with it, and then facilitates their binding and phagocytosis by macrophages • Present at concentrations 0.5 mg/ml in the serum of healthy humans • During the acute phase it is synthesized in the hepatocytes • Reaches concentrations of 50 mg/ml in serum in the acute phase of infection

45. Bears NH2-terminal sequence homologous with another LPS-binding protein BPI (bactericidal/permeability-increasing protein) • BPI is stored in the specific granules of PMN and kills gram negative bacteria by binding LPS and disrupting the permeability barrier. • In contrast to BPI, LBP is not directly cytotoxic, it binds to LPS forming an LPS-LPB complex and then it attaches to cell via a receptor.

47. Crystal structure of BPI Beamer, et al. 1997

48. CD14 • Myeloid differentiation antigen expressed primarily on peripheral blood monocytes and macrophages • The CD14 gene maps to the human chromosome 5 band q23 to q31, murine chromosome 18 (a region that encodes several myeloid growth factors and receptors) • 55 kDa, glycosylphosphatidylinositol (GPI) anchored membrane protein (mCD14)

49. Diagram of the conserved growth factor and receptor genes located on human chromosome 5 (murine 18)

50. Soluble CD14 • It is also found as a soluble protein (sCD14) lacking the GPI anchor in normal blood sera, in urine, and in culture media of cells expressing it on their surface • There are two soluble forms sCD14a (48kDa), and sCD14b (56kDa)