TOLL-LIKE RECEPTORS AND CYTOKINES IN SEPSIS

Transcription

1 TOLL-LIKE RECEPTORS AND CYTOKINES IN SEPSIS A/PROF WILLIAM SEWELL ST VINCENT S CLINICAL SCHOOL, UNSW SYDPATH, ST VINCENT S HOSPITAL SYDNEY GARVAN INSTITUTE

2 INNATE VERSUS ADAPTIVE IMMUNE RESPONSES INNATE IMMUNITY Rapid response - minutes to hours after pathogens cross epithelial barriers Responds to general molecular properties of pathogens Does not improve on repeated contact with same pathogen ADAPTIVE IMMUNITY Slower response - clonal expansion of T and B cells takes several days Responds to fine molecular detail of antigens Improves on repeated contact with same pathogen

3 PAMPs Pathogen-Associated Molecular Patterns Molecules that are recognized by the innate immune system Widely conserved across groups of pathogens PAMPs are limited to microbes and are generally not present in the human host But in unusual cases PAMPs may be linked to autoimmunity The name is not perfect because non-pathogenic microorganisms may express the same molecules.

4 DAMPs Damage-Associated Molecular Patterns Host-derived molecules released during cell injury or death Otherwise have similar roles to PAMPs

5 PRRs Pattern Recognition Receptors A set of host molecules that bind to PAMPs and DAMPs PRRs are typically conserved across many pathogens, therefore: The host is protected by a relatively small number of PRRs (compared with the huge range of antigen receptors of T and B cells). PRRs are encoded by the germ-line (compared with rearrangement of immunoglobulin and T cell receptor genes). After PRRs bind PAMPs or DAMPs, they activate signalling cascades that cause cellular activation and secretion of cytokines, chemokines (cytokines that attract leukocytes) and other factors. This drives the acute inflammatory response (innate immunity). It also drives the initiation of the adaptive response.

6 CLASSIFICATION OF PRRs Membrane-bound: Toll-Like Receptors (TLRs) C-type Lectin Receptors Group I mannose receptors Group II asialoglycoprotein receptors Cytosolic NOD-like Receptors (NLRs) NLRP3 links to inflammasome generating IL-1β, IL-18 & IL-33 RIG-1-like Receptors (RLRs) : RIG-1 & MDA5 Lead to type I interferon (IFN-α and IFN-β) response

7 BASIC STRUCTURE OF CELL CURFACE TLRs A single polypeptide chain spans the plasma membrane. intracellular extracellular leucine-rich repeat plasma membrane TIR domain

8 Toll-Like Receptors Highly conserved receptor system. First found in fruit flies when the German scientist Christiane Nüsslein-Volhard saw a mutant fruit fly that lacked a TLR in 1985, she exclaimed, "Das ist ja toll! Toll is German for cool or amazing Single membrane-spanning molecules Extracellular domains contain leucine-rich repeats. Intracellular domains contain motifs shared with the IL-1, IL- 18 & IL-33 receptors: Toll & IL-1 receptor domain (TIR domain) The extracellular domains of these cytokine receptors are entirely different from TLRs.

9 Toll-like receptors (contd) Mainly expressed on cells of the innate immune system: macrophages dendritic cells (DC) both conventional (cdc) and plasmacytoid (pdc) mast cells B cells Also some expression in epithelial cells TLRs 3, 7, 8 & 9 are intracellular and detect nucleic acids. The other TLRs are cell surface receptors and typically detect microbial cell wall-associated PAMPs. The ligands of the cell surface receptors have molecular structures that are generally very different from molecules normally present in humans.

10 INTRACELLULAR TLR STRUCTURE endosome membrane endosome lumen leucine-rich repeat cytoplasm TIR domain

11 LOCATIONS & LIGANDS OF MAJOR TLRs Heterodimers are shown. The other TLRs form homodimers. Janeway s Immunobiology

12 Nucleic acid-sensing TLRs TLR3, 7, 8, 9 TLR3 responds to dsrna; TLR9 to unmethylated CpG DNA dsrna is a common feature of viral infection but is normally absent in host cells CpG is usually methylated in host cells So autoimmune responses to nucleic acids are limited Intracellular in endosomes only, not on cell surface. Restricted location limits autoimmune responses to nucleic acids; host ssrna (ligand of TLR7 & 8) is not normally present in endosomes In macrophages and conventional DC, TLR7 & 9 give a proinflammatory response, but in plasmacytoid DC these TLRs give a Type I interferon response.

13 HUMAN TLRs TLR TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 Ligand, Notes dimerizes with TLR2 dimerizes with TLR1, TLR6 PAMPs in Gram pos and neg bacteria, mycobacteria, mycoplasma, spirochetes etc dsrna prominent in DC unlike other TLRs does not signal thru MyD88 but uses TRIF. lipopolysaccharide etc, uses MyD88 and TRIF flagellin dimerizes with TLR2 ssrna. Mediates response to imiquimod. Prominent in pdc ssrna CpG DNA. Prominent In pdc TLR10?

14 TLR SIGNALLING TLRs deliver intracellular signals following ligand binding that leads to the formation of TLR dimers. Typically, pathogens have multiple PAMPs that cross-link TLRs. Most TLRs form homodimers. TLR1, 2 & 6 form heterodimers of TLR1/2 or TLR2/6. After activation, all TLRs except TLR3 recruit the intracellular protein myeloid differentiation factor 88 (MyD88). IL-1, IL-18 & 1L-33 receptors also use MyD88 Collectively, TLRs activate the NF-κB pathway (especially for bacterial infection) and/or the Type I interferon pathway (especially for viral infection).

15 TLR4 ACTIVATION Classical ligand is LPS also binds some other PAMPs Also binds DAMPs including heat-shock protein, hyaluronan, modified LDLs The extracellular domain of TLR4 interacts with other cell surface host proteins: LPS-binding protein (LBP), CD14 and MD2. This gives rise to TLR4 homodimers. The complex recruits an intracellular complex including MyD88. This activates the NF-κB pathway. In addition, TLR4 is internalized and trafficked to endosomes, where it forms a different complex, recruiting TRAM and TRIF, that signal to produce Type I interferons.

16 TLR4 PATHWAY Maeshima N & Fernandez RC. Front. Cell. Infect. Microbiol., 2013; 3:3.

17 NF-κB pathway NF-κB is a family of transcription factors with a key role in signalling in immunity. In resting cells, NF-κB is retained in the cytoplasm by a family of inhibitory proteins, I-κB. After activation TLR4 or other receptors, the signalling cascade phosphorylates I-κB, which is removed from the complex and degraded, allowing NF-κB to translocate to the nucleus. NF-κB binds to the promoter regions of the proinflammatory cytokines TNF-α, IL-1β and IL-6, and other genes important in acute inflammation.

18 NF-κB PATHWAY 101 TLR IL-1R TNFR BCR, TCR I-κB degraded I-κB NF-κB NF-κB NF-κB promoter gene cytokine & chemokine secretion etc

19 DEFICIENCIES OF THE TLR AND RELATED PATHWAYS - 1 MyD88 deficiency is a rare autosomal recessive primary immunodeficiency disorder. Patients suffer from recurrent pyogenic bacterial infections, especially pneumococcus. However there is little evidence of impaired protection against viruses, fungi and parasites. IRAK4 deficiency gives the same phenotype. TIRAP (partner of MyD88) deficiency causes increased susceptibility to staphylococcal infections. NEMO deficiency also affects the NF-κB pathway but phenotype involves developmental defects, adaptive and innate systems.

20 PATIENTS WITH MYD88 OR IRAK-4 DEFICIENCY WHO SURVIVE CHILDHOOD HAVE A GOOD PROGNOSIS Casanova JL et al, Annu Rev Immunol :

21 DEFICIENCIES OF THE TLR AND RELATED PATHWAYS - 2 TLR3 deficiency, causing childhood herpes simplex encephalitis. Can be autosomal dominant or autosomal recessive. Limited evidence of increased susceptibility to other pathogens including viruses. Similar phenotype with several downstream members of the TLR3/Type I Interferon pathway.

22 TOO MUCH MyD88 Over 90% of cases of the uncommon B cell lymphoma lymphoplasmacytic lymphoma (Waldenstrom s macroglobulinaemia) harbour an activating mutation of the MyD88 gene. Therapeutic blockade of this signalling pathway is beneficial. Mark Benedict & S. David Hudnall. Blood 2015;126:2342

23 ACUTE INFLAMMATION Janeway s Immunobiology

24 TNF-α A key early product of the NF-κB pathway. Stimulates production of other pro-inflammatory cytokines such as IL-1β and IL-6. Stimulates production of chemokines including IL-8 which attracts neutrophils to sites of inflammation. Acts on endothelial cells and leukocytes to increase adhesion of leukocytes to endothelium, encouraging leukocyte migration into the tissues. Acts on endothelial cells to trigger coagulation, limiting the dissemination of pathogens from the initial site of infection.

25 NEUTROPHIL EGRESS FROM BLOODSTREAM Janeway s Immunobiology

26 OTHER ASPECTS OF ACUTE INFLAMMATION TNF-α, IL-1β and IL-6 cause fever by stimulating production of prostaglandin E2 in the hypothalamus. These cytokines act as endogenous pyrogens. Cytokines especially IL-6 act on liver to stimulate production of acute phase proteins, e. g. CRP, fibrinogen. Increased fibrinogen is an important factor causing elevated ESR in inflammation. Pro-inflammatory cytokines stimulate neutrophil release from the bone marrow. Other cytokines such as G-CSF and GM-CSF stimulate bone marrow generation of neutrophils.

27 CRP CRP is a member of the pentraxin family, so called because of five identical subunits. CRP binds to phosphocholine in certain bacteria and fungi, and acts as an opsonin (facilitates phagocytosis). CRP, circulating at mg/l, is much easier to measure than IL-6 which circulates at ng/l.

28 INITATION OF ADAPTIVE RESPONSE TNF-α acts on dendritic cells to stimulate their migration to lymph nodes and to increase their capacity to act as antigenpresenting cells. IL-6 stimulates differentiation of CD4 T cells into Th17 cells. IL-12 stimulates differentiation of CD4 T cells into Th1 cells.

29 IMPACT OF DIFFERENT AMOUNTS OF TNF-α PRODUCTION Abbas et al Cellular & Molecular Immunology

30 TNF-α AND SEPSIS Many features of sepsis can be attributed to the massive early over-production of TNF-α: Vasodilation and increased vascular permeability leading to hypotension & adult respiratory distress syndrome Platelet activation and up-regulation of pro-coagulant pathways leading to disseminated intravascular coagulation Metabolic disturbances including hypoglycaemia Strong evidence in animal models that blockade of TNF-α ameliorates sepsis

31 Effect of intraduodenal anti-tnf-α antibodies on survival Rats were given LPS 35 mg/kg iv at time 0. Anti-TNF was infused from 0-4 hours. Graham D. F. Jackson et al. Infect. Immun. 2000;68:

32 PRE-CLINICAL STUDIES IN SEPSIS Over 100 mediators have been implicated in rodent studies of endotoxaemia. However these studies have not led to licenced therapies. Limitations of animal studies: Young animals No co-morbidities Genetically inbred strains Identical single challenge to all Intervention commonly starts at time of or before onset of challenge. No supportive therapy

33 CONSIDERATIONS IN THE PATHOGENESIS OF SEPTIC SHOCK The TNF-α model under-estimates the complex pathophysiology of inflammation, coagulation and antiinflammation. Later in sepsis, the anti-inflammatory cytokine IL-10 is prominent. Host factors may stimulate aspects of sepsis. Neural influences on the immune system may complicate the pathogenesis. Recruitment into clinical trials may have been too general. Future trials could include only those patients with a biomarker relevant to the effect of the therapy.

34 USEFUL REVIEWS 1. Casanova JL, Abel L, Quintana-Murci L. Human TLRs and IL- 1Rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics. Annual review of immunology 2011; 29: Kieser KJ, Kagan JC. Multi-receptor detection of individual bacterial products by the innate immune system. Nature reviews Immunology 2017; 17: Marshall JC. Why have clinical trials in sepsis failed? Trends in molecular medicine 2014; 20: