Cytokine Biology Fish Lectures

Cytokines: secreted proteins that affect tissue/cell survival, growth, differentiation, and effector functions (drives nave effector). Cytokines and their role in the immune system - Inflammation and damage repair - Hematopoiesis - Immune regulation - Tumor surveillance - T cell differentiation Nave cells need a process of activation when infection occurs. Cytokines drive nave cells to a specific lineage. Cytokines bind cell surface receptors that are SPECIFIC. The receptor complex (usually multi-subunit) has an extracellular pocket. Transmembrane domains and intracellular components of surface receptors. Cytokine Receptors - cytokines USUALLY have an -helical tertiary structure - helices are rigid - contact sites on surface - high affinity binding of cytokine to its receptor triggers activation - receptors usually occur as COMPLEXES - can be homodimeric, and can have shared subunits between different cytokines Examples of cytokines/their receptors (talked about later): IL-1 cytokine with beta sheets IL-7 helical bundle, touching one receptor subunit IFN touches two receptors The JAK-STAT Pathway - very generic/common signaling pathway - triggers transcriptional activation specific to cytokine - A cytokine is secreted due to infection (cytokines dont travel far, close proximity) - Prot-prot interactions on outside cause prot-prot interactions intracellularly - Cytokine binding to surface receptor induces receptor conformational change - JAK is a kinase, intracellular on receptor - JAK becomes active with cytokine binding, trans-phosphorylates itself and tyrosine residues of the receptor, and this recruits Stat - Stat = signal transducer and activator of transcription - Jak phosphorylates Stat, causing Stat to dissociate and homodimerize with another stat it is now an active TF (transcription factor), binds promoter sequences in the nucleus

Jak Family: - four kinds of Jaks structurally related kinases (common domains) Jak1, Jak2, Jak3, Tyk2 - FERM domain mediates binding to cytokine receptor - Kinase domain - Enzymatically inactive pseudokinase domain - SH2 domain Stat Family: - 7 kinds of stats (Stats 1-6, with 5a and 5b) - Have tyrosine and serine residues that are phosphorylated - DNA-binding domain (since its a TF) - Transactivation domain has the tyrosine and serine residues that are phosphorylated - Has coiled-coil domain, SH2, and N-terminal domain (dont think this needs to be memorized)

Cytokines and T Cell Activation

Cytokines Activate T cells T helper 1 produces IFN , deals with intracellular pathogens T helper 2 produces IL-4, IL-5, deals with extracellular pathogens T reg cell suppresses regulation suppresses effector response (prevents autoimmunity) T helper 17 proinflammatory cytokines (IL-17) TH1, TH2, and TH17 = effector T helper triad Balance between Effector and Suppressor More T(reg) suppressors than T effectors = tolerance (because it suppresses immune response) More T effectors than T (reg) = graft rejection (excessive immune response) Balance between effectors and suppressors. Cytokine action following infection Virus comes in via skin, respiratory tract, whatever route Viruses replicate rapidly so they need a rapid immune response. APC (usually dendritic) takes up virus, contain TOLL-LIKE RECEPTORS (TLRs) (TLRs recognize nucleic acids of viruses). TLRs can be on cell surfaces, endosomes, or in the cytoplasm. TLR activation triggers signal cascade. APC makes IL-12, and IL-18, which are secreted.

Virus in APC Ag is presented by MHC II nave T cell is activated by presentation to TCR (expresses one of IL-12 receptor subunits on surface the other subunit is inducible, talk about later). When IL-12 binds its receptor, activates the Jak-Stat pathway. Stat4 is activated here, translocates into the nucleus, makes IFN- active, which is secreted and binds its own receptor, activating Stat-1, which activates T-bet, which drives chromatin remodeling. The point: Not a single signaling cascade, EVER. Always multiple pathways activating simultaneously. Pathways here: Presentation of Ag induces IL-12R2 receptor. Thelper1 pathway: IL-12 binds receptor stat4 IFN- IL-18 also helps in this pathway IFN- binds receptor stat1 T-bet chromatin remodeling **Must be a high affinity event with MHC II and TCR. IFN- (key in Thelper 1 pathway) negatively regulates the potential to become a Thelper 2 cell it drives Th1 commitment. The cytokines that T cells secrete drive different immune responses. Cytokines involved in speciation of different cells IFN- speciates Thelper 1 cells, secretes IFN- , LT IL-4 speciates Thelper 2 cells, secretes IL-4, IL-5, IL-T3 TGF- and IL-6 speciate Thelper 17 cells secretes IL-17A, IL-17F, IL-6 TGF- speciates T(reg) cells Thelper 17 Cells Vs. Adaptive T(reg) cells Depends on the presence of a mature or immature DC. Immature DCs cannot secrete IL-6. Mature DCs can secrete IL-6. IL-6 distinguishes whether it differentiates into a T(reg) cell or an inflammatory TH-17. The Jak-stat pathway is used here. In the presence of Ag, immature DCs make nave T cells differentiate into T(reg) cells in the presence of TGF- . In the presence of Ag, mature DCs make nave T cells differentiate into Thelper 17 cells because of IL-6, which also allows IL-23 to help. T cell lineage commitment T cells recognize MHC molecules loaded with antigen peptides on the surface of APCs (here, DCs). Immunogenic DCs stimulate CD4+ T cells into TH1/TH2, and CD8+ cells into CTLs. Regulatory DCs trigger apoptosis/anergy of Ag-specific T cells, inducing T(reg cells). DC:T cell contact factors 1. Affinity of MHC for TCR

2. Level of costimulatory molecules 3. Length of DC:T contact 4. DC:T cell ratio 5. Cytokines secreted by DCs and neighboring cells TH1 Expression A high affinity event, a lot of antigen, and a lot of IFN- TH2 Expression A lower affinity event, less antigen. TH17 Expression Mature DC, IL-6, IL-23, IL-21 Regulatory T cells Immature DC T Follicular Helper Cell Pathway IL-21 Human IL-12 Not just alpha or beta tertiary structure a mosaic structure. IL-12 receptor two subunits, 1 and 2. Remember IL-12 elicits a TH1 response. Jaks: Tyk2 and Jak2 Stats: Stat4/Stat4 When TLR recognizes virus, IL-12 made. MHC meets TCR, evokes a functional IL-12 receptor. IL-12R1 subunit is shared also in IL-23 receptor (for making TH17 cells). So nave T cell can express one receptor constitutively, and the other one must be induced to make functional receptor complex. IL-12 and IL-23 also use the same Jak kinases. Human IL-1 -sheets. Cofactor for T and B cell activation. Induces inflammatory response. Also important for breakdown of cartilage. 3 IL-1 proteins: IL-1, IL-1, and IL-ra (receptor antagonist). IL-1, IL-1 = agonists Needs to associate with IL-1RacP receptor accessory protein. Comes together with IL-1R1 (receptor) to help bind agonists. IL-1R2 the outside is like the IL-1R1 receptor but inside there is nothing, so it binds up agonist but no intracellular response. Mechanisms to suppress IL-1 response (Regulation) 1. IL-1R2 no signal

2. Receptor antagonist binding IL-1R1 receptor 3. Soluble decoy receptors that bind up agonist, so they can reach IL-1R1

Human IL-2 Helical bundles. Receptor with 3 subunits , , Promotes clonal expansion of T cells. IL-2R (receptor) - , are constitutively expressed, IL-2R is inducible (disturbs energy?) 3 sites of contact with receptor (interloop), (N-terminus), (C-terminus) Important the -chain is shared with many different receptors. This chain would recruit the same kind of factors, so the inducible chains are for specificity. The conformational change induced by cytokine binding to receptor exposes certain amino acids specificity of response. IL-2 in the Jak-Stat Pathway (inducible), (Jak-1), (Jak-3) ( is always Jak-3). Typical Jak-stat pathway activates transcription of IL-2 proteins. Dont forget it also activates translation. Downstream signals also trigger the Map-kinase pathway Interferons (IFNs) and Viruses IFNs very effective at clearing viruses. 3 types: 1. Type 1 IFN, IFN a. Inhibit viral replication b. Dendritic Cells 2. Type 2 - IFN- a. Functionally active as a dimer, 4 subunits of receptor (think of everything as double) b. Receptors ubiquitously expressed on all cells because this IFN is so important c. Macrophages express 3. Type 3 IFN-lambda a. Wont really talk about IFN- Pathway Helical bundles. Jaks: Jak1 (2 of them) and Jak2 (2 of them)

Stat: Stat 1 Typical Jak-stat pathway mechanism

Type 1 Interferon Signaling Many many different stat combinations (here can be heterodimers) Each stat complex binds two different elements (in nucleus?) 1. ISRE usually antiviral proteins a. ONLY type I activate ISGF3 (stat 2:1:IRF9) 2. GAS sequence gamma activated sequence many different functions consensus sequence TLR detects virus, binds, triggers signaling cascade Type 1 IFN. Type 1 IFN == primary response to all viruses Attacks multiple steps of replication, triggers immune response. Typical TFN Response Virus in endosomal compartment (for example)TLRsignal inducedIFN produced and secreted (cytokine)short half lifecan only interact with neighborsbinds its receptortranscriptional activationtriggers MORE IFNs for secretionthese can directly activate lymphocytesDC cellsTH1 Cell response. The IFN, in addition to triggering lymphocytes/differentiation, secretes antiviral proteins, which target viral replication, prevent viruses from coming in, block uncoating of virus, cleave viral genetic protein, etc etc. Note IFNs can inhibit growth of cells, so they are human cancer therapy targets. Importance of Stat/Jak proteins Without stats, lack of IFN response = defect in antimicrobial defenses, embyronic lethal, etc. Without Jaks, perinatal lethal, or embryonic lethal.

Cytokines and B Cell Activation

Human IL-7 Helical bundles Receptor: Specific -chain (Jak3) and common chain (Jak1) Important for stromal and epithelial cells Pre-pro-B cell pro-B cell early pre-B cel (important for first three stages) Stat5 also activated (Jak-stat signaling). Principle Cytokines for B Cells IL-4 IgE production IL-5 IgA production IL-6 B cell differentiation IL-7 survival, proliferation, differentiation Recall TH1 cells secrete IFN-. Negatively regulates TH2 production. TH2 cells releases IL-3, IL-4, IL-5, IL-6, IL-7. Negatively regulates TH1 production. Human IL-4 Helical core, loop regions determine contact with receptor. Important for differentiation and IgE switching. Produced by activated CD4+ TH2 cells, exhibits anti-tumor activity. Receptor: Specific -chain and common chain Stat6 activation. sIL-4R soluble decoy receptor that catches IL-4 so it cant bind. Remember that IFN- downregulates TH2. This is how it does it by downregulating IL-4R expression. IL-4R chain also present in IL-13 receptor.

Cytokines and Macrophage Activation

Macrophage activation IFN- is the main macrophage activating agent. Expression depends on cytokines secreted. T cell activation leads to macrophage activation. Macrophage Types Two types of macrophages: M1 and M2 (a,b,c). Type M1 classical macrophage. TH2 cell response - IFN- Type M2a TH2 cell response IL-4 Type M2b TH1 cell response IL-10 Type M2c T(reg) cell response IL-10 Can screen for certain cytokines for type identification.

Cytokines In Hematopoiesis
Lineage Determination Control mitigated by environment and stimulis. Four major CSFs involved (Colony Stimulating Factors): IL-3, GM-CSF (neutrophils), G-CSF (granulocytes), M-CSF (macrophages). Early stage: IL-3 and GM-CSF (helical bundle) Late stage: G-CSF and M-CSF. IL-5: eosinophil production IL-4: mast cell production Epo: Clinically significant, clones were developed to treat patients with kidney failure where erthyrocytes tanked. Its now used for patients on dialysis. IL-3 Secreted by CD4+ TH1 and TH2 IL-3R and c receptor (this is a shared receptor) Stat 5 activation (with IL-5 as well)

IL-5 Homodimer is active. CD4+ TH2 cells are source. Eosinophils and basophils IL-5R and c receptor (this is a shared receptor) Stat 5 activation. Alpha chains determine specificity.

Cytokines In Inflammation
Inflammatory Triad IL-1, IL-6, TNF- Rapidly produced (inflammation), a link between innate and adaptive responses. Theres ALWAYS a local inflammatory response, but sometimes theres a systemic response as well. Brain: fever response Liver: acute phase proteins (APP) associated with rapid response, triggered by inflammatory triad. Thymus: thymic evolution. Chemokines: another huge class of key players trafficking. IL-6 Helical bundles Functions: CTL differentiation, B cell differentiation, Overexpression, Induction of acute phase reactants. IL-6 is produced by a wide variety of cells. IL-6R has transmembrane receptor unit gp130. Jaks: JAK1, JAK2, TYK2, phosphorylate gp130 as well. Overproduction of IL-6: induction of TH17 cells, acute and chronic inflammation (acute phase proteins), bone and cartilige destruction. TNF Cytokine Superfamily Beta sheets. TNF only active as trimer, interacts with trimeric receptor. Important receptor subunit: TNFR1 Binding of ligand to TNFR1 activates death domains (DDs) which are recruitment sites. 3 proteins come together: TRADD, TRAF2, RIP. TRADD itself has a DD that recruits TRAF2 and RIP. NOT Jak-stat pathway. NFkB activation through NFkB element in nucleus.

There are TWO COMPLEXES that can be activated by TNFs. Complex I pathway: TRADD, TRAF2, RIP (above) - NFkB Complex II Pathway: caspases apoptosis. Pro-inflammatory IL-1 Response Unrelated to TNF family. Also uses Traf-6. Vital accessory protein IL-1R-AcP recruits traf. IL-1R-AcP IRAK (kinase) Nik (NFkB inducing kinase) NFkB activation is always linked to some pro-inflammatory response.

Autoimmunity
Autoimmunity targets specific tissues, more prevalent in women. Arthritis: bone + cartilige, etc. Anti-inflammatory meds (makes sense) also B cell, IL, Jak, inhibitors through biologicals Autoimmune diseases: RA (rheumatoid), OA (osteo), scleroderma (skin), psoriatic (skin), ankylosing spondylitis (spine). RA Rheumatoid Arthritis Swollen and fluid filled synovial fluid shows inflammatory cells. Pro-inflamm cells play a role: TNF, IL-1, IL-17, IL-6. TH17 secretes IL-17. Rheumatoid spreads over time whereas osteo is wear and tear Can score swollen and inflamed joints for deformation. Therapies for RA Multiple targets. Abatacept prevents APC from delivering to T cell, and limits co-stimulation to T cell. Anakira blocks IL-1. Rituximab blocks T cell activation Tocilizumab IL-6 inhibitor Tofacitinib JAK inhibitor (almost FDA approved) Anti-TNF inhibitors have revolutionized rheumatoid therapy as theres a central role of TNF- in RA. There is a disequilibrium of cytokines in joints of patients with RA. Need to shut down the immune response so that its not persistant. A very large subset of pro-inflamm cytokines use Jak-stat pathways, so inhibit JAK (Tofacitinib). If you have antibodies staining for STATS, there is much stain in an RA synovium, but none in a normal synovium.

The best way to attack use multiple drugs simultaenously like HIV therapy. Etanercept: human soluble receptor Infliximab: chimeric monoclonal antibody Adalimumab: human monoclonal antibody CIS/SOCs Cytokine Regulation CIS = cytokine inducible SH2 domain SOC = suppressor of cytokine signaling Family of proteins induced by cytokine itself after receptor activation, as a feedback mechanism to shut down cytokine signaling since cytokines are so powerful. SH2 domains of SOCS2, SOCS3, and Cis bind to the tyrosines on receptors that are usually phosphorylated by Jak so it shuts down Jak/Stat signalling. Methods to shut down cytokine response 1. SOCs 2. Soluble decoy receptors 3. Soluble receptor antagonists If the receptor is not used it is degraded. SOCS1 regulates IFN- signaling SOCS3 regulates IL-6, IL-12 SOCS1/5 negative regulator of IL-4, inhibits TH2 differentiation

Chemokines
Traffickers of immune cells. Dysregulated chemokines = inflammatory disease. RA T cells express CCR5 chemokine, role in inflammation Main ones CCL5, CCL4 (also in RA) Chemokine Families C-X-C (at amino terminus) C-C C C-X3-C Chemokine Function and Receptors At an area of inflammation cell creating a large amount of chemokines Chemokines activate adhesion mols T cells bind and enter site of inflammation (diapedesis) Receptors ONE subunit, spans the membrane 7 times creates extracellular pocket and intracellular domain by spanning the membrane so many times. Receptors can also bind MANY things (not just one thing like cytokines). Sometimes viruses use chemokine receptor homologues overexpression of receptors results in disease. Therapy chemokine receptor antagonists in development to block the activity.

CCR5 -

have many tyrosine residues in intracellular domain use the Jak/Stat pathway Associated with G-proteins (GTP/GDP exchange factors) A few different pathways: G-protein, Jak/Stat, Map kinase

Bone Remodelling Bone remodelling and bone loss are controlled by a balance of TNF mol OPGL and its DECOY receptor, OPG utility in osteoperosis Synthesis of bone matrix with osteoblasts and osteoclasts must be tightly which is why decoy receptor is needed. OPGL expression is induced by AG, activated T cells. OPG blocks bone loss look at this through bone density People with arthritis have little bone density. OPG prevents cartilige destruction and even restores it.