Week 2: Fusion Session Cytokines, Chemokines, & Cell Signaling PDF

1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 This is the main receptor used by IL-1 and IL-18, and this receptor primarily signals using the NF-kB signaling pathway, with crosstalk with Mitogen-Activated Prot...

1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 This is the main receptor used by IL-1 and IL-18, and this receptor primarily signals using the NF-kB signaling pathway, with crosstalk with Mitogen-Activated Protein Kinase (MAPK) signaling pathways, namely the Stress-Activated Protein Kinase/c-JUN Kinase (SAPK/JUNK) MAPK pathway and the p38 stress-activated MAPK pathway. This is the pathway used by the vast majority of chemokines; you have seen this receptor in the Cell Signaling lectures (Biochemistry) so it will not be further developed here. G-Protein Coupled Receptor Signaling Regulation through cAMP cascade: Phosphorylation (many protein substrates) Top Gene transcription Pathways are similar through the PKA step PKA can phosphorylate a nuclear transcription factor This affects gene transcription https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 21/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 This is typically a result of persistent elevated cAMP Some cAMP increases are very transient, and are more important for metabolic changes or acute regulation Transforming Growth Factors β (TGF-β) have their own receptor family, which are single-pass serine/threonine kinase receptors, and these receptors signal using SMADs (Mothers against decapentaplegic, Caenorhabditis elegans Sma genes and the Drosophila Mad). We will not elaborate further on TGF-β signaling. Note: To simplify things, consider that all cytokines signal through type I cytokine receptors and learn the exceptions: IFNs and IL-10 through type II cytokine receptors, TNF (CD40L & CD95L) through TNFRs, IL-1 and IL-18 through the IL-1 receptor, and chemokines through seven transmembrane G-protein-coupled receptors; also, because of its clinical importance, learn what type I cytokine receptors possess a common g-chain (esp. IL-2, IL-7, & IL-15).  Binding of the cytokine ligand to its receptor usually triggers the clustering of the receptor chains (most receptors are indeed composed of two or more polypeptide chains, i.e. subunits). Clustering of the receptor chains brings these closer to one another thereby bringing the signal transducers into closer proximity to allow phosphorylation events to be initiated. You have learned about several signaling pathways in previous Cell Signaling lectures in Biochemistry, and some of these also apply to immunological processes and we will consider that you have already learned them; therefore, the remainder of this section will focus on two of the most important signaling pathways in Immunology: the JAK/STAT pathway and the NF-kB pathway. Not only are these pathways quintessential to immunological processes, (1) they are also the target of many pharmacological treatments, and (2) dysregulation of these pathways are key to the pathophysiology of many immune deficiencies. As aforementioned, this is the pathway used by most cytokines. This is a very straightforward Top as previously mentioned, mainly lie in the JAK/STAT signaling pathway and its intricacies, combinations used by different receptors to generate the desired physiological (immunological) response. The following describes the events involved in JAK/STAT signaling in general: 1. Cytokine engagement of cytokine receptor chains; https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 22/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 2. Dimerization (or trimerization in the case of cytokine receptors with three receptor subunits) of receptor chains bringing receptor-associated JAK molecules in close proximity; 3. Activation of JAKs by cross-phosphorylation (sometimes called trans-phosphorylation); 4. JAK-mediated phosphorylation of receptor chain ITAMs (Inducible Tyrosine-Based Activation Motif); 5. Recruitment and docking of STATs; 6. JAK-mediated phosphorylation and dimerization of STATs; Top 7. STAT dimer nuclear translocation; 8. STAT dimer binding to promoter palindromic GAS-binding sites (Gamma Interferon Activation Site); 9. Gene transcription. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 23/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 Important Note: You don’t need to know about ITAMs and GAS-binding sites; however, one thing you do need to know is that JAK3 is the TK associated with the IL-2Rγc, and that both IL-7 and IL-15 possess an IL-2Rγc. Defects in either IL-2Rγc or JAK3 lead to a form of Severe Combined ImmunoDeficiency (SCID) marked by a near absence of both T lymphocytes and NK cells. This particular signaling pathway is almost omnipresent in immune reactions. Not only is the NF-kB pathway used for cytokine signaling, but it is also used, for example, by TLRs to inform on the characteristics of the ‘invading’ pathogen. It is used for T lymphocyte activation B lymphocyte activation, as well as for triggering apoptosis through TNFRs paired with so-called death domains. We will be referring to this signaling pathway on a regular basis, especially when addressing inflammation, innate immunity, as well as T and B lymphocyte activation. A few NF-kB pathway inhibitors are currently on the market, and many candidates are in clinical trial… Many of these are under trial for the treatment of several cancers. Given the complexity of the NF-kB pathway, in that there are several forms of the pathway, we will only hold you responsible for the so-called ‘canonical’ (i.e., general, typical, or ideal) pathway (left side of the next figure); this pathway is used by TNFRs, the IL-1R, TLRs, as well as antigen receptors later seen such as the T cell receptor and the B cell receptor. The following describes the events involved in the canonical NF-kB signaling pathway: 1. TNFR: TNFR trimerization → TRADD → TRAF2 → (RIP1)/TAK1/TAB1-2 → IKKα/IKKβ/IKKγ (NEMO) → IkBα phosphorylation and proteasomal degradation → release and translocation of NF-kB transcription factor to the nucleus where it binds NFkB-responsive elements (i.e., cytokine gene as cytokine receptor gene promoters among others). 2. Some TLRs (3, 4): TLR → Myd88 adaptor protein (TLR4) → RIP1 → TAK1/TAB12/TRAF6 → IKKα/IKKβ/IKKγ (NEMO) → IkBα phosphorylation and proteasomal degradation → release and translocation of NF-kB transcription factor to the nucleus where it binds NF-kB-responsive elements (i.e., cytokine gene as cytokine receptor gene promoters among others). 3. IL-1R and other TLRs (1/2, 2/6, 5, 7, 8, 9, 10): TLR → Myd88 adaptor protein → IRAK → TAK1/TAB1-2/TRAF6 → IKKα/IKKβ/IKKγ (NEMO) → IkBa phosphorylation and Top proteasomal degradation → release and translocation of NF-kB transcription factor to the nucleus where it binds NF-kB-responsive elements (i.e., cytokine gene as cytokine receptor gene promoters among others). https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 24/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 This image shows the details of nuclear factor kappa B (NF-κB) signaling. Two main pathways exist: the (1) canonical (i.e., typical) and (2) noncanonical (i.e., non-typical) pathways; both pathways are common throughout leukocyte signaling. The canonical pathway is characterized by the activation of the inhibitor of NF-κB (IκB) kinase-gamma (IKKγ also referred to as NEMO (NF-kappa-B essential modulator)) as part of the IKK complex (IKKα/IKKβ/IKKγ) with subsequent phosphorylation and degradation of IκB and consequent release of NF-κB which is free to translocate to the nucleus and activate NF-κB-responsive genes. The noncanonical pathway does not involve IKKγ/NEMO but rather the other two IKKs, which phosphorylate inactive NF-κB which is then ubiquitinated and processed into its active form by the proteasome; processed NF-κB can Topthen translocate to the nucleus and activate NF-κBresponsive genes. Important receptors signaling through these pathways include TNFRs, TLRs, IL-1R, IL-18R, TCRs, and BCRs. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 25/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 Watch this video about NF-κB pathway v=7F_HPRfHdDk%20) . (https://www.youtube.com/watch? Many signaling pathways crosstalk (incl. the JAK/STAT, NF-κB, & MAPK pathways), and we will return to this when we address TCR signaling in the Antigen Presentation and T Lymphocyte Biology lecture. But for the time being, we need to address crosstalk between the NF-κB and MAPK pathways since both these pathways are activated through IL-1R signaling and are important in the initiation of inflammation as well as TCR and BCR signaling. There are several IL-1 signaling inhibitors currently on the market, primarily for the treatment of inflammatory conditions, several of which you will be learning about in Pharmacology. Top https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 26/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 Top The IL-1R and many TLRs (TLRs 1, 2, 4, 5, & 6 mainly) signal through the same pathways to yield NF-κB activation and proinflammatory cytokine (and other proinflammatory genes) expression; TLRs 3, 7, 8, and 9 signal mainly through parts of the upper NF-κB signaling https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 27/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 pathway but, instead of activating NF-κB, they crosstalk and activate other transcription factors, namely Interferon Regulatory Factors (IRFs) 3 and 7, which are important in defenses against intracellular infections, especially virus infections, by promoting type 1 and type 3 interferons (IFNs α & β (type 1) and IFN-λ (type 3) – we will return to this when we address the antiviral state). Toll-like receptor 4 yields the activation of both NF-κB and IRF transcription factors. The dichotomy in IL-1R signaling occurs at the level of TAK1/TAB1-2/TRAF6 activation, where the signal gets split between 1. the NF-κB signaling pathway (as seen in the last section on NF-κB signaling – #3) to yield NF-κB activation and 2. the SAPK/JNK, Extracellular Signal-Regulated Kinase (ERK), and p38 MAPK pathways to activate the Activator Protein 1 (AP-1) and the cAMP Response Element-Binding protein (CREB) transcription factors. Top https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 28/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 MyD88-related signaling Top M D88 l t d i li i k l l th i th i t i https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling t i il 29/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 MyD88-related signaling is a key molecular pathway in the innate immune system, primarily responsible for initiating the cellular response to various pathogens and activating proinflammatory genes through the activation of MyD88 adaptor protein. Which transcription factor would fail to be activated in a patient with a NEMO (IKKγ) deficiency?  NF-κB  AP-1  IkBα  IRF5  STAT  Check  Question 1 Which transcription factor would fail to be activated in a patient with a NEMO (IKKγ) deficiency? IkBα AP-1 NF-κB (Correct answer) IRF5 STAT Question 2 Which TK is responsible for the phosphorylation of STATs? Top JAKs (Correct answer) NEMO (IKKγ) IRAKs MEKKs https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 30/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 STAT Question 3 Table Although cytokines function as part of normal physiological processes and are important in controlling infections, they are sometimes involved in the pathophysiology of many diseases and conditions. Sometimes cytokines can be expressed in an exaggerated fashion such as in sepsis, acute respiratory distress syndrome, toxic shock, COVID-19, multisystem inflammatory syndrome in adults (MIS-A), multisystem inflammatory syndrome in children (MIS-C, or Kawasaki syndrome or disease), hemorrhagic fevers, etc. Sometimes cytokines can be expressed in situations they should not be expressed, e.g., autoimmune and inflammatory diseases and, finally, some immune responses engender aberrant cytokine expression in some individuals, e.g., lepromatous leprosy (severe form) versus tuberculoid leprosy (mild form) and in some HIV-infected persons who progress faster to AIDS. You will see many of these examples as you progress through your medical education.  Top https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 31/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 1. A graduate student conducting research on hyperlipidemia evaluates lipolytic activation (as measured by glycerol release) in adipocytes treated with proinflammatory cytokines, and obtains the following results: What kind of cytokine interaction(s) is(are) occurring in this example? Additivity Antagonism Pleiotropism Redundancy (Correct answer) Synergy (Correct answer) Top Feedback: About 35% more than sum of IL-6 and TNF-α. Strictly speaking, based on the definition, this would be an example of cytokines acting in synergy (and this is what would constitute a correct answer if such a question were to be asked on an exam). In practice however, the combination of both cytokines barely exceeds the sum of their individual action, meaning that, in biological terms, and given standard errors on measurements, we are probably https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 32/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 more within the realm of additivity than synergism. This would actually be a good discussion point. This example also shows that IL-6 and TNF-α possess redundant functions since both stimulate lipolysis in adipocytes. 2. Taken together, and knowing the functions of IL-1b, TNF-a, and IL-6, how can these cytokines be qualified? Additive Antagonistic Pleiotropic (Correct answer) Redundant (Correct answer) Synergistic Feedback: All of these cytokines exhibit different functions, and all have functions that overlap (e.g., fever); consequently, they can all be qualified as pleiotropic and redundant. No concentrations are given for the effects of individual cytokines or any of their combinations so no conclusions can be drawn as to whether they act additively or synergistically; they certainly are not antagonistic though, as we will later see while addressing inflammation 3. What type of signaling is exemplified by IL-12 and IFN-g in the figure below? Autocrine Endocrine Paracrine (Correct answer) 4. A 5-year-old boy with leukemia receives a hematopoietic stem cell transplant (bone marrow transplant). Which of the following cytokines would promote the production of new leukocytes following the bone marrow transplant? IL-2 IL-3 (Correct answer) IL-10 IL-12 TGF-b A 5-year-old boy presents with his mother to his physician with ulcers in the buccal mucosa and skin around the mouth. Patient history is significant for recurrent bacterial and fungal skin infections, periodontitis, impaired wound healing, as well as delayed separation of the umbilical cord. Laboratory investigations are significant for a leukocytosis with a predominance of PMNs (polymorphonuclear leukocytes). The accompanying figure shows how periodontitis normally presents in an immunocompetent individual. As shown in A, one feature of periodontitis is inflamed and swollen gums, which usually contain a number of leukocytes including PMNs (shown in B). A feature of this boy's disease, however, is the rarity of granulocytes in the Top inflamed periodontium (the epithelia, connective tissue, and bone surrounding teeth), as compared to an immunocompetent patient with periodontitis (B). A defect in the expression of which molecule most likely accounts for this feature? CCL11 https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 33/35 1/24/24, 1:03 PM Week 2: Fusion Session | Workshop: Cytokines, Chemokines, & Cell Signaling: Hemtlgy Onclgy Infectn Imm - January 2024 CXCL8 (Correct answer) IL-1β TGF-β TNF-α 6) A 25-year-old woman presents to the Emergency Department with severe pain on the right side of the lower abdomen, nausea, and fever. The cytokines responsible for eliciting the fever in this patient is an example of what type of cytokine signaling? Autocrine Endocrine (Correct answer) Paracrine 7) A 7-month-old child has a genetic deficiency in which her IkB kinase-g (IKKg, syn. NEMO) is not functional. This child's cells will be unresponsive to the stimuli of what receptor(s)? IL-1R IL-12R IFNGR TLR (Correct answer) TNFR (Correct answer) 8) A 7-month-old child has a genetic deficiency in which her JAK2 is not functional. This child's cells will be unresponsive to the stimuli of what receptors? IL-1R IL-12R (Correct answer) IFNGR (Correct answer) TLR TNFR 9) The immune system evolved to react to non-self molecules. Food is non-self… What cytokine expression allows for tolerance for innocuous molecules such as those found in food? IL-1b IL-6 IL-12 TGF-b (Correct answer) TNF-a 10) The key cytokine responsible for initiating inflammation is IL-1β. What transcription factors are activated downstream of IL-1β signaling? Top AP-1 (Correct answer) CREB (Correct answer) NF-κB (Correct answer) SP1 https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-cytokines-chemokines-and-cell-signaling 34/35