Cell, Inflammation, Wound healing 8th December 2020 Cell Mitochondrial function/dysfunction ROS Hypoxia Lysosomal function/dysfunction Cell death 2 Mitochondria ̶ production of ATP for cellular energy needs ̶ metabolism of amino acids ̶ regulation of the redox state of cells ̶ heme synthesis ̶ differentiation and activation processes of immune cells ̶ crucial functions in the cell death program 3 Mitochondrial network Mitochondrial fusion and fission ̶ processes occur in response to various extra- or intracellular changes ̶ changes in nutrient supply, energy or redox status, during cell differentiation in a cell-type dependent manner 4 Mitochondrial fusion and fission ̶ response to metabolic/pathogenic conditions ̶ FUSION = autonomously integrate ̶ 1. fusion of the outer membrane between 2 adjacent mitochondria ̶ mediated by mitofusin 1 and 2 ̶ 2. fusion of the inner membrane ̶ cardiolipin, dynamin-like GRPase optic atrophy (OPA) ̶ important for maintenance of mitochondrial DNA integrity and cellular respiration5 Research Reports in Clinical Cardiology 2014(default):111 Mitochondrial fusion and fission ̶ FISSION ̶ important to allow inheritance of mitochondria by daughter cells during cell division ̶ when damaged and deleted - damaged mitochondria facilitates their removal by mitophagy 6 Research Reports in Clinical Cardiology 2014(default):111 Mitochondria and ROS ̶ production of reactive oxygen species ̶ generated by mitochondria via the electron transport chain ̶ byproduct during mitochondrial energy production, consequence of fatty acid β-oxidation, exposure to radiation, light, metals, and redox drugs ̶ ROS function: ̶ second messengers in various signaling pathways in immune cells: Ca2+-NFAT signaling pathway, which is critical in T cell activation. ̶ ROS can also damage bacterial pathogens, but ̶ if produced excessively - damage the producing cell or neighboring cells. 7 Int. J. Mol. Sci. 2019, 20(18), 4407 Sites of ROS production ̶ mitochondrial ROS (mROS) are basically produced as byproducts of this bioenergetic metabolism ̶ Cyt c, cytochrome c; MAO, monoamine oxidase; NOX-4, NADPH oxidase 4; VDAC, voltage-dependent anion channel 8 Mitochondria, ROS and immune cells ̶ pro-inflammatory cells - activated monocytes and activated T and B cells – glycolysis ̶ regulatory cells - regulatory T cells or M2 macrophages - increasing mitochondrial function and beta- oxidation 9 Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease Volume 1866, Issue 10, 1 October 2020, 165845 Adaptation of immune cells to the local microenvironment 10 factors that contribute to the adaptation of cells to their dynamic tissue environment (e.g. T cells): (1) functional status of T cells, (2) local factors unique to the tissue niche, (3) type of inflammation, and (4) time spent in a specific tissue Oxidative stress ̶ result of imbalance between ROS production and antioxidation ̶ pathological defects in living organisms ̶ cancer, atherosclerosis, neurological diseases, aging, and diabetes, damage of cellular components (DNA, RNA, lipids, and proteins) ̶ non-enzymatic defense: ̶ flavonoids, vitamins (A, C, and E), and glutathione ̶ enzymatic antioxidants: ̶ Superoxide dismutase (SOD), superoxide reductase, catalase, glutathione peroxidase, glutathione reductase, peroxiredoxins (Prdxs), and thioredoxins (Trx) 11 Int. J. Mol. Sci. 2019, 20(18), 4407 Effect of mitochondria in immune reaction ̶ mitochondrial DAMPs ̶ in extracellular space and circulation. ̶ mitochondrial proteins ̶ FRP receptors - production of chemoattractants. ̶ mitochondrial ROS ̶ intracellular signaling, damage cells. ̶ mitochondrial ATP and cardiolipin ̶ activate the NLRP3 inflammasome or TLR4 production of pro-inflammatory cytokines. ̶ mitochondrial DNA ̶ activate TLR9, NLRP3 inflammasomes or the cGAS pathway - production of pro-inflammatory cytokines.12 Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease Volume 1866, Issue 10, 1 October 2020, 165845 Mitochondria – induction of immune response ̶ mitochondrial danger associated molecules (DAMPs) that resemble structures of bacterial derived pathogen associated molecular patterns (PAMPs) ̶ mitochondrial DAMPS - mitochondrial DNA with hypomethylated CpG motifs, specific lipid present in prokaryotic bacteria and mitochondria, i.e. cardiolipin. ̶ via DAMPs mitochondria guide the immune response ̶ mitochondrial DAMPs - negative impact- released by damaged cells, without the presence of an infection - undesired inflammatory response, resulting in tissue damage and organ dysfunction ̶ after a trauma 13 Clin Microbiol Rev. 2012 Oct; 25(4): 609–634. Mitochondrial dysfunction 15 Nature Reviews Molecular Cell Biology volume 17, pages308–321(2016) Mitochondrial dysfunction ̶ mutations in mitochondrial DNA and oxidative stress – risk factor for neurodegenerative diseases ̶ strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis ̶ disease-specific proteins interact with mitochondria 16 Nature volume 443, pages787–795(2006) Cardiolipin ̶ Myocardial Ischemia/Reperfusion injury ̶ loss in the CL abundance and an increase in CL oxidation have been reported in rat heart mitochondria during ischemia and ̶ additional CL loss upon reperfusion ̶ alterations in bioenergetics parameters: decreased rate of O2 consumption, lower activity of complexes I, III and IV and increased basal rate of H2O2 mPTP plays an important role in cardiomyocytes death occurring during myocardial I/R 17 Cells. 2019 Jul; 8(7): 728 Mitochondrial dysfunction and heart 18 European Society of Cardiology Journals 23. 10. 2018 Mitochondria and hypoxia 19 Nature Reviews Cancer volume 2, pages38–47(2002) Mitochondria and hypoxia ̶ Acute/chronic ̶ decreased flux through the tricarboxylic acid (TCA) cycle ̶ activity of the electron transport chain (ETC) ̶ hypoxia-induced ROS, … ̶ low vs. no oxigen 20 Cell death Definujte zápatí – název prezentace nebo pracoviště21 Current Aging Science Volume 10 , Issue 1 , 2017 Cell death Signals from the lysosome: a control centre for cellular clearance and energy metabolism ̶ degradation and recycling of cellular waste ̶ via endocytosis and autophagy ̶ Lysosomal and autophagy dysfunction in ̶ lysosomal storage diseases (LSDs) and ̶ common neurodegenerative diseases ̶ defective cellular clearance and accumulation of toxic material 23 Nature Reviews Molecular Cell Biology volume 14, pages283–296(2013) Lisosoms and starvation ̶ limited nutrient availability and mediates the starvation response by regulating lipid catabolism ̶ used also by tumor cells 24 Nature Reviews Molecular Cell Biology volume 14, pages283–296(2013) Defective cellular clearance in neurodegenerative diseases ̶ loss-of-function mutations of genes involved in the lysosomal–autophagic pathway ̶ gain-of-function mutations of aggregate-prone proteins ̶ enhanced protein aggregation and impairment of lysosomal–autophagic pathways 25 Nature Reviews Molecular Cell Biology volume 14, pages283–296(2013) Inflammation Inflammation Acute fase reaction Cytokines, chemokines 26 Inflammation  Inflammation is the response of living tissue to damage. The acute inflammatory response has 3 main functions: The affected area is occupied by a transient material called the acute inflammatory exudate. The exudate carries proteins, fluid and cells from local blood vessels into the damaged area to mediate local defences. If an infective causative agent (e.g. bacteria) is present in the damaged area, it can be destroyed and eliminated by components of the exudate. The damaged tissue can be broken down and partially liquefied, and the debris removed from the site of damage. Inflammation ̶ In all these situations, the inflammatory stimulus will be met by a series of changes in the human body; it will induce production of certain cytokines and hormones, which in turn will regulate haematopoiesis, protein synthesis and metabolism. ̶ Most inflammatory stimuli are controlled by a normal immune system. The human immune system is divided into two parts which constantly and closely collaborate - the innate and the adaptive immune system. Inflammation – innate system ̶ The innate system reacts promptly without specificity and memory. Phagocytic cells are important contributors in innate reactivity together with enzymes, complement activation and acute phase proteins. ̶ When phagocytic cells are activated, the synthesis of different cytokines is triggered. These cytokines are not only important in regulation of the innate reaction, but also for induction of the adaptive immune system. There, specificity and memory are the two main characteristics. Inflammation – adaptive immune response ̶ In order to induce a strong adaptive immune response, some lymphocytes must have been educated to recognize the specific antigen on the antigenpresenting cell (APC) in context of self major histocompatibility molecules. The initial recognition will mediate a cellular immune reaction, production of antigen-specific antibodies or a combination of both. Some of the cells, which have been educated to recognize a specific antigen will survive for a long time with the memory of the specific antigen intact, rendering the host "immune" to the antigen. Non-specific Immunity Differences between innate (non-specific) and specific (adaptive) immunologic reaction of organism Specific Immunity ̶ Response is antigen-independent ̶ There is immediate maximal response ̶ Non-antigen-specific ̶ Exposure results in no immunologic memory ̶ Response is antigen-dependent ̶ There is a lag time between exposure and maximal response ̶ Antigen-specific ̶ Exposure results in immunologic memory Types of inflammation ̶ Acute ̶ Chronic ̶ Local ̶ Systemic Acute inflammation ̶ can be caused by agents such as ̶ infectious inflammatory stimuli (viruses, bacteria, fungi and parasites) ̶ by non-infectious inflammatory stimuli, as in rheumatoid arthritis and graftversus host disease ̶ by tissue necrosis as in cancer ̶ by burns and toxic influences caused by drugs or radiation Early Stages of Acute Inflammation The acute inflammatory response involves three processes: ̶ changes in vessel caliber (= vasodilation) and, consequently, slower blood flow ̶ increased vascular permeability and formation of the fluid exudate ̶ formation of the cellular exudate by emigration of the neutrophil polymorphs into the extravascular space. Early Stages of Acute Inflammation The steps involved in the acute inflammatory response are: • Small blood vessels adjacent to the area of tissue damage initially become dilated with increased blood flow, then flow along them slows down. • Endothelial cells swell and partially retract so that they no longer form a completely intact internal lining. • The vessels become leaky, permitting the passage of water, salts, and some small proteins from the plasma into the damaged area (exudation). One of the main proteins to leak out is fibrinogen. • Circulating neutrophil polymorphs initially adhere to the swollen endothelial cells (margination), then actively migrate through the vessel basement membrane (emigration), passing into the area of tissue damage. • Later, small number of blood monocytes (macrophages) migrate in a similar way, as do Iymphocytes. Changes compared with normal state Increase Decrease Cellular phagocytic cells (in circulation and at the site of inflammation) erytrocytes Metabolic acute phase proteins serum Cu protein catabolism gluconeogenesis serum Fe serum Zn albumin synthesis transthyretin transferrin Endocrine glucagon insulin ACTH GH T4 cortisol aldosterone vasopressin T3 TSH Systemic manifestation of inflammation • Increase of body temperature (fever) • Acute phase reaction Justin Root Acute phase reaction The acute phase reaction is the body's first-line inflammatory defense system, functioning without specificity and memory, and in front of, and in parallel with, the adaptive immune system. In the acute phase reaction, several biochemical, metabolic, hormonal and cellular changes take place in order to fight the stimulus and re-establish a normal functional state in the body. An increase in the number of granulocytes will increase the phagocytotic capacity, an increase in scavengers will potentiate the capability to neutralize free oxygen radicals, and an increase in metabolic rate will increase the energy available for cellular activities, despite a reduced food intake. Some of these changes can explain the symptoms of an acute phase reaction, which are typically fever, tiredness, loss of appetite and general sickness, in addition to local symptoms from the inducer of the acute phase. Canadian Medical Association Journal 182(18):E834-8 General and local clinical symptoms of the acute phase reaction General symptoms Local symptoms fever calor tachycardia rubor hyperventilation dolor tiredness tumor Loss of appetite functio laesa Systemic effects of acute/chronic inflammation  Pyrexia Polymorphs and macrophages produce compounds known as endogenous pyrogens, which act on the hypothalamus to set the thermoregulatory mechanisms at a higher temperature. Release of endogenous pyrogen is stimulated by phagocytosis, endotoxins and immune complexes.  Constitutional symptoms Constitutional symptoms include malaise, anorexia and nausea. Weight loss is common when there is extensive chronic inflammation.  Local or systemic Iymph node enlargement commonly accompanies inflammation, while splenomegaly is found in certain specific infections (e.g. malaria, infectious mononucleosis). Mol Cancer Res; 11(9); 967–72. ©2013 AACR. Systemic effects of inflammation Haematological changes ̶ Increased erythrocyte sedimentation rate. An increased erythrocyte sedimentation rate is a non-specific finding in many types of inflammation. ̶ Leukocytosis. ̶ Neutrophilia occurs in pyogenic infections and tissue destruction; ̶ eosinophilia in allergic disorders and parasitic infection; ̶ Iymphocytosis in chronic infection (e .g. tuberculosis), many viral infections and in whooping cough; and ̶ monocytosis occurs in infectious mononucleosis and certain bacterial infections (e.g. tuberculosis, typhoid). ̶ Anaemia. ̶ blood-loss in the inflammatory exudate (e.g. in ulcerative colitis), ̶ haemolysis (due to bacterial toxins), and ̶ 'the anemia of chronic disorders' due to toxic depression of the bone marrow. Amyloidosis ̶ Longstanding chronic inflammation (for example, in rheumatoid arthritis, tuberculosis and bronchiectasis), by elevating serum amyloid A protein (SAA), may cause amyloid to be deposited in various tissues resulting in secondary (reactive) amyloidosis. Difference between anaemia of chronic disease and irondeficiency anaemia Anemia of Chronic Diseases Iron Deficiency Anemia Seru Iron Reduced Reduced Transferrin Reduced to normal Increased Transferrin Saturation Reduced Reduced Ferritin Normal to increased Reduced Soluble transferrin receptor Normal Increased Cytokine level Increased Normal Hepcidin Increased Reduced Bone marrow iron stores Normal to increased Reduced Ery Normal, microcytes Microcytes J Clin Invest. 2007;117(7):1755-1758. https://doi.org/10.1172/JCI32701. Acute phase proteins ̶ Induction of the acute phase reaction - changes in synthesis of many proteins in the liver ̶ measured in plasma. ̶ Regulation of protein synthesis - at the level of both transcription (DNA, RNA) and translation to protein. ̶ The cells have intricate systems for up- and down-regulation of protein synthesis, initiated by a complex system of signals induced in the acute phase reaction. Biotechnology Advances Volume 34, Issue 3, May–June 2016 Acute phase proteins Function related to ̶ limiting the negative effects of the acute phase stimulus or ̶ repair of inflammatory induced damage. Examples are enzyme inhibitors limiting the negative effect of enzymes released from neutrophils, scavengers of free oxygen radicals, increase in some transport proteins and increased synthesis and activity of the cascade proteins such as coagulation and complement factors. The protein synthesis may be upregulated even if plasma levels are normal, due to increased consumption of acute phase proteins. N Engl J Med 1999; 340:448-454 DOI: 10.1056/NEJM199902113400607 Function Positive acute phase protein Increase up to Protease inhibitors Alfa 1-antitrypsin Alfa 1-antichymotrypsin 4 x 6 x Coagulation proteins (serin proteinases) fibrinogen prothrombin factor VIII plasminogen 8 x Complement factors C1s C2b C3, C4, C5 C9 C5b 2 x Transport proteins haptoglobin hemopexin ferritin 8 x 2 x 4 x Scavenger proteins ceruloplasmin 4 x Others alfa1-acid glycoprotein (orosomukoid) serum amyloid A protein C-reactive protein 4 x 1000 x 1000 x 45 Biochemistry and physiology of the acute phase reaction ̶ CRP is a major acute phase protein acting mainly through Ca2+-dependent binding to, and clearance of, different target molecules in proteins, having evolved almost unchanged from primitive to advanced species. microbes, cell debris and cell nuclear material. In an acute phase reaction there may be a more than 1000-fold increase in the serum concentration of CRP. CRP is regarded as an important member of the family of acute phase, having evolved almost unchanged from primitive to advanced species. Nature Reviews Rheumatology volume 7, pages282–289 (2011) C-reactive protein Most functions of CRP are easily understood in the context of the body's defenses against infective agents. • The bacteria are opsonized by CRP and increased phagocytosis is induced. • CRP activates complement with the split product being chemotactic, increasing the number of phagocytes at the site of infection. Enzyme inhibitors protect surrounding tissue from the damage of enzymes released from the phagocytes. • CRP binds to chromatin from dead cells and to cell debris which are cleared from the circulation by phagocytosis, either directly or by binding to Fc-, C3b- or CRP-specific receptors. Platelet aggregation is inhibited, decreasing the possibility of thrombosis. • CRP binds to low density lipoprotein (LDL) and may clear LDL from the site of atherosclerotic plaques by binding to cell surface receptors on phagocytic cells. N Engl J Med 1999; 340:448-454 DOI: 10.1056/NEJM199902113400607 Biologically active products of complement activation Chemotactic factors C5a and MAC (membrane attack complex C5b67) are both chemotactic. C5a is also a potent activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules on vascular endothelial cells. Opsonins C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis. Other biologically active products of C activation Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their function. Biologically active products of complement activation Activation of complement results in the production of several biologically active molecules which contribute to resistance, anaphylaxis and inflammation. Kinin production C2b generated during the classical pathway of C activation is a prokinin which becomes biologically active following enzymatic alteration by plasmin. Anaphylotoxins C4a, C3a and C5a (in increasing order of activity) are all anaphylatoxins, which cause basophil/mast cell degranulation and smooth muscle contraction. Negative proteins of acute phase ̶ Decreases in albumin, transferrin, cortisol-binding globulin, transthyretin and vitamin A binding protein temporarily lead to an increased supply of free hormones, which usually bind to these proteins. ̶ Transthyretin (pre-albumin binding thyroxine, transports thyroid hormones from the plexus choroideus to the cerebrospinal fluid) inhibits the production of IL-1β by monocytes and endothelial cells. Its decline can thus be considered as a pro-inflammatory mechanism. These changes in blood protein profiles appear to be partly related to muscle starvation and catabolism. It is also an offer of amino acids for the production of positive acute phase proteins. 50 Cytokines ̶ generic name for a diverse group of soluble proteins and peptides ̶ act as humoral regulators at nano- to picomolar concentrations under normal or pathological conditions ̶ modulate the functional activities of individual cells and tissues. ̶ These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Nature Reviews Immunology volume 19, pages205–217(2019) Cytokine network This term essentially refers to the extremely complex interactions of cytokines by which they induce or suppress their own synthesis or that of other cytokines or their receptors, and antagonize or synergies with each other in many different and often redundant ways. These interactions often resemble Cytokine cascades with one cytokine initially triggering the expression of one or more other cytokines that, in turn, trigger the expression of further factors and create complicated feedback regulatory circuits. Mutually interdependent pleiotropic cytokines usually interact with a variety of cells, tissues and organs and produce various regulatory effects, both local and systemic. International Journal of Oral Science volume 11, Article number: 30 (2019) “cytokine storm” ̶ extreme increase in inflammatory cytokines, including IL-1β, IL-2, IL- 6, IL-7, IL-8, IL-10, granulocytecolony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-inducible protein-10 (IP10), monocyte chemotactic protein 1 (MCP1), macrophage inflammation protein-1α, IFN-γ, and TNF-α. 54 Signal Transduction and Targeted Therapy volume 5, Article number: 128 (2020) 55 Journal of Infection 80 (2020) 607-613 Chemokines Generic name given to a family of pro-inflammatory activationinducible cytokines. These proteins are mainly chemotactic for different cell types. All chemokines possess a number of conserved cysteine residues involved in intramolecular disulfide bond formation, which allows chemokines to be grouped into families according to the presence or absence of one or more conserved cysteine residues. Chemokines ̶ According to their mode of expression and function, chemokines have been categorized as inflammatory chemokines and homeostatic chemokines. ̶ Inflammatory chemokines are expressed usually by leukocytes or related cells only upon cell activation. These factors mediate emigration of leukocytes. ̶ Homeostatic chemokines are expressed constitutively and are involved usually in relocation of lymphocytes or other cell types. ̶ Dual-function chemokines can act as inflammatory cytokines or homeostatic cytokines. Wound healing 59 Wound healing ̶ Wound healing is the process of repair that follows injury to the skin and other soft tissues. ̶ Healing is the interaction of a complex cascade of cellular events that generates resurfacing, reconstitution, and restoration of the tensile strength of injured tissue. ̶ Under the most ideal circumstances, healing is a systematic process, traditionally explained in terms of 3 classic phases: inflammation, proliferation, and maturation. Wound healing ̶ The inflammatory phase: ̶ a clot forms and cells of inflammation debride injured tissue. ̶ The proliferative phase: ̶ epithelialization, fibroplasia, and angiogenesis occur; additionally, granulation tissue forms and the wound begins to contract. ̶ The maturation phase: ̶ Collagen forms tight cross-links to other collagen and with protein molecules, increasing the tensile strength of the scar. I. Inflammatory Phase Immediate to 2-5 days ̶ Hemostasis ̶ Vasoconstriction ̶ Platelet aggregation ̶ Thromboplastin makes clot ̶ Inflammation ̶ Vasodilation ̶ Phagocytosis ̶ Fibrin products ̶ essential to wound healing and ̶ primary component of the wound matrix into which inflammatory cells, platelets, and plasma proteins migrate. ̶ Removal of the fibrin matrix impedes wound healing. II. Proliferative Phase 2 days to 3 weeks ̶ B) Granulation ̶ Fibroblasts lay bed of collagen - scaffold for migration and further fibroblast proliferation ̶ C) Contraction ̶ Wound edges pull together to reduce defect ̶ D) Epithelialization ̶ Crosses moist surface ̶ Cell travel about 3 cm from point of origin in all directions vascular network is also re-established through angiogenesis - main regulator of angiogenesis is the vascular endothelial growth factor (VEGF) family, which includes VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF) Journal of Theoretical Biology 459, 2018, 1-17 III. Maturation Phase ̶ During the maturation phase, fibroblasts leave the wound and collagen is remodeled into a more organized matrix. ̶ Tensile strength increases for up to one year following the injury. While healed wounds never regain the full strength of uninjured skin, they can regain up to 70 to 80% of its original strength. Acute vs. chronic wound ̶ Acute wounds ̶ adequate angiogenesis promotes re-epithelialization, fibroblasts' proliferation, and neutrophils' antiinfection activities. ̶ Chronic wounds ̶ persistent local bacterial infections hinder the formation of novel blood vessels. The restricted angiogenesis hampers fibroblasts' proliferation and the neutrophils' anti-infection activities. 66 Front. Bioeng. Biotechnol., 11 June 2020 67 Wound healing in DM International Journal of Research in Medical Sciences 2017: 5(10):4206 Thank you for attention Regenerative medicine 70 Nature volume 453, pages314–321(2008)