1191CHAPTER 100 Innate Immunity an immune response Similarly, it does not explain how a develop ing fetus is protected from the maternal immune system The danger hypothesis suggests that the immune sy[.]
CHAPTER 100 Innate Immunity an immune response Similarly, it does not explain how a developing fetus is protected from the maternal immune system The danger hypothesis suggests that the immune system does not simply detect self from nonself but instead detects danger or tissue damage As the first line of defense, the innate immune system must this rapidly against an almost infinite number of pathogens or noxious molecules This response is not learned, and it is conserved across phyla How does the immune system differentiate between endogenous and pathogenic molecules? The danger hypothesis suggests that it does not The innate immune response is triggered by molecular patterns that are associated with pathogenic organisms or tissue damage, or both These signals, known as danger-associated molecular patterns (DAMPs), are a diverse set of molecules (or parts of molecules or polymeric components) sharing recognizable common features that identify them as pathogenic.1 For example, nucleic acids or mitochondrial proteins released from their intracellular compartment during tissue damage or invading organisms are typical DAMPs Similarly, hydrophobic protein moieties, or hyppos, are also often hidden within tertiary and quaternary protein structures When they are exposed, they become DAMPs and trigger an innate immune response When associated with pathogens, these molecular patterns signaling danger are known as pathogen-associated molecular proteins (PAMPs) Lipopolysaccharide is an example of PAMPs within the bacterial cell wall, where the hydrophobic part of lipopolysaccharide is hidden When released by pathogens, this becomes exposed and acts as a PAMP All hyppos can be DAMPs, including lipid particles and nucleic acids Protein misfolding, damage, or binding to other molecules (e.g., lipopolysaccharidebinding protein [LBP] binding to lipopolysaccharide) can lead to hyppos being exposed, and normally functional endogenous proteins may become DAMPs That not all hyppos are always immunostimulatory may be down to the level of aggregation or quantity (evolved from quorum sensing used by eukaryotic colonies such as bacteria—a certain concentration of exposed hyppos will alert the colony of impending danger).2 DAMPs released following cell or tissue damage trigger responses from the innate immune system, which are similar to those produced by PAMPs.3 These endogenous signals are known as alarmins High-mobility group protein B1 (HMGB-1) is one of the best described alarmins In an intact cell, HMGB-1 is a histone-associated chromatin protein involved in DNA structural modulation, thereby regulating transcription However, following necrosis, the intranuclear HMGB-1 is released and recognized by receptors for advanced glycated end products (RAGE), which initiates an immune response aimed to contain damage Cells have also evolved to release HGMB-1 in response to other signs of danger, which, in the case of ischemia/reperfusion, may lead to actively amplifying the immune response.4 Signal Recognition Pattern recognition receptors (PRRs), a group of molecules with repeating sugars or specific amino acid motifs, recognize the typical molecular patterns of DAMPs PRRs can be soluble circulating molecules, bound to cell surfaces, or intracellular There is crossover between the types of PRRs: some PRRs, such as CD14, can operate both as a cell surface–bound receptor and in soluble form Circulating PRRs include sugar-recognizing collectins, ficolins, and small peptides called antimicrobial peptides Mannosebinding lectin (MBL) is produced in the liver and activates the 1191 complement system after binding typical repeated sugar patterns on pathogen cell walls The association between MBL levels and the risk of critical illness has not been consistently demonstrated— while some authors describe an increase in risk of the systemic inflammatory response with low levels of MBL, others have not.5,6 Indeed, in some diseases characterized by immune-mediated damage such as mycobacterial infection, moderately low levels of MBL are associated with better protection.7 Bacterial permeability increasing protein (BPI) is an antimicrobial protein It acts against Gram-negative bacteria by increasing cell wall permeability and has been investigated as a therapeutic agent in meningococcal sepsis in children Although there was no mortality benefit, there was a tendency toward a reduction in sequelae.8 Toll-like receptors (TLRs) are the best characterized group of membrane-bound PRRs There are at least 10 related receptors in humans and 12 in mice (Fig 100.1) TLRs are evolutionarily preserved from the worm Caenorhabditis elegans and strikingly homologous to toll, a gene product essential to Drosophila immunity CD14 LPS LPS LBP TLR4 Cell membrane A TIR CD14 LPS LBP LPS TLR4 MD2 Cell membrane TIR MyD88 Cascade of reactions leading to NF-κB activation and cytokine transcription Nucleus NF-κB B • Fig 100.1 Pathogen-associated molecular proteins and the toll-like receptor (TLR) pathway (A) TLR4 is bound to the cell surface TLR forms a dimeric complex along with MD2 to bind lipopolysaccharide (LPS) (B) The LPS-TLR4-MD2 complex causes a conformational change that allows TIRAP to bind to the TIR domain of TLR4 MyD88 binds to TIRAP, which eventually leads to early NF-kB activation and transcription of inflammatory cytokines LBP, Lipopolysaccharide-binding protein; TIR, toll/interleukin-1 receptor; MyD88, myeloid differentiation factor 88; TIRAP, TIR domain containing adaptor protein; NF-kB, nuclear factor kB 1192 S E C T I O N X I Pediatric Critical Care: Immunity and Infection and dorsoventral patterning.9 They are characterized by the presence of three distinct domains The TLR family can be divided into two subgroups depending on location TLRs 1, 2, 4, 5, and are bound to cell surfaces and recognize microbial membrane components The remaining TLRs 3, 7, 8, and are expressed in intracellular membrane-bound organelles and vesicles, such as the endoplasmic reticulum, endosomes, and lysosomes, where they typically recognize nucleic acids from intracellular pathogens TLR4 is the best described of the cell surface TLRs given its central role in the pathogenicity of Gram-negative septic shock as a key part of the lipopolysaccharide (LPS) recognition apparatus In order to bind LPS, TLR4 forms a dimeric complex in association with MD2 This complex formation exposes the binding site that recognizes LPS LBP binds to LPS, which allows recognition by soluble CD14 CD14 is instrumental in delivering the LPSbound complex to the TLR4/MD2 complex on the cell surface TLR4 can also bind to the streptococcal toxin pneumolysin, respiratory syncytial virus (RSV) fusion protein, and paclitaxel, the chemotherapeutic taxol used to treat ovarian, breast, and certain lung cancers (Fig 100.2) TLR2 recognizes lipoteichoic acid from gram-positive bacteria, lipoarabinomannan from mycobacteria, zymosan from fungi, and hemagglutinin from measles viruses, among others TLR2 forms complexes with TLR1 and TLR6 to mediate DAMP recognition TLR5 is present mainly in gut mucosal dendritic cells and Dying cell Signal Transduction HMGB-1 Cell debris Cell membrane Soluble HMGB-1 RAGE complex RAGE TLR2/4/6 MAL Signaling cascade leading to cytokine transcription Endolysosome TLR7/9 Nucleus NF-κB MyD88 recognizes bacterial flagellin In doing so, it can activate the adaptive immune system to mount a more specific response to pathogenic bacteria in the gut The intracellular membrane-bound TLRs3,7–9 are instrumental in defending against invasive pathogens They recognize nucleic acids, especially pathogen-associated nucleic acids such as doublestranded ribonucleic acid (dsRNA) and unmethylated CpG deoxyribonucleic acid (DNA) motifs not seen in mammalian cells TLR3 recognizes dsRNA during the replication of single-stranded RNA viruses, including RSV TLR3 deficiency in humans is associated with a susceptibility to infection with herpes simplex virus type TLR7 recognizes RNA viruses, as they are transported in autophagosomes, as well as bacterial RNA from group B Streptococcus TLR8 is also active against bacteria, and expression can be upregulated in bacterial infection TLR9 senses bacterial DNA (unmethylated CpG motifs) but also recognizes hemozoin, which is generated after the digestion of hemoglobin by Plasmodium falciparum (Fig 100.3) Three major classes of cytoplasmic PRRs are not membrane bound: nucleotide-binding and oligomerization domain-like receptors (NLRs), RIG-I–like receptors (RLR), and absent in melanoma 2–like receptors NLRs are the best described cytoplasmic PRRs Similar to TLRs, they have three distinct domains: Knockout mice deficient in one domain of NLRs (NOD-12/2 mice) are susceptible to Staphylococcus aureus and Helicobacter pylori infections; NOD-2–deficient mice are prone to infections from Toxoplasma gondii.10 SARM binds TLR complex to negatively regulate signaling cascade Recognition of self nucleic acids can lead to autoimmune reaction • Fig 100.2 Danger-associated molecular patterns and the toll-like recep- tor (TLR) pathway Cell debris released from a necrotic cell binds to soluble and cell surface receptors (e.g., HMGB-1 binds to RAGE) Debris may also directly bind to TLR receptors 2/4 or 6, leading to an inflammatory cascade Nucleic acids, in particular, and HMGB-1 are internalized into endolysosomes, where they bind to TLR7 and TLR9 This also leads to an inflammatory cascade, including an autoimmune reaction The cascade is negative regulated by the TIR domain adaptor protein SARM to prevent overwhelming inflammation HMGB-1, High mobility group box-1; RAGE, receptor for advanced glycation products; MAL, MyD88 adaptor-like protein; SARM, sterile a and armadillo motif containing proteins Once PRRs recognize a signal, they need to trigger a response In order to so, they send signals to effector pathways using second messengers Circulating PRRs can directly activate the complement system via the lectin pathway MBLs form complexes with MBLassociated serine proteases, which cleave C4 and C2 to form a C3 convertase C3 convertase cleaves C3 into active components C3a and C3b This leads to an antiinflammatory cascade, opsonization of phagocytic cells, and the formation of the complement membrane attach complex Other soluble-protein PRRs such as defensins can act directly as opsonins and chemotactic particles Cell surface and intracellular PRRs have Toll/interleukin-1 receptor (TIR)-containing domains that interact with intracellular signaling molecules Following TLR recognition of DAMPs, the cytosolic TIR-containing domains undergo structural reorganization, which provides a platform for the interaction with a family of adaptor molecules These include myeloid differentiation primary response 88 (MyD88), the prototypic adaptor molecule involved in the LPS-TLR4 pathway When bound to activated TLR4, MyD88 recruits a family of kinases, interleukin-1R (IL-1R)–associated kinases (IRAKs), in a complex Following a cascade of interactions, nuclear factor kB (NF-kB) is activated The result is the transcription of cytokines leading to an inflammatory response (Figs 100.2 and 100.3) As with so many other arms of the innate immune system, there is significant redundancy among adaptor molecules and the signaling pathway However, MyD88-deficient mice show reduction of NF-kB activation in all TLR pathways apart from TLR3 and TLR4, underlining its importance The adaptor proteins also have a regulatory function: The adaptor protein sterile-a- and armadillo motif– containing protein (SARM) negatively interacts with the adaptor protein TRIF, limiting the downstream activation of NF-kB.11 CHAPTER 100 Innate Immunity 1193 Soluble Mediator Response TLR5 TLR4 TLR9 TLR8 TLR7 TLR6-2 TLR3 TLR1-2 TLR2 Gram-negative bacteria Fungi Gram-positive bacteria RNA viruses Mycoplasma Mycobacteria DNA viruses Parasites (Trypanosoma) Parasites (Plasmodia) • Fig 100.3 Danger-associated molecular patterns and the toll-like recep- tor (TLR) pathway Cell debris released from a necrotic cell binds to soluble and cell surface receptors (e.g., HMGB-1 binds to RAGE) Debris may also directly bind to TLR receptors 2/4 or 6, leading to an inflammatory cascade Nucleic acids in particular and HMGB-1 are internalized into endolysosomes, where they bind to TLR7 and TLR9 This also leads to an inflammatory cascade, including an autoimmune reaction The cascade is negative, regulated by the TIR domain adaptor protein sterile a- and armadillo motif–containing proteins (SARM) to prevent overwhelming inflammation HMGB-1, High-mobility group protein B1; MAL, MyD88 adaptorlike protein; RAGE, receptor for advance glycated end products Effector Pathways For the innate immune system to be effective, it must respond to danger rapidly The aim of the response is to neutralize the threat where possible, raise the alarm, and seek reinforcements The effector response can be broadly divided into two arms: the soluble mediator response, which has both widespread systemic effects and more targeted local effects, and the cellular response, which aims to neutralize directly danger through phagocytosis and self-destruction The signal transduction pathways described activate nuclear transcription factors such as NF-kB Cytokine production is an end point of activation of NF-kB The inflammatory cytokines include tumor necrosis factor–a (TNF-a), IL-1b, IL-4, Il-6, IL-10, IL-12, IL-18, CCL4-RANTES, and transforming growth factor-b The specific subset of cytokines that is produced and released depends on the innate immune cell type involved Although NF-kB is the prototypic transcription factor downstream of the innate immune signaling pathways, more recently, transcription factor EB (TFEB) has been described as a potentially important mammalian factor in innate immunity Unlike NF-kB, TFEB exists in the cytoplasm of macrophages Upon activation, TFEB moves to the nucleus and leads to transcription of proinflammatory genes TFEB is increasingly being recognized as a key mediator in both phagocytosis and autophagy (self-destruction) Therefore, TFEB mediates macrophage responses to mycobacteria and bacteria such as S aureus but also is vital in regulating the response to cellular stress seen in critical illness states.12 The soluble mediator arm has a widespread systemic effect as well as a local effect to contain and neutralize the danger signal Systemic cytokine effects: Systemic effects rapidly follow the exposure to danger These include the production of fever: TNF-a and IL-1B, among other cytokines, act on the hypothalamus to increase the temperature set point The fever response is likely to have a beneficial effect in the immune response In several species, controlling this fever response has a detrimental effect on survival Observational evidence in humans suggests that the lack of a fever response in critical illness is associated with an increase in mortality.13 The exact mechanisms of how fever lends itself to immunity are not yet fully elucidated, but it is likely to be multimodal: from decreased pathogenicity of microorganisms to improved immune function The effects of fever on the immune system include (a) the increased release of neutrophils from the bone marrow; (b) improved localization of neutrophils to tissues; (c) improved phagocytic and cytotoxic activities of neutrophils, macrophages, and natural killer (NK) cells; and (d) increased antigen presentation to T cells by dendritic cells within lymph nodes.14 Cytokines also have effects on the circulatory system and nervous system through providing nociceptive signals This mobilizes energy sources to vital organs to fight the danger and may restrict spread between organisms, respectively Nervous system involvement may also change behavior to avoid noxious stimuli; for example, TNF-a knockout mice have a decreased perception of bitter taste, implying that the TNF-a sensitizes animals to bitter substances, which may be deemed as noxious.15 Local neutralization: The complement system, as described earlier, plays an important role in the effector arm of the innate immune system PRRs activate complement through the lectin pathway This directly attacks pathogens via the membrane attack complex but also via opsonization and chemokinesis.16 Cytokines also have an important role in attracting immune cells to the site of danger; for example, CCL4-RANTES attracts macrophages and NK cells to the site of danger to neutralize the threat This can lead to a positive feed-forward loop: Cytokines released from local tissue, such as endothelial cells, initially attract immune cells, such as macrophages, to the site of danger The immune cells then produce more cytokines to amplify the signal and get further reinforcements 1194 S E C T I O N X I Pediatric Critical Care: Immunity and Infection Cellular Response The cellular response involves two processes—phagocytosis and self-destruction Both aim to neutralize the threat of danger where possible and recruit other immune mechanisms, such as the adaptive immune system Phagocytosis: DAMP recognition by cell surface PRRs sets into motion cytoskeletal rearrangements These are achieved by adaptor proteins activating cascades of kinases and guanosine triphosphatases (GTPases), leading to actin polymerization This results in a phagosome forming and pinching off the cell surface Activated macrophages are able to internalize 100% of their surface area within 30 minutes Once internalized, the phagosome may fuse with lysosomes to form a phagolysosome Here, the DAMPs may be neutralized or processed and relocated to the surface, where they can be presented to T cells to promote an adaptive immune response Reactive oxygen species (ROS) and nitric oxide are both involved in the neutralization of DAMPS within the phagolysosome Both are produced during an innate immune response.17 Self-destruction: Because phagocytosis is the process by which cells internalize and neutralize extracellular material, autophagy is the analogous process for cytoplasmic protein and organelles: the controlled destruction of cellular material following damage Autophagy is an essential part of cell maintenance and survival: multiorgan failure seen in an inflammatory response is postulated as not being sequelae of disease but rather an adaptive mechanism in response to a reduction in available energy.18 Autophagy is initiated following damage-induced expression of a family of autophagy-related genes (ATGs) The ATG-encoded proteins assemble to form an autophagosome, a double-membrane structure that envelopes the damaged proteins or organelles The autophagosome eventually fuses with a lysosome, where the protein or organelle gets enzymatically degraded.19 Autophagy may result in cell death once all of the organelles undergo autophagy and the autophagic cell eventually undergoes phagocytosis This is particularly important following damage, or infection with intracellular pathogens, when the pathogen cannot be controlled Traditionally, cell death has been described as apoptosis (regulated cell death) and necrosis (accidental cell death) However, as the mechanisms behind cell death have been discovered, this distinction has proved to be an oversimplification There are currently 10 nonapoptotic described pathways that result in regulated cell death! Some of these occur during normal physiologic processes of development, while others may be triggered by very specific stimuli, such as toxins It is important to note that regulated cell death can be classified according to whether it is immunogenic (leads to an immune response) or tolerogenic (inhibits an immune response) This is closely related to the release of DAMPs Many chemotherapeutic agents work by initiating an immunogenic cell death—releasing DAMPs that are then presented to cytotoxic T cells to harness the immune system to kill tumor cells There is an added degree of control imposed by the post-release modification of DAMPs that can switch an immunogenic cell death to a tolerogenic one HMGB-1, described earlier, provides a classic example: extracellular HMGB-1 initiates and amplifies an immune response as a DAMP Once oxidized, HMGB-1 becomes tolerogenic, limiting the anticancer immunogenic response Similarly, the abundance of HMGB-1, controlled through cleavage and degradation, can also determine the type of immune response it generates.19 Crosstalk Between Systems The innate and adaptive immune systems interact continuously Considering them as separate systems is an enormous simplification Coagulation, neuroendocrine, cardiovascular, and autonomic nervous systems all influence, and are influenced by, immune responses At the simplest level, this is shown by many molecules having important properties in multiple systems (e.g., acetylcholine is a neurotransmitter and a paracrine regulator of lymphocytes, epinephrine has profound cardiovascular effects but also stimulates the bone marrow to release neutrophils into the circulation) High plasma glucose levels arising from the stress response and insulin resistance during critical illness may inhibit complement binding to and killing of microorganisms The interaction between the innate immune system and coagulation cascade is particularly important in critical illness, especially in the context of sepsis and trauma Plasminogen activator inhibitor (PAI-1) is a potent inhibitor of fibrinolysis It achieves this response by inhibiting both tissue- and urinary-type plasminogen activator Levels of PAI-1 are increased after trauma and sepsis, especially so in severe meningococcal sepsis Inflammatory mediators TNF-a, IL-1 and IL-6, complement 5a, and LPS all act to increase PAI-1 production In turn, PAI-1 contributes to a procoagulant state and inhibits neutrophil apoptosis Although this may help to contain inflammation at the site of infection, genotypes associated with high levels of PAI-1 production are associated with worse outcome in septic shock Thus, there is a direct link between how readily the immune system triggers an increased clotting tendency in critical illness and poor outcome.20 Similarly, inflammatory mediators such as IL-6 and HMGB-1 stimulate release of the potent coagulation activator tissue factor (TF) from activated monocytes, macrophages, and endothelial cells Small membrane vesicles from apoptotic cells known as microparticles bind to cell surfaces through specific receptors, expressing TF This promotes thrombin formation, which, in turn, converts fibrinogen to fibrin Thrombin and fibrin generation are increased in inflammation, in part because fibrinolysis is impaired due to increased activity of PAI-1 but also secondary to diminished activated protein C (APC) and tissue factor pathway inhibitor (TFPI) These processes have been the targets for numerous clinical trials of drugs with anticoagulant/profibrinolytic actions—all aiming to achieve antiinflammatory effects by targeting coagulation systems APC may also have other antiinflammatory actions, downregulating inflammatory cytokines, preventing the loss of the endothelial barrier, and acting as an antioxidant and antiapoptotic agent Although initial trials suggested therapeutic benefits of APC in sepsis, this has since been contradicted in larger trials in both children and adults.21–23 Another example of a novel interaction between the coagulation and immune systems is that TLR4-activated platelets interact with neutrophils to trap and kill bacteria in so-called neutrophil extracellular traps (Fig 100.4) In vitro studies showed that LPS, as well as plasma from septic adults, could induce this phenomenon.24 Regulation of the Innate Immune Response As with any physiologic response, the innate immune response needs regulation to balance the sensitivity of the response against the potential to overreact and cause harm to the host Key regulatory mechanisms for the innate immune system are summarized in Table 100.1 CHAPTER 100 Innate Immunity Clinical Manifestations of the Innate Immune Response in the Intensive Care Unit The innate immune system is the body’s front-line defense patrol against danger All patients admitted to the ICU will have activated an innate immune response In some cases, this response may itself be deleterious either through amplification in the face 100.00 àm Y 100.00 àm X TRENDS in Microbiology ãFig 100.4 A scanning electron microscope image of a neutrophil extra- cellular trap Neutrophils release an extracellular trap made of histones and DNA, along with proteases and antimicrobial peptides They entrap pathogens, with the DNA exhibiting intrinsic antimicrobial properties (From Baums CG, von Köckritz-Blickwede M Novel role of DNA in neutrophil extracellular traps Trends Microbiol 2015;23:330–331.) 1195 of an insurmountable threat or through dysregulation of the response In other cases, the critical illness may be a result of an inadequate response The prototypic description of the innate immune response is based on the sepsis model However, many other pathologic processes in the ICU start with an innate immune response: Hypoxic ischemic injury: Hypoxia—either systemic, such as at high altitude, or localized, such as in solid tumors—leads to the activation of an innate immune response Oxygen status is sensed by proline hydroxylases, which act on the proline residues of hypoxia-inducible factors (HIFs) 1a and 2a This allows the binding of an E3 ubiquitin ligase complex, which inactivates HIFs Hypoxia releases this inactivation HIFs set off a cascade of downstream pathways, including the activation of NF-kB, the transcriptional regulator that is a key factor in the TLR response to LPS The overall effect in hypoxia is for the innate immune cells to increase phagocytosis and antimicrobial killing, prevent apoptosis of neutrophils, increase antigen presentation, and increase endothelial adhesion and cytokine release HIF-deficient myeloid cells are unable to overcome pathogens effectively but form chronic ulcerative lesions However, adaptive immunity pathways are downregulated to prevent an overactive response.32 In addition to phagocytosis, hypoxia also triggers autophagy Importantly, hypoxia can lead to the adaptive destruction of mitochondria, known as mitophagy While mitochondria act as the cell’s energy powerhouse, they also produce ROS While low levels of ROS act as signaling molecules in various cellular processes, during hypoxia, mitochondria can produce high levels of ROS This can lead to more widespread cellular damage Therefore, one of the adaptive responses to hypoxia is mitophagy, to regulate the levels of ROS generation HIF can upregulate several components involved in the mitophagy pathway, although the process is complex, with variable responses to levels of hypoxia.33 Trauma: Trauma leads to varying degrees of cell necrosis, which is inflammatory Cell necrosis releases alarmins, such TABLE Regulatory Mechanisms Controlling the Innate Immune Response: Loss of Regulation Can Lead to Diseases 100.1 Such as Hemophagocytic Lymphohistiocytosis Regulation Example Negative feedback Antiinflammatory arachidonic acid–derived lipoxin released after prostaglandin to inhibit granulocyte migration and promote phagocytosis25 Cellular compartmentalization LPS-stimulated migration of TLR7 and TLR9 from the endoplasmic reticulum to the endolysosome9 Tissue dependence IL-10 on mucosal surfaces has baseline antiinflammatory effect, preventing response to microbiota; released by LPSstimulated TLR426 Redox state In apoptosis, the mitochondrial outer membrane is permeabilized, arresting the respiratory chain Reactive oxygen species generate oxidize HMGB-1, making it nonimmunostimulatory20 Energy state mTOR phosphorylated in states of energy abundance inhibiting autophagy; this is released in energy-depleted states27 Hormonal control Vitamin–dependent defensin and cathelicidin production (both PRRs)28 Steroid and catecholamine up/downregulate cytokine production29,30 Microbiome interactions Gut microbiome activates the NLR3-inflammasome to induce pro IL-1b, but not its cleavage into IL-1b–pathogenic bacteria invading the microbiome and then induce the cleavage of pro IL-1b, producing a rapid inflammatory response31 HMGB-1, High-mobility group protein B1; IL, interleukin; LPS, lipopolysaccharide; mTOR, mammalian target of rapamycin; PRR, pattern recognition receptor; TLR, toll-like receptor.\ ... to T cells by dendritic cells within lymph nodes.14 Cytokines also have effects on the circulatory system and nervous system through providing nociceptive signals This mobilizes energy sources... to actin polymerization This results in a phagosome forming and pinching off the cell surface Activated macrophages are able to internalize 100% of their surface area within 30 minutes Once internalized,... of hypoxia-inducible factors (HIFs) 1a and 2a This allows the binding of an E3 ubiquitin ligase complex, which inactivates HIFs Hypoxia releases this inactivation HIFs set off a cascade of downstream