Asthma is an inflammatory disorder of the airways resulting in their hyperreactivity. In the United States, approximately 25.7 million people have been diagnosed with asthma by a healthcare professional . Asthma initially starts out as an episodic disease in which repetitive environmental insults such as viral infection or allergen exposure result in a period of wheezing and airflow obstruction followed by resolution of the symptoms. After several bouts of wheezing, the threshold for airway reactivity is reduced and patients begin to experience mild symptoms between episodes . The persistence of symptoms and airway hyperreactivity between episodes suggest the existence of a memory-driven process (Fig. 1). Given that most asthma exhibits an immunologically based T-helper-2-type (Th2) inflammatory response with extensive upregulation of the interleukin (IL)-4 gene cluster, it has been difficult to discern whether this phenotype is mediated by an immunological or a tissue-based memory . Here, we present a hypothesis of disease-related memory and its relevance for the development and persistence of chronic asthma.
CONCEPT OF MEMORY IN BIOLOGICAL SYSTEMS
In physiological systems, memory is the retention and retrieval of an acquired or learned signal. Memory occurs through encoding of information, storage and then retrieval. Persistence of memory usually requires consolidation and reconsolidation (repetition of the encoding process). Retrieval of the memory may occur in various forms. In psychological terms, declarative (explicit) memory requires a conscious recall. On the contrary, procedural (implicit) memory does not require conscious recall but is based upon implicit learning through repetition (reviewed in ). Outside the central nervous system, the term memory is often applied to T and B cells, and more recently natural killer (NK) cells of the immune system. The T and B cell memory is induced and maintained through the expansion and persistence of antigen-specific clones. The immunological memory is akin to episodic/explicit memory, as it requires an act of ‘conscious recall’ through re-exposure to the inciting antigen [5,6].
NEUROLOGICAL SIGNALLING MECHANISMS OF MEMORY FORMATION
Neurological memory can be formed by a variety of short-term and long-term facilitation, augmentation and potentiation mechanisms. The order, frequency and duration of stimulation can alter the neuronal system such that the magnitude and frequency of the presynaptic input and the resulting postsynaptic output are no longer in a one-to-one correspondence [7,8]. This ability is termed synaptic plasticity. Synaptic plasticity alters the N-methyl-D-aspartate (NMDA) receptor, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and calcium that are directly required for synaptic signalling, as reviewed in . In addition, evidence demonstrates shifts in the signalling outputs including cyclic AMP (cAMP) (reviewed in [10,11]), mitogen-activated protein kinase (MAPK) amplification , and protein kinase C (PKC) and phosphatidylinositol-3-kinase (PI3K) [13▪]. Secondary activation of the systems produces neurotrophin-like neuropeptide in a PKC-dependent manner, thus increasing overall signalling during synaptic plasticity.
Coordinated activation of AMPA receptors in combination with the metabotropic glutamate receptors (mGluRs) or NMDA glutamate receptors results in sustained intracellular signalling. This coordinated receptor stimulation leads to activation of the Ca2+–PLC–PKC pathway, the PI3K pathway  and the ERK1/2 MAPK pathways . Many signalling pathways ultimately converge on ERK signalling, resulting in translational and posttranslational effects that maintain altered signalling patterns despite a lack of new activation triggers. Activation of cyclic AMP response element-binding protein downstream of cAMP and ERK1/2 can specifically increase immediate early genes including c-Fos during plasticity. Activation of immediate early genes can perpetuate intracellular signalling and prolong phenotypes following cessation of activation signals. c-Fos dimerizes with c-jun to form the activator protein 1 transcription factor, which is crucial for long-term memory and synaptic plasticity .
ROLE OF ENDOSOMAL RECYCLING OF RECEPTORS IN SUSTAINED SIGNALLING AND MEMORY GENERATION
Repeated stimulation of neurons leads to long-term potentiation, which manifests with prolonged and exaggerated outputs of action potentials. This eventually increases signalling output and expression of immediate early genes. Endosomal trafficking of certain receptors such as AMPA and NMDA receptors is critical, given that during long-term potentiation, AMPA receptors that become internalized will undergo preferential sorting via early endosomes and recycling endosomes to return to the plasma membrane. In contrast, long-term depression, which results in decreased excitatory signalling, will preferentially target AMPA receptors to the lysosomes (reviewed in [17,18]). These differentially regulated processes suggest that recycling of neurotransmitter receptors plays a seminal role in synaptic plasticity. Homer is a recently identified protein, whose deficiency impairs memory. Homer is an ERK1/2-inducible protein, and it functions to facilitate AMPA receptor recycling . Outside the nervous system, an interleukin 2 receptor α mutant with increased recycling causes sustained T cell signalling and proliferation . Similarly, reduced degradation and increased recycling of IL4RA in Dock2-/- mice lead to sustained signal transducers and activators of transcription 6 (STAT6) signalling and heightened Th2 response . The mechanisms surrounding receptor recycling are likely to be complex and involve many factors, but appear to be conserved between physiological systems.
ROLE OF HYSTERESIS AND BISTABILITY OF SIGNALLING MOLECULES IN MEMORY FORMATION
Many biological processes manifest a behaviour known as hysteresis. Hysteresis in biology means that a cellular behaviour and a response are shaped not only by the current environment but also by the past history/experience. Biological processes manifesting hysteresis can toggle between two alternative stable steady states: bistability. A bistable system can exist in three states: an ‘on’ state, an ‘off’ state and an unstable intermediate state . Early examples of biological bistable systems include the lambda phage lysis-lysogeny switch and the hysteretic lac repressor system . Lisman in 1985  first suggested that a bistable system could serve as a self-sustaining biochemical memory. Bistability arises from a positive feedback loop or a mutually inhibitory, double-negative feedback loop .
Ferrell and associates have mathematically shown that when the strength of positive feedback exceeds a certain limit, the system shows hysteresis and becomes bistable . In confirmation, they have shown that progesterone-induced activation of MAPK and Cdc2 induces a self-sustained activation mechanism in frog oocytes, which is dependent upon a positive feedback loop . Disruption of the positive feedback loop at the level of c-Mos (Raf-1) abrogates the signalling memory. The concentration and activity of the kinases of the ERK module and their phosphatases play a pivotal role in bistability . ERK can exist in an ‘off’ state or an ‘on’ state on the basis of the concentrations of its immediate activator mitogen-activated protein kinase kinase (MEK) and its inactivating enzyme mitogen-activated protein kinase phosphatase 3. Within a narrow range of concentrations, an intermediate unstable state exists. Thus, the ERK pathway could function as a bistable module and the ‘on’ state of a bistable module could function as memory. In summary, there is strong theoretical, mathematical and experimental evidence that the ERK1/2 module can function as a system memory.
EVIDENCE FOR MEMORY-DRIVEN PROCESSES IN ASTHMA
In a similar fashion to synaptic plasticity in the central nervous system, a primed cell and tissue are able to respond faster and stronger because of prior experience. Tissue priming in asthma manifests as airway hyperreactivity, one of the most reliable features of asthma and detectable when the patient is asymptomatic. The mechanism of airway hyperreactivity is complex and likely involves memory-driven processes in the airway smooth muscle and epithelium. Priming of epithelium is also manifested through increased permeability and increased secretion of mucus, cytokines and other epithelial mediators.
EXTRACELLULAR SIGNAL REGULATED KINASE 1/2 MANIFESTS SUSTAINED ACTIVATION IN ASTHMA
Sustained ERK1/2 activation is critical for asthma. Pharmacological inhibition of ERK1/2 blocks airway inflammation, mucus production and airway hyperreactivity in a mouse model of asthma [26,29,30]. We have further shown that ERK1 knockout mice are unable to generate a Th2 immune response and features of asthma in the mouse model. Finally, we have demonstrated sustained expression of pERK1/2 in airway epithelium and smooth muscle biopsy samples from asthmatic patients. The expression level of the activated ERK1/2 correlated with the clinical severity of asthma and eosinophilic infiltrates. We have also reported increased expression of MEK and sustained levels of pERK1/2 in blood CD4 T cells from asthmatic patients. This increased pERK1/2 conferred T cells’ resistance against inhibition by glucocorticoids, transforming growth factor (TGF)-beta and IL-10 .
EXTRACELLULAR SIGNAL REGULATED KINASE 1/2 ACTIVATION IS SELF-SUSTAINED (MEMORY-DRIVEN) FOLLOWING REPETITIVE STIMULATION AND IS ASSOCIATED WITH EPITHELIAL CELL PRIMING
In epithelial cells, a rapid activation and a return to baseline of ERK1/2 result following a single stimulation with IL-4, IL-6, IL-13, eotaxin or epidermal growth factor (EGF). In contrast, repetitive stimulation with any of these cytokines results in sustained activation of ERK1/2 lasting more than 7 days after the last stimulation . This provides a strong evidence for ERK1/2 bistability and suggests that airway epithelial cells can maintain activated ERK1/2 through a memory-driven process. As the airways of a sensitized subject are repeatedly exposed to environmental triggers during the natural course of asthma, we speculate that this generates a cellular memory sustaining ERK1/2 activation at times when there are no apparent environmental triggers.
There is a spatial difference in ERK1/2 subcellular localization between acute/single and repetitive stimulation. Nuclear translocation of pERK1/2 dominates following an acute stimulation. In contrast, pERK1/2 primarily localizes to Rab4+ recycling endosomes and Rab5+ early endosomes with modest translocation to the nucleus in the repeated stimulation model. The nuclear localized pERK1/2 drives transcription of many genes and induces an acute state of cellular activation. In contrast, the endosomally localized pERK1/2 behaves more like a latent activator of the cell, which is akin to memory storage (Fig. 2). The endosomally localized pERK1/2 primes epithelial cells for heightened production of cytokines and chemokines and is associated with epithelial resistance against growth factor withdrawal induced apoptosis .
We have shown in vivo that repetitive intranasal exposure to multiple allergens (dustmite, ragweed, asperguillus) results in sustained ERK1/2 activation in the airway tissue that persists greater than 12 weeks after the last allergen exposure. This sustained ERK1/2 activation is associated with the development of persistent asthma (heightened airway hyperreactivity, sustained inflammation, mucus production and airway remodelling) despite the cessation of allergen exposure 3 months earlier . In accordance with prior in-vitro data, a significant portion of the pERK1/2 was localized to the endosomal compartment in airway epithelial cells. We have reported a similar endosomal localization of pERK1/2 in airway epithelial cells from human asthma. The results suggest that the sustained ERK1/2 activation in asthma is, at least, in part memory-driven.
MECHANISM OF MEMORY-DRIVEN ACTIVATION OF EXTRACELLULAR SIGNAL REGULATED KINASE 1/2
Several potential mechanisms have been proposed to explain the sustained pERK signalling following repeated stimulation. In cultured cells, EGF stimulation activates ERK1/2 prior to Cbl-dependent ubiquitination and downregulation of epidermal growth factor receptor (EGFR). However, when sprouty 2 (Spry 2) is overexpressed, there is direct binding and sequestration of Cbl by Spry 2 . As a result, EGFR is rescued from degradation and recycled via endosomes back to the cell surface producing sustained activation of EGFR and ERK1/2 signalling. Indeed, we have demonstrated that spry 2 is increasingly induced at a high level during repetitive stimulation of epithelial cells in a Fyn kinase-dependent manner. Spry 2 plays an essential role in sustained ERK1/2 activation, as its knockdown in epithelial cells and its biallelic deletion in fibroblasts result in inability to sustain ERK1/2 activation in this model. Our results indicate the development of multiple positive feedback and double-negative feedback loops, which lead to ERK1/2 bistability in our model (Fig. 3).
In addition to spry 2, other molecules that have been reported to promote sustained ERK1/2 signalling include B-Raf , Rap1  and beta-arrestin . The involvement of beta-arrestin is of particular interest, as mice with beta-arrestin deficiency are unable to manifest features of asthma. Subcellular localization of ERK1/2 is also regulated by phosphatidylinositol-4-phosphate 5-kinase and related FYVE finger-containing proteins 1 and neurofibromin 1 . An imbalance between ERK1/2 and its phosphatases in favour of ERK1/2 also promotes sustained ERK1/2 signalling [38▪].
OTHER EXAMPLES OF MEMORY-DRIVEN SIGNALLING PROCESSES IN ASTHMA
A classic example of memory-driven signalling processes is the auto-induction of GATA-binding protein 3 (GATA3) during a Th2 immune response. Following an initial induction of GATA3 by IL4-driven STAT6, the induced GATA3 binds to its own promoter and drives its transcription . This positive feedback loop establishes a bistable system, which functions as a memory for sustained GATA3 induction.
ROLE OF AUTOCRINE AND PARACRINE SECRETION IN GENERATION OF CELLULAR MEMORY
Positive feedback loops can be established not only through intracellular signalling networks but also through autocrine and paracrine secretory processes. Eosinophils partially regulate their own survival and activation through autocrine secretion of pro-survival IL-5 and pro-apoptotic TGF-beta . A similar promotion of survival and priming occurs in basophils through autocrine production of IL-3 . IL-17 induces sustained mucus production by autocrine secretion of epithelial IL-6 . It is unclear whether endosomal recycling of receptors and ligands is involved in the foregoing processes.
Utilization of learned skills to gain evolutionary advantage is critical in human development. The learning of these skills and their application are driven by memory. This memory allows adaptation to the environment. Many human diseases including asthma and allergic diseases are elicited by environmental factors in genetically susceptible individuals. We believe that the disease-inducing environmental factors generate a tissue memory upon multiple iterations. This tissue memory develops independent of the immunological memory. The immunological memory facilitates a robust response but requires the presence of the inciting antigen. In contrast, the tissue memory can sustain the pathological process in the absence of the environmental trigger. In the natural course of a disease such as asthma, we speculate that these two mechanisms complement each other. Allergens (and other nonallergenic asthma triggers) elicit asthma through direct activation of a sensitized immune system in a periodic manner. When allergens are absent in the environment, the tissue memory maintains the pathologic processes, albeit at a low intensity, until the next exposure to the allergen. Repetitive allergen exposure not only reinvigorates the disease process but also reinforces the disease memory and the cycles continue (Fig. 1).
System bistability provides a mathematical explanation for a tissue-based memory. In order to take advantage of the mathematical understanding of system bistability and memory, we will have to generate quantitative data about the involved biochemical reactions (substrates, products, dissociation constants of the reactions). Only then will we be able to understand and interfere with the disease memory and curtail the persistence of the disease.
This work is supported by NIH funding AI091614, HL 36577 and HHSN272200700048C.
Conflicts of interest
There are no conflicts of interest.
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