key: cord-324788-echu0zmf
authors: Aich, Palok; Potter, Andrew A; Griebel, Philip J
title: Modern approaches to understanding stress and disease susceptibility: A review with special emphasis on respiratory disease
date: 2009-07-30
journal: Int J Gen Med
DOI: nan
sha: 
doc_id: 324788
cord_uid: echu0zmf

Studies in animals and humans link both physical and psychological stress with an increased incidence and severity of respiratory infections. For this manuscript we define stress as the physiological responses an individual undergoes while adjusting to a continually changing environment. It is known that stressors of various types (psychological/physical) can alter the physiological levels of certain hormones, chemokines and cytokines. These alterations send information to the central nervous system to take necessary action which then sends messages to appropriate organs/tissues/cells to respond. These messages can either activate or suppress the immune system as needed and failure to compensate for this by the body can lead to serious health-related problems. Little is known how stress affects disease susceptibility, yet understanding this mechanism is important for developing effective treatments, and for improving health and food quality. The current review focuses on (a) the effects of psychological stressors in humans and animals, (b) various methodologies employed to understand stress responses and their outcomes, and (c) the current status of the attempts to correlate stress and disease with respiratory disease as model system. The methodologies included in this review span traditional epidemiological, behavioral and immunological studies to current high throughput genomic, proteomic, metabolomic/metabonomic approaches. With the advent of various newer omics and bioinformatics methodologies we postulate that it will become feasible to understand the mechanisms through which stress can influence disease onset. Although the literature in this area is limited because of the infancy of this research area, the objective of this review is to illustrate the power of new approaches to address complex biological questions. These new approaches will also aid in our understanding how these processes are related to the dynamics and kinetics of changes in expression of multiple genes at various levels.

From the preceding discussion it is clear that stress can be defi ned in many different ways depending upon the objectives or perspective of the researcher. All these defi nitions, however, share a common component of adaptive physiological responses following challenges to homeostasis. The adaptive reactions to stressors may involve mobilization of a wide variety of physiological responses including the immune response. Stress responses usually include physical perturbations that can encompass either the entire body or specifi c cellular compartments. Considering the volume of work in various areas of stressors and their effects the main objective of the current review is to focus on one type of stress, which includes psychological stressors.

For our purposes, stress can be defi ned as a psychologically perturbing condition occurring in response to adverse external infl uences capable of affecting physical health. Transportation, fear (ie, fright and fl ight response), overcrowding and weaning in the form of social reorganization are a few of the important types of psychological stressors identifi ed in the literature. These stressors have been linked to many conditions including immune suppression, disease susceptibility, hypertension and reproductive dysfunctions. [4] [5] [6] Stress is a major concern because it is ubiquitous, recurring in nature and has detrimental effects on health. In this review we will primarily deal with important psychological stressors and their infl uence on respiratory disease which is one of the most widely studied models.

For many years, psychological stress has been shown to signifi cantly increase disease susceptibility. 6, 7 Until 20 years ago, researchers investigating the psychological factors contributing to human disease focused primarily on coronary heart disease and cancer and neglected studies on infectious diseases. 8 However, interest in this area started to shift with the publication of evidence that psychological factors infl uenced immune function. 9 Furthermore, there was an increasing awareness that stress and other psychological factors played a role in the onset and progression of acquired immunodefi ciency syndrome (AIDS). 10 These studies demonstrated a signifi cant role for psychological stressors in compromising immunity and interest in the effects of stressors in other diseases was initiated. Considerable emphasis has been placed on respiratory diseases in understanding the onset and severity of the disease as a result of psychological stress. 11

Increased risk of fatal bacterial respiratory infections following a primary viral infection has been observed in a wide variety of species. This phenomenon is called viral-bacterial synergy and was fi rst established following human infl uenza epidemics when a variety of secondary bacterial respiratory infections were associated with increased mortality. 12 Studies have also linked a variety of psychological stressors with an increased incidence and severity of respiratory infections in humans 13, 14 and animals. 15, 16 It is known that respiratory disease has a huge economic impact in the areas of human health care, animal welfare and the food industry. 17, 18 To focus the review, we will restrict our discussion to research related to a comparative analysis of the effects of psychological stress on respiratory disease in humans and cattle.

There are reports which have shown direct connections between stress and immune system function. 19 Similarly, other studies have shown that social stressors could also increase the risk for upper respiratory infection. 20 A viral challenge study provides the strongest evidence for a link between stress and susceptibility to the common cold. 21 Other studies have extended these results by considering a wider range of psychosocial factors. 22 The effects of stress on health are often mediated by a number of psychological factors. Social support can often act as a buffer against the effects of stress as Cohen and colleagues showed that social support reduced viral replication rate and increased mucociliary clearance of infection. 11, 13 In another report, they examined the effects of stress and social support in a routine study of upper respiratory tract illnesses. 23 Under low levels of stress, high levels of social support were associated with a decreased risk of infection, whereas social support had no effect when levels of stress were high.

A separate study was done to examine the associations between psychosocial factors (stress, social support, fl uctuations in mood) and viral exacerbations of asthma. The study involved naturally occurring illnesses rather than experimentally-induced infections but it maintained several important features of the methodology used by Cohen and colleagues. 21, 24 For example, stress was measured at the start of the study by measuring the immune responses in terms of leukocytes present in peripheral blood in order to determine the extent to which stress could predict subsequent illness. In addition, effects of personality, 25 smoking status, and alcohol consumption 21 were also studied as possible predictors of susceptibility to respiratory viral infections.

Before considering the effects of psychosocial factors on respiratory virus-induced exacerbations of asthma, it is essential to have strong evidence that these viruses play a direct role in asthma. Until recently, it appeared to be only a weak association between asthma and upper respiratory tract infection in adults. 26, 27 The absence of a stronger association in these epidemiological studies of adult asthmatics could, at least in part, have been due to diffi culties in isolating human rhinoviruses and coronaviruses. Indeed, results from a study using enzyme-linked immunosorbent assays for antibodies to human coronavirus and semi-nested reverse transcriptase polymerase chain reactions for detections of rhinovirus suggested a stronger association between these viruses and asthma in adults. 28 In summary, this study confirmed that psychosocial factors and health-related behaviors were associated with increased susceptibility to colds, which then led to an exacerbation of asthma. This conclusion was made in the context of a study where both diseases (cold and asthma) were verifi ed using objective measures. The well established buffering effect of social support was observed in the high stress group and possible confounders such as demographics, health-related behaviors or personality could not account for this effect. Alcohol consumption, personality and demographic factors were also shown to be important predictors of susceptibility. In contrast smoking was related to illness severity but not disease susceptibility. These results show that one must consider a range of psychosocial factors, personality traits, demographic factors, and health-related behaviors in studies of individual differences in susceptibility and severity of upper respiratory tract infections.

While studies have correlated stress-related behavior with disease susceptibility, research has also been performed to determine the immunological basis of the relationship between stress and disease. Exposure to viral agents that cause upper respiratory disease provokes illness in some individuals but not others. Moreover, the severity of clinical symptoms among those who develop illness can vary substantially. Evidence from prospective epidemiological studies 20, 23, 29 and from experimental viral-challenge studies 21, 30 show that individuals reporting greater psychological stress have both a higher incidence and a greater severity of illness. However, past attempts to identify the behavioral patterns and biological responses linking psychological stress with upper respiratory viral illness have been unsuccessful. 20, 21, 30 Other studies reported that psychological stressors acutely activate the production of interleukin-6 (IL-6), a pro-inflammatory cytokine. IL-6 release in response to infection is thought to be mediated by glucocorticoids, thus providing a hypothetical pathway by which stressors (via the induction of glucocorticoid production) could control cytokine release. 31 In addition, IL-6 triggers additional release of glucocorticoids, possibly exacerbating the stress response through positive feedback. At least one source of IL-6 is epithelial cells as evidenced by their production of IL-6 in vitro and in vivo when exposed to rhinovirus. 32 Because a local increase in the concentration of this pro-infl ammatory cytokine precedes the development of acute signs and symptoms of illness, it has been implicated as a mediator in the pathway for symptom expression. In fact, IL-6 concentrations in nasal secretions were associated with upper respiratory tract symptoms among persons infected with infl uenza A virus (A/Texas and A/Kawasaki) and rhinovirus (strain Hanks' and type 39). 13 Cohen and colleagues addressed the hypothesis that IL-6 production in response to infl uenza A virus infection represents a viable pathway through which psychological stress infl uences the severity of illness. To achieve that goal, they measured levels of psychological stress in a group of adult subjects before experimentally infecting them with influenza A virus. Self-reported respiratory symptoms, mucus weights, and local IL-6 concentrations were then measured on the day before and for seven days after virus exposure. The data collected supported the conclusion that the level of psychological stress predicts the severity of illness and also the magnitude of the cytokine response. The data were then examined for evidence that IL-6 mediated the association between stress and illness. These analyses suggested that most of the effects of psychological stress on clinical symptoms and mucus weights could be accounted for by changes in IL-6. However, it is possible that IL-6 itself is not the causal link but rather just a marker (covariate) for other pro-infl ammatory cytokines which were elevated during the course of experimental infection. For example, in another related study 33 reported that interferon α and IL-6 levels (but not tumor necrosis factor [TNF], IL-8, IL-1, or IL-2) increased early in the course of infection and both correlated with viral titers, increases in body temperature, mucus production and symptom scores. There is also an issue regarding the correlational nature of the mediational analysis. Although consistent with the hypothesis that the association between stress and illness was mediated by IL-6, the data do not permit causal inference. For example, this pattern of data is also consistent with increases in IL-6 occurring in response to tissue damage associated with illness symptoms. Even with these reservations, this was the fi rst Aich et al study to provide evidence consistent with the hypothetical model that psychological stress infl uences upper respiratory infections through a biological pathway. 13 

There are two parts to the stress response: sympatheticadrenal-medullary (SAM) and the hypothalamic-pituitaryadrenal axis (HPA). The HPA is the core stress axis in mammals and together with the SAM system co-ordinates response to the diverse range of stressors from psychological to physical. There is considerable interplay between both neuronal systems especially between the noradrenergic nucleus locus ceruleus which provides central regulation of the SAM and the parvocellular neurones which regulate the HPA. The SAM, by triggering catecholamine release, provides the acute stress response whilst the HPA governs longer term stress defence mechanisms. Together those systems regulate energy utilisation and metabolic activity throughout the body. 34 The SAM system produces the immediate shock response by acting on the hypothalamus, which activates the adrenal medulla and the sympathetic autonomic nervous system (ANS) (Figure 1 ). The SAM produces the "fi ght-or-fl ight" response which increases alertness, blood fl ow to muscles, heart rate, blood pressure, respiration rate, etc. and might decrease activity in the digestive system. The HPA system regulates release of the hormone CRF to activate the anterior pituitary and uses another hormone, adrenocorticotropic hormone (ACTH), to activate the adrenal cortex to release a group of hormones including cortisol ( Figure 2 ). Cortisol and other glucocorticoid hormones have various effects such as conservation of glucose for neural tissues, elevation or stabilization of blood glucose levels, mobilization of protein reserves, conservation of salts and water, suppression of wound healing and the immune system. According to Seyle's general adaptation syndrome (GAS) theory the general adaptation syndrome divides the stress response into three stages: Stage 1: Alarm reaction (SAM and HPA activity increases and result in the "fi ghtor-fl ight" response); Stage 2: Resistance (HPA activity takes over, bodily resources are at maximum and if the stress is experienced for short term the body returns to normalcy); Stage 3: Exhaustion (with very prolonged stress, bodily systems are ineffective. Sympathetic ANS action reappears. Adrenal cortex damage causes parasympathetic action, Omics approaches in understanding stress and disease susceptibility eg, energy storage failure. The immune system collapses, and stress-related diseases increase).

While it has been assumed that psychological (psychogenic) and physical (neurogenic) stressors are most closely aligned with depression, the suspicion has arisen that systemic stressors, including immune alterations, may also act in such a provocative capacity. 35 Communication occurs between the immune, endocrine, autonomic and central nervous systems 36 such that psychological events that affect central neurochemical processes may affect immune activity. Conversely, immune activation may affect hormonal processes and the activity of central neurotransmitters. Thus, by virtue of the neurochemical effects imparted, immune activation may affect behavioral outputs and may even be related to behavioral pathology such as depressive illness. 37 The hypothesis that altered immune activation may occur as a result of various stressors emerged over time. The initial theory came from Hans Seyles' GAS, which was derived from observation and experimentation on laboratory animals. Using a variety of stressors (ie, pain, thermal extremes and starvation), Seyle described a common nonspecifi c stress response pathway. 38 After initial perception of a stressor, the animal mounts an emergency alarm or fi ght-or-fl ight response. This catecholamine-driven reaction results in increased cardiovascular function and an overall increase in metabolism. If the stressor persists, the resistance phase or 'conservation withdrawal reaction' is initiated, which is a physiological coping reaction to the increased demands of maintaining homeostasis. Chronic stress leads to the exhaustion phase and may lead to pathology. Seyle's theory unifi ed the stress phenomena because it provided a common response pathway to all the varied stressors encountered.

This pathway, the HPA axis, involves perception in the brain with release of hypothalamic corticotropin-releasing factor (CRF) and vasopressin which stimulates the anterior pituitary to secrete ACTH ( Figure 2 ). Circulating ACTH causes the adrenal cortex to produce glucocorticoids (GC). Glucocorticoids cause gluconeogenesis with conversion of lipid to glucose for the central nervous system (CNS) and other functions. Thus, GAS allowed the identifi cation of stressors (and presumably the status of well being of the animal) by measurement of GC levels. It was an attractive theory because GC levels were relatively easy to measure. 2 Unfortunately the GAS theory has proven to be too simplistic. Mason's experiments with Rhesus monkeys Aich et al exposed to different types of stressors revealed a disparity in neuroendocrine responses provided by specifi c stressors. 38 For instance, monkeys subjected to emotional stress had elevated serum GC levels but those subjected to a heat-stress regime failed to show GC elevation. In addition to GC, other neuroendocrine mediators were characteristically produced in response to specifi c stressors. 38 Emerging information on the response to stressors suggests that there are at least four different avenues of neuroendocrine responses. These involve the autonomic nervous system, the HPA axis, neuropeptides, neurotransmitters and neuroimmunological peptides and receptors. 2

Interactions between the immune and nervous systems play an important role in modulating host susceptibility and resistance to infl ammatory disease. Neuroendocrine regulation of inflammatory and immune responses as well as onset of disease can occur at multiple levels: (a) systemically through the anti-infl ammatory action of GCs released via HPA axis stimulation, (b) regionally through production of GCs within the affected tissue as well as by sympathetic enervation of immune organs such as the thymus and (c) locally at sites of infl ammation. Estrogens also play an important role in immune modulation and contribute to the approximately 2-to 10-fold higher incidence of autoimmune/infl ammatory diseases in females of all mammalian species. 39 During infl ammation, cytokines from the periphery activate the central nervous system through multiple routes. This results in stimulation of the HPA axis which in turn, through the immunosuppressive effects of the glucocorticoids, generally inhibits infl ammation. Recent studies indicate that physiological levels of glucocorticoids are immunomodulatory rather than solely immunosuppressive causing a shift in patterns of cytokine production from a TH1-to a TH2-type pattern. Interruptions of this loop at any level and through multiple mechanisms, whether genetic, or through surgical or pharmacological interventions, can render an infl ammatory resistant host susceptible to infl ammatory disease. Over-activation of this axis, as occurs during stress, can also affect severity of infectious diseases through the immunosuppressive effects of the glucocorticoids. These interactions have been clearly demonstrated in many animal models using a variety of species and infectious agents. 39 The results from these models are also relevant to human infl ammatory, autoimmune and allergic illnesses including rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, allergic asthma and atopic skin disease. While many genes and environmental factors contribute to susceptibility and resistance to autoimmune/infl ammatory diseases, a full understanding of the molecular mechanisms by which a combination of neuropeptides, neurohormones and neurotransmitters can modulate immune responses is essential for effective design of future interventions. 39

It is clear from the previous discussion and review of previous research that the relationship between stress and disease susceptibility is very complex and intertwined with cascades of events. In order to understand or characterize stress-induced disease susceptibility one needs to identify various biological or functional pathways and interaction networking involved at any given time. To follow the cascade of molecular events happening over time one needs to employ methodologies which are holistic in nature and can provide global information related to the kinetics of multiple changes in gene expression and multiple biomolecular interactions. With the advent of various high-throughput genomic approaches it has become possible to explore complex biological processes and in one step obtain information about gene expression at the transcriptional (genomics) and translational (proteomics) level, as well as to identify metabolites arising from these responses (metabolomics/metabonomics). These methodologies together are often referred to as 'omics' approaches. Although these methodologies are still in their infancy they have started showing promise in understanding systems' biology and various disease processes. Work employing omics approaches to understand mechanism of stress and disease susceptibility is limited at this time. There are reports which describe the effects of oxidative stress on cellular apoptosis 40 but there are no reports on the effects of psychological stressor and disease susceptibility in animals using holistic methodologies such as various omics approaches. The literature in this area is very scarce because these methodologies are very new and few results are currently available. The main studies to correlate stress and disease susceptibility was done either in plant systems 41 or a correlation between oxidative stress and infectious disease. 42 The review by Hirai and Saito established, using liquid chromatogram based mass spectrometric proteomic and metabolomic and cDNA microarray based transcriptomic analyses, that these omics studies can reveal the genomic networking involving several pathways related to oxidative stress response and key metabolic pathways. 41 Bioinformatics and statistical approaches specifi c for detailed transcriptomics or cluster analyses of expressed genes have been developed and are useful for extracting information on various functional genomic responses. [59] [60] [61] It is important, however, to understand the translation of transcriptomic information into protein expression and modifi cations since it is the protein that acts as biomarkers for various disease processes or conditions. Several proteomic tools have been developed. These include gel-based two-dimensional (2D) gel electrophoresis (GE), 47 2D fluorescence difference gel electrophoresis (2DIGE) 62 or gel-free multidimensional protein identifi cation technology (MudPIT), 63,64 isotope-coded affinity tag (ICAT™), 65 Surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) MS 66,67 or isobaric tag for relative and absolute quantitation (iTRAQ) 68 methodologies. While these technologies are being matured more appropriate biological questions can be addressed.

In 2D-GE whether traditional or newer DIGE system, proteins are fi rst focused (fi rst dimension) in terms of their pI values followed by SDS-PAGE (second dimension). In contrast to traditional 2D-GE, DIGE system utilizes the fl uorescence labeling of protein samples and two-to-three different samples can then be run on a single gel which makes it superior to traditional 2D-GE in terms of eliminating gel to gel variation and comparative analysis. Although 2D-GE is a very good way of separating complex protein mixtures, it is limited in terms of high-throughput analysis and sensitivity. Because of low sensitivity it is mostly limited to the analysis of high abundant proteins of a system. Efforts have been made to improve the sensitivity and analysis capacity of proteomics techniques. As a result, other methodologies such as 2D-HPLC, MudPIT, ICAT, and iTRAQ have been developed and are gaining popularity. Although various bioinformatic and data analyses approaches have been developed to analyze gel images and mass spectrometric based protein characterization a highthroughput methodology in this area has yet to be developed.

Functional genomic techniques of transcriptomics and proteomics and available bioinformatic and statistical analyses promise unparalleled global information during the analyses of complex biological responses. However, if these technologies are used in isolation, the large multivariate data sets produced are often diffi cult to interpret and have the potential to ignore key metabolic events to understand the true biology. High resolution 1 H NMR spectroscopy used in conjunction with pattern recognition provides one such tool for defi ning the dynamic phenotype of a cell, organ, or organism in terms of a metabolic phenotype. In a recent review the benefi ts of this metabonomics/metabolomics approach to problems in toxicology have been discussed. 69 One of the major benefi ts of this approach is its high-throughput nature and cost-effectiveness on a per sample basis. Using such a method, the consortium for metabonomic toxicology (COMET) is currently investigating approximately 150 model liver and kidney toxins. This investigation will allow the generation of expert systems where liver and kidney toxicity can be predicted for model drug compounds, providing a new research tool in the fi eld of drug metabolism. The review also included how metabonomics may be used to investigate co-responses with transcripts and proteins involved in metabolism and stress responses such as during drug-induced fatty liver disease. By using data integration to combine metabolite analysis and gene expression profi ling, key perturbed metabolic pathways can be identifi ed.

Detailed omics-based bioinformatics studies, to correlate stress-dependent disease susceptibility, have yet to analyze this complex biological response. A few preliminary studies have been reported in the literature. A recent study has shown the adverse effects of using mechanical ventilators for respiratory support. 70 This acute lung injury because of mechanical stretch can lead to high mortality and this study has utilized recent advances in bioinformatic-intense candidate gene searches to correlate the lung injury and gene expression profi les in a rodent model analyzed with microarrays. 70 The authors used 2,769 mouse/rat orthologous genes identifi ed on RG_U34A and MG_U74Av2 arrays and the expression profi les were simultaneously analyzed by signifi cance analysis of microarrays. This combined ortholog and signifi cance analysis of microarrays approach identifi ed 41 up-and 7 downregulated ventilator-induced stress-related candidate genes. Results were validated by comparable expression levels obtained by either real-time or relative RT-PCR for 15 randomly selected genes. K-mean clustering of 48 candidate genes clustered several well-known lung injury associated genes (IL-6, plasminogen activator inhibitor type 1, CCL-2, cyclooxygenase-2) with a number of stressrelated genes (Myc, Cyr61, Socs3). The only unannotated member of this cluster (n = 14) was RIKEN_1300002F13 EST, an ortholog of the stress-related Gene33/Mig-6 gene. The authors speculated that the ortholog-Signifi cant Analysis of Microarray approach is a useful, time-and resourceeffi cient tool for identifi cation of candidate genes in a variety of complex disease models such as ventilator induced lung injury. 70 Using microarray-based genomic approaches work has also been initiated to identify hypertension-related genes in rat model. 71

Though it is a common belief that the stressors mentioned earlier can compromise host immune responses and enhance susceptibility to various diseases, very little work has been done in this area to understand the mechanism of stress dependent disease susceptibility in mammals. Kelley in 1980 identifi ed eight stressors that typically occur in modern livestock production units: heat, cold, crowding, mixing, weaning, controlled-feeding, noise and restraint. All these stressors were shown to alter components of the immune system in animals and these changes in immune function may ultimately explain the physiological basis of diseaseenvironment interactions. 72 Another study in 1993 showed that neurotransmitters and neuroendocrine hormones can modify the function of immune cells. Conversely, cytokines produced by immune cells can alter brain homeostasis. 73 This connection is further manifested by experimental studies showing a relationship between stress and resistance to infection. Human subjects under high stress were shown to be more susceptible to infection with common cold viruses. Furthermore, a diversity of experimental animal models confi rmed that laboratory stressors such as forced exercise, avoidance learning, restraint, isolation and cold exposure made animals more susceptible to primary infection with a variety of viruses and bacteria.

The cellular and molecular basis for the observed modulation of host resistance is not fully understood but involves altered functioning of both T-lymphocytes and cells of the hypothalamic-pituitary adrenal axis. Also involved is the altered production of cytokines and hormones produced by the immune system and brain. 73 Emau and colleagues in 1987 showed that the onset of immunodepression by stress or viral infection in the pathogenesis of bovine pneumonia permits super infection of the lungs with Mannheimia haemolytica (formerly called Pasteurella haemolytica) which results in exudative fi brinous pneumonia. 74 Although these studies provide a preliminary basis of stress-induced bovine respiratory disease (BRD), there was a lack of necessary information to determine the molecular basis of such dependence, which can be found by studying changes at the level of gene expression (transcriptional and proteomic) and metabolites. A recent study compared proteomics of epithelial lining fl uid from lungs of weaned and nonweaned cattle. 75 The study was done over a shorter period of time (36 h) with a combination of stressors (weaning, transportation) and important questions such as to determine specifi city and duration (stability) of the protein biomarkers are yet to be addressed. The group also studied the proteomic profi le of bronchoalveolar lung lavage fl uid following treating the calves with dexamethasone to determine the effects of glucocorticoids, which is elevated following induction of stress. 76, 77 More studies are however needed (a) to fi nd biomarkers associated with stressors and infection, (b) to determine the duration of the biomarkers so that these are sustainable enough to identify and predict stressed animals (which might be more susceptible to infection in real life situation such as in feedlot cattle).

Disease models have been developed to study the molecular mechanisms underlying the viral-bacterial synergy which results in fatal BRD infections. [78] [79] [80] We have confi rmed with the infectious BRD model that the stress of weaning 81 significantly enhances the viral-bacterial synergy leading to fatal bacterial respiratory infection. 82 The major stressors young Omics approaches in understanding stress and disease susceptibility cattle experience include maternal separation or weaning, dietary changes, transportation, social reorganization and other environmental effects (Figure 3 ). With this in mind, we focused our initial studies to understand the role of weaning on the clinical response to BRD. Fifteen suckling calves were removed from their mothers 24 h prior to viral infection (abruptly weaned/stressed, AW). A second group of 15 calves was weaned 2 wks prior to viral infection (pre-conditioned/ control, PC); 2 wks was chosen as an appropriate interval to eliminate psychological and physiological effects associated with breaking the maternal/nutritional bond and to adapt to the dietary change and social re-organization that follows weaning ( Figure 4 ). All calves were transported to Vaccine and Infections Disease Organization VIDO, and aerosol challenged with bovine herpesvirus-1 (BHV-1) followed by M. haemolytica; this combined viral-bacterial infection induces fatal pneumonia in 50%-70% of calves. 83 Our clinical analyses revealed a signifi cant difference in BRD clinical disease when comparing freshly weaned calves (80% mortality) versus pre-conditioned calves (40% mortality). 84, 85 Increased mortality associated with fresh weaning was characterized by a decrease in both survival time post-infection and, interestingly, decreased lung pathology which suggested a systemic reaction. Contrary to expectations, transportation induced a signifi cant cortisolemia in pre-conditioned but not freshly weaned calves ( Figure 5 ). Transportation is known as an important stressor which increases blood cortisol level. [86] [87] [88] [89] However, the stressor combination of weaning and transportation may have different physiological markers than commonly expected. Cortisol is a potent regulator of pro-infl ammatory cytokine transcription by acting as a negative regulator of NFκB activation, and can be used as an indicator of stress. 90 Fatal bacterial respiratory infection in calves was usually associated with an accelerated decline in serum cortisol levels. Therefore, we hypothesized that increased respiratory disease susceptibility was associated with enhanced pro-infl ammatory responses in freshly weaned calves. Elevated body temperature and interferon-gamma (IFN-γ) levels in nasal secretions during BHV-1 infection of freshly weaned calves were similar to previous reports, 82, 91 supporting this hypothesis. These immune responses were signifi cantly increased despite no signifi cant difference in virus shedding. Furthermore, increased IL-10 expression, during BHV-1 infection of pre-conditioned calves, was consistent with reduced pro-infl ammatory responses. 82

We conducted bovine microarray analyses of RNA isolated from blood mononuclear cells to determine if changes in gene expression correlated with either stress or the severity of BRD infection; results support the conclusion that differential regulation of pro-infl ammatory responses is a major mechanism contributing to increased disease susceptibility. Conserved responses included an enhanced potential to Aich et al respond to pathogen-associated molecular patterns through increased expression of toll-like receptors (TLRs). TLR induction of innate immune responses is mediated primarily through activation of nuclear factor kappaB (NFκB). We also observed differential expression of β-defensin 5 (a host defense peptide induced by TLR4 signaling) in freshly weaned calves, consistent with reduced cortisol levels. 92 In addition, when we compared expression of select innate immune genes (eg, TLR2, TLR4, IFN-γ and 2'5' O-adenylate synthetase) between day 4 (post BHV-1 infection) and day 0 (prior to BHV-1 infection) for AW-and PC-groups, results revealed a trend of increased expression of the select genes on day 4 irrespective of the stress situation (Table 1 ). This trend of activation of select immune genes clearly revealed that following BHV-1 infection the innate immune response was enhanced. This observation contradicts the current hypothesis that the primary viral enhances the secondary bacterial infection by compromising the immune system. 78, 93 Our studies revealed that the anti-infl ammatory gene IL-10 was upregulated on day 4 in PC groups while β-defensin was activated on day 4 in AW groups (Table 1) . IL-10 which acts as an anti-infl ammatory gene should be activated on day 4 to control pro-infl ammatory responses as a result of BHV-1 infection in either AW or in both groups; instead it was only over expressed in control (PC) groups. On a similar token β-defensin on day 4 should also be observed in both groups to control BHV-1 infection. These results are particularly interesting to explore further to understand the detailed mechanism of stress-induced disease correlation.

Although advances have been made in understanding various disease processes, successful intervention often depends upon the stage at which disease is detected. Thus, identifying markers for disease or its cause, as well as understanding the mechanism of disease onset and susceptibility, are very important. Systems biology approaches such as omics (genomic, proteomic, metabonomic) and multivariate analysis to understand mechanisms and to identify biomarkers provide potential tools to address these questions. 69, [94] [95] [96] It is particularly important to employ a combination of molecular approaches such as omics to understand complex processes such as stress and its correlation with disease susceptibility. With such goals in mind, we identifi ed and characterized a group of protein, metabolite and elemental biomarkers using serum samples from BHV-1 infected as well as from stressed animals. The trend of each group of biomarkers distribution was analyzed to correlate with the groupings based on stress condition or infection. 53 Multivariate analysis revealed distinct differential trends in the distribution profi le of proteins, metabolites and elements following a stress response both before and after primary viral infection. A group of acute phase proteins, metabolites and elements could be specifi cally linked to either a stress response (decreased serum amyloid A and Cu, increased apolipoprotein CIII, amino acids, low-density lipoprotein [LDL], P and Mo) or a primary viral respiratory infection (increased apolipoprotein A1, haptoglobin, glucose, amino acids, LDL and Cu, decreased lipid and P). Thus, combined OMICS analysis of serum samples revealed that multimethod analyses could be used to discriminate between the complex biological responses to stress and viral infection. There are reports which suggest that transport can be an important stressor which alters blood leukocyte populations and sets the stage for BRD. 97, 98 Currently we are conducting experiments in which transportation of the animals is eliminated, such that stressors related to a new environment are removed. It was documented that abrupt weaning (separation of suckling calves from their dams) causes a prolonged psychological stress response. 81 This stress response manifests as an increase in vocalization by both calves and cows and a significant increase in time spent walking by the calf. Consequently, time spent eating and resting also decreases for abruptly weaned calves. Statistically, these changes in behavior return to baseline values after four to fi ve days of weaning.

As is evident from the review of the literature that traditionally effects of stress have been studied by attempting to understand behavioral patterns of the subject and cellular immune response of the host. It is, however, clear that amount of data to understand such a complex condition (such as stress and stress-induced diseases) is not adequate and enough. Behavioral studies may not always be correlated to a particular stressor as there might have been other parameters affecting the observations. Attempts have also been made to use cortisol as a unique biomarker for stress, but cortisol is diurnal hence cannot be a reliable marker. While it is important to understand cellular immunology to establish the mechanism of stress-induced disease susceptibility, however without identifying and establishing the immune markers specifi c for stressor it will not lead into any meaningful inference. Systems biology approaches using various omics and bioinformatics methodologies can identify a group of biomarkers at various levels of host immune response, gene expression and metabolism. Association of the identifi ed biomarkers and the patterns in changes in their expression level above or below a determined threshold level with specifi c stressor(s) could then be used to defi ne stress situation, identif ication of individuals susceptible to stressinduced disease. Moreover, establishing a pattern in changes of biomarkers is more reliable than a single biomarker. Duration of identifi ed biomarkers can further strengthen the methodology for applying in real life situation.

The current review established the importance of psychological stress and its effects on infectious disease severity and susceptibility with emphasis on respiratory disease. As evident from the literature, initial work focused mainly on understanding the roles of various stressors on physiology and behavior. Theories emerged as a consequence of these studies which implicated the HPA axis and the sympathetic nervous system (SNS) in altered immunity. However, physiological responses studied in a laboratory may not necessarily be consistent with real life phenomena, especially when attempting to analyze heterogeneous populations such as cattle or humans. Experiments with human subjects cannot always be properly controlled because of ethical concerns and therefore using appropriate animal models to mimic natural disease outcomes may be more informative. With the use of high-throughput omics approaches it should be possible to extract more detailed information out of these complex biological systems and understand the complex biology and mechanisms by which stress augments disease susceptibility. More studies are needed to understand the physiological differences between acute versus chronic stress as well as the combined effects of various stressors. It is becoming more apparent that stress not only makes an individual more susceptible to a disease but can also enhance disease severity. Therefore, it is very important to understand the kinetics, specifi city and stability or duration of biomarkers associated with a specifi c stressor or combination of stressors before we can predict disease susceptibility in individual people or animals.

The concept of stress and its historical development

Stress and immunity: A unifying concept. Veterinary Immunology and Immunopathology

The term 'stress' in the veterinary context

Modeling and predicting stress-induced immunosuppression in mice using blood parameters

On the interactions of the hypothalamicpituitary-adrenal (HPA) axis and sleep: normal HPA axis activity and circadian rhythm, exemplary sleep disorders

Stress, immune reactivity and susceptibility to infectious disease

Stress-induced immunomodulation and the implications for health

Behavioral health: A handbook for health enhancement and disease prevention

Psychosocial factors, immunologic mediation, and human susceptibility to infectious diseases: how much do we know?

Health psychology

Chronic social stress, social status, and susceptibility to upper respiratory infections in nonhuman primates

The impact of infl uenza epidemics on hospitalizations

Psychological stress, cytokine production, and severity of upper respiratory illness

Family functioning and stress as predictors of infl uenza B infection

Restraint stress differentially affects the pathogenesis of an experimental infl uenza viral infection in three inbred strains of mice

Restraint stress differentially affects anti-viral cellular and humoral immune responses in mice

The clinical and health economic burden of respiratory syncytial virus disease among children under 2 years of age in a defi ned geographical area

Psychosocial factors, stress, disease, and immunity

Stress and acute respiratory infection

Psychological stress and susceptibility to the common cold

Psychological stress and susceptibility to upper respiratory infections

Psychosocial stress and susceptibility to upper respiratory tract illness in an adult population sample

Stress and infectious disease in humans

Some further studies on the prediction of experimental colds in volunteers by psychological factors

Viral respiratory tract infection and exacerbations of asthma in adult patients

Viral and bacterial infections in adults with chronic asthma

Respiratory viruses and exacerbations of asthma in adults

Streptococcal infections in families. Factors altering individual susceptibility

Types of stressors that increase susceptibility to the common cold in healthy adults

Omics approaches in understanding stress and disease susceptibility

Neuroendocrine regulation of cytokine production during experimental infl uenza viral infection: effects of restraint stress-induced elevation in endogenous corticosterone

Rhinovirus stimulation of interleukin-6 in vivo and in vitro . evidence for nuclear factor kappa B-dependent transcriptional activation

Local and systemic cytokine responses during experimental human infl uenza A virus infection. Relation to symptom formation and host defense

Novel approaches to the treatment of depression by modulating the hypothalamic -pituitary -adrenal axis

Major depression and activation of the infl ammatory response system

Cytokines and the nervous system II: actions and mechanisms of action

The role of infl ammatory mediators in the biology of major depression: central nervous system cytokines modulate the biological substrate of depressive symptoms, regulate stress-responsive systems, and contribute to neurotoxicity and neuroprotection

Autonomic control of heart rate in the neonatal rhesus monkey

Neuroendocrine regulation of autoimmune/infl ammatory disease

Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis

Post-genomics approaches for the elucidation of plant adaptive mechanisms to sulphur deficiency

Nitric oxide and Mycobacterium leprae pathogenicity

Multiplexed biochemical assays with biological chips

Parallel synthesis and analysis of large numbers of related chemical compounds: applications to oligonucleotides

Quantitative monitoring of gene expression patterns with a complementary DNA microarray

Serial analysis of gene expression

High resolution two-dimensional electrophoresis of proteins

Within the fold: assessing differential expression measures and reproducibility in microarray assays

The use and analysis of microarray data

DNA arrays and neurobiology -what's new and what's next?

Cholesterol-lowering statins possess antiinfl ammatory activity that might be useful for treatment of MS

Molecular analyses of disease pathogenesis: Application of bovine microarrays

Comparative approaches to the investigation of responses to stress and viral infection in cattle

Comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus and bovine coronavirus

Functional genomics of the chicken-a model organism

Genome-wide association studies: progress in identifying genetic biomarkers in common, complex diseases

Genomics applied to the improvement of farm animals: a survey of the research projects funded in the frame of the French programme

Bridging bench to clinic: roles of animal models for post-genomics drug discovery on metabolic diseases

Computational gene prediction using multiple sources of evidence

Data standards for 'omic' science

Analysis of cDNA microarray images

Detection technologies in proteome analysis

Multidimensional protein identifi cation technology (MudPIT): technical overview of a profi ling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue

An automated multidimensional protein identifi cation technology for shotgun proteomics

Fractionation of isotopically labeled peptides in quantitative proteomics

SELDI-TOF MS for diagnostic proteomics

New approaches to proteomic analysis of breast cancer

For quantitative proteomics using iTRAQ

NMRbased metabonomic approaches for evaluating physiological infl uences on biofl uid composition

Bioinformatic identifi cation of novel early stress response genes in rodent models of lung injury

Genetic models of hypertension in experimental animals

Stress and immune function: a bibliographic review

Stress and infections

Viral-bacterial pneumonia in calves: effects on plasma eicosanoids and long chain fatty acids

Stress alters the cellular and proteomic compartments of bovine bronchoalveolar lavage fl uid

Altered protein expression in neutrophils of calves treated with dexamethasone

Alterations in the bovine bronchoalveolar lavage proteome induced by dexamethasone

Effect of bovine alpha 1 interferon on bovine herpesvirus type 1-induced respiratory disease

Use of recombinant bovine alpha 1 interferon in reducing respiratory disease induced by bovine herpesvirus type 1

Prevention of experimental bovine pneumonic pasteurellosis with an extract of Pasteurella haemolytica

The effects of regrouping on behavioral and production parameters in finishing swine

Effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate infl ammation

Bovine herpesvirus-1 vaccination against experimental bovine herpesvirus-1 and Pasteurella haemolytica respiratory tract infection: onset of protection

Effect of stress on viral-bacterial synergy in bovine respiratory disease: Novel mechanisms to regulate infl ammation

Comparative approaches to the investigation of responses to stress and viral infection in cattle

Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress

Acute phase proteins in cattle after exposure to complex stress

Effects of road transportation on lymphocyte subsets in calves

Transportation stress alters the circulating steroid environment and neutrophil gene expression in beef bulls

Hypercortisolemia in acute stroke is related to the infl ammatory response

Induction of interferon activity in nasal mucus by association of levamisole by systemic route -inactivated virus by local route

Effect of stress on viral-bacterial synergy in bovine respiratory disease: A review of potential mechanisms

Immune mechanisms of pathogenetic synergy in concurrent bovine pulmonary infection with Haemophilus somnus and bovine respiratory syncytial virus

Proteomic characterization of the interstitial fl uid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery

Unlocking the mysteries of virus-host interactions: does functional genomics hold the key?

A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cells

Effect of transport stress on respiratory disease, serum antioxidant status, and serum concentrations of lipid peroxidation biomarkers in beef cattle

Effect of transportation on blood serum composition, disease incidence, and production traits in young calves. Infl uence of the journey duration

This project was funded by the Saskatchewan Agriculture and Food Rural Revitalization (SAFRR), Genome BC and Genome Prairie for the 'Pathogenomics of Innate Immunity' research program and the Ontario Cattleman's Association (OCA). This manuscript is published with permission of the Director of Vaccine and Infectious Disease Organization as article number 512. A.A.P. is the holder of an NSERC Senior Industrial Research Chair.
