3.1.1. Stress-induced Effects on Innate Immune Responses to Infection with Influenza Virus
In the initial studies of stress and the immune response, a murine model of influenza viral infection was initiated by intranasal challenge of C57BL/6 male mice; physical restraint (RST) was selected as the stressor (Sheridan et al., 1991). Our first observations showed that RST reduced the accumulation of cells in the lungs of influenza-infected mice (Hunzeker et al., 2004). In addition to having effects in the lungs, RST was also responsible for a reduction in lymphadenopathy in the draining lymph nodes (Hermann et al., 1995). The cells whose numbers were suppressed by RST included macrophages, NK cells, and T and B lymphocytes. During the past 15 years or so, our studies have revealed that this effect of RST has a substantial impact on both the innate and adaptive immune response to influenza virus infection.
As mentioned above, the first, innate responses to influenza virus involve the ligation of Toll-like receptors (TLRs) and the subsequent production of the type I interferons (IFN-a and IFN-P), proinflammatory cytokines (IL-1, IL-6, and TNF-a), and chemokines (MCP-1 and MIP-1a).Together these TLR-driven responses are targeted toward creating an antiviral state in the infected tissue and toward recruiting inflammatory cells to the lung and draining lymph nodes.
The antiviral state is created by the type I interferons which play a major role in restricting the spread of virus during the early phase of the infection. As would be predicted, infection with influenza A/PR8 virus induced the expression of both of the type I interferons in the lungs of control mice. However, somewhat unexpectedly, expression of the genes coding for IFN-a and IFN-P was enhanced by RST (Hunzeker et al., 2004). It is not clear whether this reflected an adaptive response to the stressor or whether it was a compensatory response in an attempt to control viral replication while other aspects of the innate response were suppressed.
The development of an activated endothelium, critical to the local inflammatory response during infection, is dependent on the proinflammatory cytokines. Our data have showed that RST has pronounced effects on proinflammatory cytokine responses to influenza infection. For example, lung IL-1a responses in RST/infected mice were suppressed (values were similar to uninfected control responses), and treatment with RU486 resulted in a blockade of the type II steroid receptor and failed to restore the response. In nonstressed control mice, influenza A/PR 8 infection elevated the lung IL-6 response by 24 h postinfection (p.i.), with the peak occurring at 48 h and levels remaining above background at 72h. Interest ingly, RST did not affect the magnitude or the kinetics of the lung IL-6 response during the infection (Konstantinos and Sheridan, 2001). Because two cytokines associated with the early inflammatory responses to infection were differentially regulated in restraint-stressed mice, it appeared that the stress effect on cytokine production was specific for particular cytokines.
The actual transmigration of cells into tissue is dependent on the chemokine signal at the reactive endothelium in the lung. Again, during influenza infection, the P-chemokines are key molecules that aid in the accumulation of mononuclear cells in the lungs of infected mice. In studies in which MIP-1a was knocked out by targeted mutation, infection with influenza virus resulted in reduced pneumonitis and delayed clearance of the virus. Histological analysis of the infected lung tissues showed a significant reduction in the inflammatory infiltrate in the KO mice when compared with the wild type (Cook et al., 1995). In experiments to examine the effects of stress on P-chemokine responses during influenza infection, RST suppressed monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1a) (Hunzeker et al., 2004). Suppression occurred early (before day 3 p.i.) and remained below control levels at day 5 p.i. (Hunzeker et al., 2004). Thus, reduction in the expression of proin-flammatory cytokine and P-chemokine responses by RST were likely contributors to the diminished inflammatory response observed in the lungs of infected RST mice.
In sum, the data show that although TLR-mediated IFN-a and IFN-P responses are intact if not elevated, in RST-mice, the proinflammatory cytokine and chemokine responses are both impaired. Presumably, this would impact the recruitment of cells to both the lung and regional lymph nodes. In fact, enumeration of mononuclear cells in the infected lungs confirmed that fewer cells accumulated during A/PR8 infection when the mice were stressed. Subsequent histological studies documented reduced cellu-larity up to and beyond 7 days postinfection in the infected RST mice.
Of interest to us was the observation that cellularity was restored to RST-stressed animals by treating them with the glucocorticoid-antagonist RU486 (Hermann et al., 1995). Thus, because activation of the HPA axis, and the resultant elevation of plasma corticosterone, was known to affect cell trafficking, studies were performed to examine the effect of RST on circulating plasma corticosterone levels. Samples were obtained before the start of an RST cycle (6 p.m.), at 30min into the cycle (6:30 p.m.), and again at the end of the cycle (10 a.m.). Three groups were compared including RST/infected, no RST/infected, and no RST/not infected. Both infection and RST individually elevated corticosterone. However, together, RST plus influenza infection had a synergistic effect on corticosterone levels which increased more than sevenfold in comparison with non-stress non-infected controls (Sheridan et al., 1991). Further studies of HPA activation showed that four or more consecutive cycles of restraint broke the circadian rhythm and resulted in persistent high levels of plasma corticosterone throughout the day. Moreover, loss of the HPA rhythm correlated with increased pathophysiology after influenza infection (Hermann et al., 1993).
Natural killer cells play an important role in the early innate defenses to influenza infection as they seek to limit the spread of virus (Leung and Ada, 1981). They respond rapidly in the early phase of the infection to kill virus-infected cells and when activated produce cytokines that initiate and enhance subsequent, specific antiviral immune responses (Biron et al., 1999). It is generally believed that during a viral infection, NK cells limit viral spread until a virus-specific CD 8+ cytotoxic T-cell response can be mounted. In fact, NK cells not only are important in innate resistance to infection but also are required for development of anti-influenza cytotoxic T-cell responses. Mice depleted of NK cells had increased mortality during an influenza viral infection (Stein-Streilein and Guffee, 1986).
Infection of C57BL/6 male mice with influenza A/PR8 virus resulted in an NK response in the lungs that was detectable on day 3 p.i., peaked on day 5 p.i., and was still present in lung tissue 7 days p.i. RST suppressed NK cell cytotoxic activity in the lungs of influenza-infected mice throughout the course of infection. This reduced NK cytotoxic activity was, in part, due to RST-induced suppression of NK cell trafficking to the lungs (Hunzeker et al., 2004). Specifically, RST suppressed IL-1a, MCP-1, and MIP-1a responses at the time that peak NK infiltration was observed in infected control mice. These data suggest that, in stressed animals, NK cells were not accumulating in the lungs to fight infection (Hunzeker et al., 2004).
Restraint-induced reduction in NK cell trafficking was the result of elevated corticosterone levels, as evidenced by the finding that NK trafficking to the infected lungs of RST mice was restored by blockade of the type II glucocorticoid receptor with RU486 treatment. Concomitantly, receptor blockade restored expression of MIP-1a and MCP-1 chemokine genes in the lungs of stressed, infected mice. However, blockade of the glucorticoid receptor failed to restore NK cell cytotoxicity (Tseng et al., 2005). Thus, although glucocorticoids induced by stress diminished cell trafficking to the lungs, they were not involved in suppression of NK cytotoxicity. This observation suggested that another "stress mediator" might be involved in regulation of natural resistance. Studies conducted by Tseng and colleagues (2005) demonstrated conclusively that suppression of NK cytotoxicity was restored by pharmacologic blockade of the ^-opioid receptor in the stressed animals. Interestingly, NK cells do not appear to have ^-opioid receptors, suggesting that the opioids are acting indirectly rather than directly on the NK cells (Tseng et al., 2005).
When taken together, these studies create a picture in which restraint stress alters three major components of natural resistance to viral infection: the proinflammatory cytokine IL-1a response, the P-chemokine response, and natural killer cell activity. Responses were altered at the site of virus replication and in secondary lymphoid tissues for all three responses. Stress-induced corticosterone reduced lymphadenopathy in draining lymph nodes and diminished mononuclear cell trafficking to the infected lung. In addition, stress-induced corticosterone suppressed cytokine gene expression for some cytokines that were studied (IL-1 a, MCP-1, and MIP-1a) while it enhanced, or had no effect on, gene expression of other cytokines induced during infection (e.g., type 1 IFN-a/p and IL-6). Moreover, suppression of NK cell trafficking during stress was corticosterone-dependent, whereas NK cell cytoxicity was suppressed by an opioid response. The question of whether the opioid effect on NK during an influenza infection is mediated centrally or peripherally remains unanswered, but strong evidence to support a centrally mediated mechanism for opioid-associated modulation of immune function has been published (Mellon and Bayer, 1998; Nelson and Lysle, 2001).
3.1.2. Stress-induced Effects on Adaptive Immunity to Infection with Influenza Virus
Previous studies have shown that stress, in addition to affecting natural resistance, also modulates virus-specific T- and B-cell responses during an influenza viral infection (Bonneau et al., 1991; Sheridan et al., 1991; Dobbs et al., 1996). RST reduced both virus-specific CD4+ T-cell cytokine responses and CD8+ cytolytic T-cell responses during infection. Studies of stress-induced neuroendocrine responses showed that sustained, elevated levels of corticosterone-suppressed T-cell cytokine responses reduced the accumulation of T cells in the draining lymph nodes and altered the trafficking of T cells to the lungs of virus-infected animals. Pharmacologic blockade of type II steroid receptors, using RU486 treatment, restored lymphadenopa-thy, cell trafficking to the lungs, and the expression of T-cell cytokine genes (Dobbs et al., 1996). However, as was the case with suppression of NK cell cytotoxicity, virus-specific cytolytic T-cell responses remained suppressed after RU486 treatment. Subsequent experiments demonstrated that suppression of CD8+ T-cell cytolytic activity was associated with stress-induced activation of the sympathetic nervous system and release of cate-cholamines; blocking P-adrenergic receptors (with nadolol) restored T-cell cytolytic activity. Thus, RST-induced activation of the sympathetic nervous system results in regulation of virus-specific T-cell cytotoxicity (Dobbs et al.,1993).
Elevated serum corticosterone induced by RST suppressed CD4+ T-cell responses after influenza viral infection. RST suppressed the production of IL-2, IFN-y, and IL-I0 by mononuclear cells from the regional lymph nodes and spleen of A/PR8-infected mice (Dobbs et al., 1996). Because IFN-y and
IL-10 were both suppressed, this finding suggested that RST suppressed both Th1 and Th2 CD4+ T-cell responses. Thus, during an A/PR8 infection, RST suppressed both CD4+ subsets without biasing the direction of the cytokine response. Diminished cytokine gene expression during infection and stress was restored by RU486 treatment, thus confirming a role for cor-ticosterone in mediating the effect (Dobbs et al., 1996).
To further examine the role of HPA activation in modulating cytokine responses, experiments were conducted using androstenediol (AED; a metabolite of dehydroepiandrosterone; DHEA), which is known to coun-terregulate glucocorticoid modulation of the immune response (Padgett and Loria, 1994). AED treatment blunted RST-induced HPA activation resulting in lower plasma corticosterone levels during infection. Moreover, the reduced plasma corticosterone levels in AED-treated mice correlated with restoration of lymphadenopathy to draining lymph nodes, enhanced IFN-y production, and elevated IL-10 gene expression on day 7 p.i. (Padgett and Sheridan, 1999). AED functioned in opposition to corticosterone by regulating T-helper cytokine secretion.
The finding of significant suppression of antiviral T-cell responses by RST suggested that T-cell help for antibody production might be limited and therefore that the kinetics and/or magnitude of antibody responses to influenza might also be affected. As hypothesized, RST affected the development of virus-specific B-cell antibody responses by delaying seroconver-sion in the restrained mice infected with A/PR8 virus. In addition, antibody class switching from IgM to IgG to IgA was delayed. After resolution of the infection, during the memory phase, stressed mice eventually achieved virus-specific antibody titers similar to those found in non-stressed infected control mice (Feng et al., 1991).
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