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Our knowledge regarding immune-protective and immunopathogenic events in severe acute respiratory syndrome coronavirus (SARS-CoV) infection is limited, and little is known about the dynamics of the immune response at the primary site of disease. Here, an African green monkey (AGM) model was used to elucidate immune mechanisms that facilitate viral clearance but may also contribute to persistent lung inflammation following SARS-CoV infection. During primary infection, SARS-CoV replicated in the AGM lung for up to 10 days. Interestingly, lung inflammation was more prevalent following viral clearance, as leukocyte numbers peaked at 14 days postinfection (dpi) and remained elevated at 28 dpi compared to those of mock-infected controls. Lung macrophages but not dendritic cells were rapidly activated, and both cell types had high activation marker expression at late infection time points. Lung proinflammatory cytokines were induced at 1 to 14 dpi, but most returned to baseline by 28 dpi except interleukin 12 (IL-12) and gamma interferon. In SARS-CoV homologous rechallenge studies, 11 of the 12 animals were free of replicating virus at day 5 after rechallenge. However, incidence and severity of lung inflammation was not reduced despite the limited viral replication upon rechallenge. Evaluating the role of antibodies in immune protection or potentiation revealed a progressive increase in anti-SARS-CoV antibodies in lung and serum that did not correlate temporally or spatially with enhanced viral replication. This study represents one of the first comprehensive analyses of lung immunity, including changes in leukocyte populations, lung-specific cytokines, and antibody responses following SARS-CoV rechallenge in AGMs.

Emerging strains of influenza, such as avian H5N1 and 2009 pandemic H1N1, are more virulent than seasonal H1N1 influenza, yet the underlying mechanisms for these differences are not well understood. Subtle differences in how a given strain interacts with the immune system are likely a key factor in determining virulence. One aspect of the interaction is the ability of T cells to locate the foci of the infection in time to prevent uncontrolled expansion. Here, we develop an agent based spatial model to focus on T cell migration from lymph nodes through the vascular system to sites of infection. We use our model to investigate whether different strains of influenza modulate this process. We calibrate the model using viral and chemokine secretion rates we measure in vitro together with values taken from literature. The spatial nature of the model reveals unique challenges for T cell recruitment that are not apparent in standard differential equation models. In this model comparing three influenza viruses, plaque expansion is governed primarily by the replication rate of the virus strain, and the efficiency of the T cell search-and-kill is limited by the density of infected epithelial cells in each plaque. Thus for each virus there is a different threshold of T cell search time above which recruited T cells are unable to control further expansion. Future models could use this relationship to more accurately predict control of the infection.

BackgroundMany respiratory viruses disproportionately impact the elderly. Likewise, advanced age correlated with more adverse disease outcomes following severe acute respiratory syndrome coronavirus (SARS-CoV) infection in humans. We used an aged African green monkey SARS-CoV infection model to better understand age-related mechanisms of increased susceptibility to viral respiratory infections. Nonhuman primates are critical translational models for such research given their similarities to humans in immune-ageing as well as lung structure.ResultsSignificant age- and infection-dependent differences were observed in both systemic and mucosal immune compartments. Peripheral lymphocytes, specifically CD8 T and B cells were significantly lower in aged monkeys pre- and post- SARS-CoV infection, while neutrophil and monocyte numbers were not impacted by age or infection status. Serum proinflammatory cytokines were similar in both age groups, whereas significantly lower levels of IL-1beta, IL-18, IL-6, IL-12 and IL-15 were detected in the lungs of SARS-CoV-infected aged monkeys at either 5 or 10 days post infection. Total lung leukocyte numbers and relative frequency of CD8 T cells, B cells, macrophages and dendritic cells were greatly reduced in the aged host during SARS-CoV infection, despite high levels of chemoattractants for many of these cells in the aged lung. Dendritic cells and monocytes/macrophages showed age-dependent differences in activation and chemokine receptor profiles, while the CD8 T cell and B cell responses were significantly reduced in the aged host. In examination of viral titers, significantly higher levels of SARS-CoV were detected in the nasal swabs early, at day 1 post infection, in aged as compared to juvenile monkeys, but virus levels were only slightly higher in aged animals by day 3. Although there was a trend of higher titers in respiratory tissues at day 5 post infection, this did not reach statistical significance and virus was cleared from all animals by day 10, regardless of age.ConclusionsThis study provides unique insight into how several parameters of the systemic and mucosal immune response to SARS-CoV infection are significantly modulated by age. These immune differences may contribute to deficient immune function and the observed trend of higher SARS-CoV replication in aged nonhuman primates.

Macrophages are the primary lung phagocyte and are instrumental in maintenance of a sterile, noninflamed microenvironment. IFNs are produced in response to bacterial and viral infection, and activate the macrophage to efficiently counteract and remove pathogenic invaders. Respiratory syncytial virus (RSV) inhibits IFN-mediated signaling mechanisms in epithelial cells; however, the effects on IFN signaling in the macrophage are currently unknown. We investigated the effect of RSV infection on IFN-mediated signaling in macrophages. RSV infection inhibited IFN-b-and IFN-g-activated transcriptional mechanisms in primary alveolar macrophages and macrophage cell lines, including the transactivation of important Nod-like receptor family genes, Nod1 and class II transactivator. RSV inhibited IFN-b-and IFN-g-mediated transcriptional activation by two distinct mechanisms. RSV impaired IFN-b-mediated signal transducer and activator of transcription (STAT)-1 phosphorylation through a mechanism that involves inhibition of tyrosine kinase 2 phosphorylation. In contrast, RSV-impaired transcriptional activation after IFN-g stimulation resulted from a reduction in the nuclear STAT1 interaction with the transcriptional coactivator, CBP, and was correlated with increased phosphorylation of STAT1b, a dominantnegative STAT1 splice variant, in response to IFN-g. In support of this concept, overexpression of STAT1b was sufficient to repress the IFNg-mediated expression of class II transactivator. These results demonstrate that RSV inhibits IFN-mediated transcriptional activation in macrophages, and suggests that paramyxoviruses modulate an important regulatory mechanism that is critical in linking innate and adaptive immune mechanisms after infection. Keywords: macrophages; IFN; signal transduction; transcriptional activationRespiratory syncytial virus (RSV) is an important causative agent of severe respiratory tract infections in pediatric, immunocompromised, and elderly populations (1-5). RSV infection elicits a poor adaptive immune response; therefore, infections occur repeatedly throughout life (6, 7). This common paramyxovirus has also been associated with secondary bacterial infections of the lung (3, 8-10); however, as with other viralinduced secondary bacterial infections, the underlying mechanisms are not well understood.IFN-a and IFN-b (type I) and IFN-g (type II) are produced in the lung in response to microbial infection, and are potent activators of macrophage innate antimicrobial immunity; they also induce pathways that promote efficient antigen processing and presentation to cells of the adaptive immune system (11, 12). The requirement of IFN for the efficient clearance of pathogens is evident in IFN-b 2/2 , IFN-g 2/2 , IFN-a receptor 2/2 , and IFN-g receptor 2/2 mice, as these mice, lacking IFN signaling, display severe impairment in natural resistance to a variety of viral, bacterial, and parasitic infections (13-20). IFN-a or IFN-b ligation of the IFN-a/b receptor results in the phosphorylation and activation of the ...

The immune mechanisms for neonatal susceptibility to respiratory pathogens are poorly understood. Given that mucosal surfaces serve as a first line of host defense, we hypothesized that the innate immune response to infectious agents may be developmentally regulated in airway epithelium. To test this hypothesis, we determined whether the expression of IL-8 and IL-6 in airway epithelium after LPS exposure is dependent on chronological age. Tracheas from infant, juvenile, and adult rhesus monkeys were first exposed to LPS ex vivo, and then processed for air-liquid interface primary airway epithelial cell cultures and secondary LPS treatment in vitro. Compared with adult cultures, infant and juvenile cultures expressed significantly reduced concentrations of IL-8 after LPS treatment. IL-8 protein in cultures increased with animal age, whereas LPS-induced IL-6 protein was predominantly associated with juvenile cultures. Toll-like receptor (TLR) pathway RT-PCR arrays showed differential expressions of multiple mRNAs in infant cultures relative to adult cultures, including IL-1a, TLR10, and the peptidoglycan recognition protein PGLYRP2. To determine whether the age-dependent cytokine response to LPS is reflective of antecedent exposures, we assessed primary airway epithelial cell cultures established from juvenile monkeys housed in filtered air since birth. Filtered air-housed animal cultures exhibited LPS-induced IL-8 and IL-6 expression that was discordant with age-matched ambient air-housed animals. A single LPS aerosol in vivo also affected this cytokine profile. Cumulatively, our findings demonstrate that the innate immune response to LPS in airway epithelium is variable with age, and may be modulated by previous environmental exposures.

To define the possible impact of T-lymphocyte trafficking parameters on simian immunodeficiency virus (SIV) pathogenesis, we examined migratory profiles of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T lymphocytes in acutely SIV mac251 -infected and uninfected macaques within 48 h after autologous transfer. Despite significant upregulation of homeostatic chemokine CCL19/macrophage inflammatory protein 3␤ and proinflammatory chemokine CXCL9/monokine induced by gamma interferon in secondary lymphoid tissue in SIV infection, no differences in CFSE ؉ T-lymphocyte frequencies or cell compartmentalization in lymph nodes were identified between animal groups. By contrast, a higher frequency of CFSE ؉ T lymphocytes in the small intestine was detected in acute SIV infection. This result correlated with increased numbers of gut CD4 T lymphocytes expressing chemokine receptors CCR9, CCR7, and CXCR3 and high levels of their respective chemokine ligands in the small intestine. The changes in trafficking parameters in SIV-infected macaques occurred concomitantly with acute gut CD4 T-lymphocyte depletion. Here, we present the first in vivo T-lymphocyte trafficking study in SIV infection and a novel approach to delineate T-lymphocyte recruitment into tissues in the nonhuman primate animal model for AIDS. Such studies are likely to provide unique insights into T-lymphocyte sequestration in distinct tissue compartments and possible mechanisms of CD4 T-lymphocyte depletion and immune dysfunction in simian AIDS.

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