Distinct Chemokine Triggers and In Vivo Migratory Paths of Fluorescein Dye-Labeled T Lymphocytes in Acutely Simian Immunodeficiency Virus SIV(mac251)-Infected and Uninfected Macaques

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 3β 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

Severe acute respiratory syndrome-coronavirus infection in aged nonhuman primates is associated with modulated pulmonary and systemic immune responses

BACKGROUND: Many 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. RESULTS: Significant 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. CONCLUSIONS: This 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

Innate Immune Response to LPS in Airway Epithelium Is Dependent on Chronological Age and Antecedent Exposures

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-1α, 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

Discharged patients and prognoses of caregivers after two years - Part III of the Berlin deinstitutionalisation study

<p>Airway epithelial cells harvested from one-year-old juvenile rhesus monkeys with prior ozone and/or LPS exposure, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090401#pone-0090401-g001" target="_blank">Figure 1</a> were cultured under air-liquid interface conditions and subsequently treated with increasing doses of LPS <i>in vitro</i>. Cultures were evaluated for IL-8 mRNA (A, C) and protein (B, D) expression at 24 h post-treatment. Results show the average +/− SE. *p<0.05, **p<0.01 by two-way ANOVA comparing <i>in vivo</i> exposure and <i>in vitro</i> LPS concentration (n = 4 for each group except filtered air controls n = 5).</p

Early Life Ozone Exposure Results in Dysregulated Innate Immune Function and Altered microRNA Expression in Airway Epithelium

<div><p>Exposure to ozone has been associated with increased incidence of respiratory morbidity in humans; however the mechanism(s) behind the enhancement of susceptibility are unclear. We have previously reported that exposure to episodic ozone during postnatal development results in an attenuated peripheral blood cytokine response to lipopolysaccharide (LPS) that persists with maturity. As the lung is closely interfaced with the external environment, we hypothesized that the conducting airway epithelium of neonates may also be a target of immunomodulation by ozone. To test this hypothesis, we evaluated primary airway epithelial cell cultures derived from juvenile rhesus macaque monkeys with a prior history of episodic postnatal ozone exposure. Innate immune function was measured by expression of the proinflammatory cytokines IL-6 and IL-8 in primary cultures established following <i>in vivo</i> LPS challenge or, in response to <i>in vitro</i> LPS treatment. Postnatal ozone exposure resulted in significantly attenuated IL-6 mRNA and protein expression in primary cultures from juvenile animals; IL-8 mRNA was also significantly reduced. The effect of antecedent ozone exposure was modulated by <i>in vivo</i> LPS challenge, as primary cultures exhibited enhanced cytokine expression upon secondary <i>in vitro</i> LPS treatment. Assessment of potential IL-6-targeting microRNAs miR-149, miR-202, and miR-410 showed differential expression in primary cultures based upon animal exposure history. Functional assays revealed that miR-149 is capable of binding to the IL-6 3′ UTR and decreasing IL-6 protein synthesis in airway epithelial cell lines. Cumulatively, our findings suggest that episodic ozone during early life contributes to the molecular programming of airway epithelium, such that memory from prior exposures is retained in the form of a dysregulated IL-6 and IL-8 response to LPS; differentially expressed microRNAs such as miR-149 may play a role in the persistent modulation of the epithelial innate immune response towards microbes in the mature lung.</p></div

Effect of postnatal ozone exposure on miR-149, miR-202, miR-410 and miR-let7a expression in juvenile monkey airway epithelial cell cultures.

<p>Comparison of constitutive (0 µg/ml) and LPS-induced (1 µg/ml, 6 h post-treatment) microRNA expression in primary airway epithelial cell cultures derived from one-year-old juvenile rhesus monkeys with prior ozone and/or LPS exposure as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090401#pone-0090401-g001" target="_blank">Figure 1</a>. (A) miRNA-149, (B) miRNA-202, (C) miRNA-410, (D) miRNA-let7a. Graphs show the average +/− SE of the 2?-(delta CT) where the delta CT =  (microRNA of interest – endogenous control microRNA (RNU-6b)). microRNAs were extracted from n = 3–5 animals per group. Results from Student's t-tests comparing microRNA expression at baseline and post- <i>in vitro</i> LPS treatment are shown with horizontal bars. *p<0.05, **<i>p</i><0.005, ***<i>p</i><0.0003.</p

Assessment of microRNA regulation of IL-6 in human airway epithelial cell cultures.

<p>To evaluate the ability of the putative IL-6-targeting microRNAs to regulate expression of IL-6, the BEAS-2B cell line was transfected with a negative control (NegCO) microRNA or mimics for miR-149, miR-202 or miR-410. (A) Expression of microRNAs in BEAS-2B 24 h post-transfection with a NegCO microRNA or mimics for miR-149, miR-202, or miR-410. Values are reported as the average +/− SE of the 2?-(delta CT) relative to the endogenous control microRNA RNU-6b. (B) IL-6 mRNA and (C) IL-6 protein expression was evaluated in transfected BEAS-2B cells after 24 h. Data are reported as the fold-change in IL-6 mRNA or IL-6 protein as compared to no mimic controls for each experiment. (D) Binding of microRNA mimics to IL-6 mRNA was tested in the HBE1 cell line with the IL-6 3′UTR cloned into a firefly/renilla luciferase plasmid reporter system. For transfection normalization across samples, the relative luciferase units are reported for firefly versus renilla luciferase. Data are from 4 separate experiments. *p<0.05, **p<0.005 by Student's t-test.</p