Prostaglandin D2 delays CD8+ T-cell responses and respiratory syncytial virus clearance in geriatric cotton rats

Abstract

Respiratory syncytial virus (RSV) infection is associated with increased rates of severe disease, hospitalization, and death in elderly individuals. Clearance of RSV is frequently delayed within this demographic, contributing to the more severe disease course. Geriatric cotton rats mimic this prolonged clearance kinetic and serve as a useful animal model for studying age-associated immunological deficits during RSV infection. Treatment with the cyclooxygenase (COX) inhibitor ibuprofen restores RSV clearance, indicating that inflammation contributes to impaired clearance in geriatric cotton rats. Here, we further characterize a compromised immune response in geriatric cotton rats and identify an inflammatory pathway that contributes to this deficiency. Dendritic cell (DC) activation and migration to mediastinal lymph nodes are decreased during early infection in geriatric cotton rats, resulting in delayed generation of cytotoxic T cells and virus clearance. Prostaglandin D₂ (PGD2), which reduces DC migration through the elevation of D-type prostanoid 1 receptor (DP1 receptor), is elevated in the airways of infected geriatric cotton rats. Reducing PGD2 production by inhibiting COX-2 or PGD2 synthase improves RSV clearance kinetics through DC activation and RSV-specific CD8+ T-cell responses in geriatric cotton rats, whereas activation of DP1 receptor through an agonist resulted in delayed viral clearance in adult cotton rats. These results indicate that PGD2 contributes to delayed antigen presentation and CD8+ T-cell responses to RSV in geriatric cotton rats. Inhibiting PGD2 generation or signaling may be a useful mechanism of therapeutic intervention in elderly individuals.IMPORTANCEElderly adults are at increased risk of severe disease resulting from infection with respiratory syncytial virus (RSV), characterized in part by delayed clearance (removal of the virus from airways). Understanding the immunological factors that lead to this delayed clearance may allow for the development of therapies to improve disease outcomes in elderly individuals infected with RSV and other respiratory viruses. Here, we describe an inflammatory pathway in geriatric cotton rats, the preferred small animal laboratory model for RSV, that impairs the generation of an effective immune response. We show that inhibiting this inflammatory pathway in geriatric cotton rats improves immune parameters and speeds clearance of RSV. These results contribute to our understanding of delayed RSV clearance in elderly individuals with possible applications for improving immune responses to RSV in clinical settings.

Keywords: aging; cotton rat; prostaglandin D2; respiratory syncytial virus.

Fig 1

RSV-specific CD8+ T-cell responses in the lungs, spleen, and MLN are delayed in geriatric cotton rats. At the indicated days post-RSV infection, cotton rats were euthanized, and the tissues were collected. Single-cell suspensions were plated overnight with Brefeldin A and in the presence (stimulated) or absence (unstimulated) of immunodominant MHC-I-restricted peptides from the RSV F and NP proteins. Flow cytometry was performed measuring the percentage of CD8+ T cells expressing IFN-γ. The percentage of CD8+ T cells expressing IFN-γ from matching unstimulated samples (indicative of background/nonspecific labeling) was subtracted from stimulated samples. RSV-specific CD8+ T cells were first detectable in lung of adult animals, followed by appreciable populations in the mediastinal lymph nodes and spleen. RSV-specific CD8+ T cell percentages in geriatric animals were significantly lower (*P < 0.01) until after day 8 post-infection in lung, and until after day 12 post-infection in MLN and spleen. (n = 3 for geriatric day 21 in B and C. n = 5 for all other data points.)

Fig 2

Lung DC activation and migration to lymph nodes are impaired in geriatric cotton rats. Tissues were collected at the indicated time points post-RSV infection, with 0 hours post-infection representing uninfected cotton rats. (A) A significantly lower (*P < 0.01) percentage of lung DCs (CD11c+/MHC-II+/Siglec F-) express CCR7, a marker of DC activation and migration to lymphoid tissues, at 24 and 48 hours post-infection in geriatric cotton rats compared with adults. (B) The proportions of DCs within MLN of adult cotton rats increase to significantly higher percentages (*P < 0.01) at 48 and 72 hours post-infection compared with geriatric animals, suggesting decreased DC migration from the lung in geriatric cotton rats. (n = 4 for adult 0 hours p.i. in A. n = 5 for all other data points.)

Fig 3

PGD2 is elevated in the airways of geriatric cotton rats. At the indicated time point post-RSV infection, adult and geriatric cotton rats were euthanized, and airways were sampled by BAL with 1 mL PBS. PGD2 concentrations were measured by ELISA. Day 0 values represent cotton rats administered PBS intranasally and euthanized at 2 days post-inoculation. PGD2 concentrations are significantly higher in geriatric cotton rats than in adults from days 4–6 post-infection (***P < 0.005). (n = 5 for all time points).

Fig 4

Prostaglandin D₂ receptor 1 (DP1) agonism delays RSV clearance in adult cotton rats. The DP1 receptor agonist BW245C was administered intranasally to adult cotton rats daily from days 3–5 post-RSV infection. RSV titers were measured in lung (A) and nasal turbinates (B) by TCID₅₀ assay at the indicated time points. Untreated adult cotton rats clear the virus below detectable thresholds by day 6 post-infection in both lung and nasal turbinate. Treatment with BW245C results in delayed clearance in both tissues, similar to geriatric cotton rats. (n = 5 for all adult untreated and DP1 agonist-treated time points. n = 3 for geriatric days 6 and 7. n = 4 for geriatric day 8.)

Fig 5

Inhibiting PGD2 production improves RSV clearance in geriatric cotton rats. From days 0–4 post-RSV infection, geriatric cotton rats were treated intraperitoneally with the COX-2 inhibitor NS-398, the PGDS inhibitor TFC 007, or PBS (no treatment control). At day 6 post-infection, RSV titers in lung and nasal turbinate were measured by TCID₅₀ assay. Mean titers for groups treated with either the COX-2 inhibitor or PGDS inhibitor were below the sensitivity threshold of the assay, whereas titers in untreated geriatric cotton rats were still readily detectable. (n = 5 for No Treatment and COX-2 Inhibitor groups. n = 3 for PGD2 Synthase Inhibitor group. Data points displayed below the x axis represent animals with titers lower than the detectable threshold of the assay, with values of 0 used for mean and standard deviation calculation.)

Fig 6

COX-2 inhibition increases pulmonary DC CCR7 expression and RSV-specific CD8+ T cell responses. RSV-infected geriatric cotton rats were treated daily with the COX-2 inhibitor NS-398 intraperitoneally from days 0–1 (A) or 0–4 (B). (A) At day 2 post-infection, a cohort was euthanized, and the lungs were collected to measure CCR7 expression in DCs (CD11c+/MHC-II+/Siglec F-) by flow cytometry. Compared with untreated geriatric cotton rats, NS-398 treatment resulted in higher CCR7-expressing lung DCs (*P < 0.01). (B) At day 8 post-infection, lung and MLN were collected to measure RSV-specific CD8 T-cell responses. The percentage of RSV-specific CD8+ T cells was calculated as the increase in the percentage of IFN-γ expressing CD8+ T cells in peptide-stimulated samples from unstimulated matching controls. COX-2 inhibition resulted in an increased RSV-specific CD8+ T-cell response in MLN (*P < 0.01) but not the lung. (n = 5 for all data points).

Fig 7

COX-2 and hPGDS genes are not upregulated in geriatric cotton rats compared with adults. The left lung lobe was collected from adult and geriatric cotton rats at the indicated time points post-RSV infection. Absolute quantification of IL-6, COX-2, and hPGDS copies was measured by ddPCR of 300 ng of cDNA for each sample. IL-6, a cytokine known to be upregulated early in RSV infection in cotton rats, displays an expected spike in expression at day 1 post-infection. Although an approximately 2-fold increase is seen at day 1 over day 0 for COX-2, neither COX-2 nor hPGDS differs significantly in expression throughout the course of infection compared with baseline (day 0). At baseline and all measured time points following infection, geriatric and adult cotton rats express similar concentrations of COX-2 and hPGDS mRNA. (n = 5 for all adult time points and geriatric day 6. n = 5 for geriatric days 0, 1, and 4.)