Host-directed therapy in foals can enhance functional innate immunity and reduce severity of Rhodococcus equi pneumonia

Abstract

Pneumonia caused by the intracellular bacterium Rhodococcus equi is an important cause of disease and death in immunocompromised hosts, especially foals. Antibiotics are the standard of care for treating R. equi pneumonia in foals, and adjunctive therapies are needed. We tested whether nebulization with TLR agonists (PUL-042) in foals would improve innate immunity and reduce the severity and duration of pneumonia following R. equi infection. Neonatal foals (n = 48) were nebulized with either PUL-042 or vehicle, and their lung cells infected ex vivo. PUL-042 increased inflammatory cytokines in BAL fluid and alveolar macrophages after ex vivo infection with R. equi. Then, the in vivo effects of PUL-042 on clinical signs of pneumonia were examined in 22 additional foals after intrabronchial challenge with R. equi. Foals infected and nebulized with PUL-042 or vehicle alone had a shorter duration of clinical signs of pneumonia and smaller pulmonary lesions when compared to non-nebulized foals. Our results demonstrate that host-directed therapy can enhance neonatal immune responses against respiratory pathogens and reduce the duration and severity of R. equi pneumonia.

Figure 1

Study design. (A) Ex vivo study; All foals in this study were nebulized with either PUL-042 or placebo, and Rhodococcus equi infections occurred ex vivo in alveolar macrophages (AMs) obtained through broncho-alveolar lavage (BAL). Control group (n = 12) received 2.8% glycerol, the vehicle diluent for PUL-042; PUL-042 2× (n = 12) received 46 µg Pam2 and 68 µg ODN; PUL-042 4× (n = 12) 92.8 µg Pam2 and 136 µg ODN; PUL-042 6× (n = 12) 139.8 µg Pam2 and 204 µg ODN. BAL procedures for collection of fluid and AM were performed on ages 2 (before nebulization) and 22 days (24 h following the last nebulization). Nebulizations started 1 week after the first BAL (approximately day 9 of age), and each foal received 6 nebulizations in a 2-week interval. Infections were performed ex vivo in AMs. (B) In vivo study; PUL-042 6× (n = 7) received 139.8 µg Pam2 and 204 µg ODN; control-nebulized group received 2.8% glycerol (n = 3); and control non-nebulized group (n = 12) did not receive nebulization. Infections were performed in vivo on day 28 of age, and foals were in the study until approximately 12 weeks of age. Nebulized foals received 9 nebulizations: 4 before and 5 after challenge at 28 days of age. Following challenge, twice daily physical examination, and weekly thoracic ultrasounds (TUS) were performed in all foals. At 12 weeks of age or whenever the foal recovers from clinical pneumonia if after 12 weeks of age, a tracheal wash was performed to confirm complete recovery of pneumonia by demonstrating absence of cultured R. equi and evidence of normal cytology on tracheal-wash fluid. Created with BioRender.com.

Figure 2

Broncho-alveolar lavage (BAL) fluid differential cytology (A–C) and serum amyloid A (SAA; (D)) of foals either before (Pre-nebulization) or after (Post-nebulization) serial nebulization with PUL-042 at various concentrations: 0 (n = 12), 2× (n = 12), 4× (n = 12), or 6× (n = 12). All foals were nebulized and no foals were infected in vivo with Rhodococcus equi. BALs and blood collection were performed pre- (day 2 after birth) and post-nebulization (day 22, 1 day after the last nebulization). Mean is represented by the symbol (circle for pre-nebulization; diamond for post-nebulization) and 95% confidence interval (whiskers). Percentage of macrophages (A) in BAL increased significantly (P < 0.0001) while percentage of neutrophils (B) decreased (P < 0.0001) after treatment (post) in all groups. The percentage of lymphocytes (C) did not vary significantly. Serum amyloid A (D) significantly decreased post-nebulization in all groups irrespective of treatment (P < 0.0001).

Figure 3

Concentration of pro- and anti-inflammatory cytokines in serum (left panel) or broncho-alveolar lavage (BAL; right panel) fluid of foals either before (Pre-nebulization) or after (Post-nebulization) serial nebulization with PUL-042 at various concentrations: 0 (n = 12), 2× (n = 12), 4× (n = 12), or 6× (n = 12). Cytokines were measured per ml of either serum or BAL fluid (TNF-α = pg/ml; IL-6 = pg/ml; IL-10 = ng/ml). All foals were nebulized and no foals were infected in vivo with Rhodococcus equi. BALs were performed pre (day 2 after birth) and post (day 22, 1 day after the last nebulization). In serum, TNF-⍺ (A) and IL-6 (B) significantly decreased with age, but only IL-6 had 1 group (PUL-042 2X) that was significantly different than PUL-042 0. Serum concentration of IL-10 (C) did not significantly differ among treatment groups or age. Tumor necrosis factor-alpha significantly increased in BAL fluid (D) for all groups, but effects were higher in BAL fluid of PUL-042 treated animals. The concentration of IL-6 (E) and IL-10 (F) in BAL fluid also did not significantly change with either age or treatment. Mean is represented by the symbol (circle for pre-nebulization; diamond for post-nebulization) and 95% confidence interval (whiskers).

Figure 4

Concentration of pro- and anti-inflammatory cytokines in supernatants obtained from cultured alveolar macrophages (AMs) from foals before (age 2 days; Pre-nebulization) and after (age 28 days; Post-nebulization) nebulization with glycerol 2.8% (PUL-042 0; n = 12), and varying doses of PUL-042 (2X, n = 12; 4×, n = 12; 6×, n = 12). All foals were nebulized and no foals were infected in vivo with Rhodococcus equi. Broncho-alveolar lavage was performed pre- (day 2 after birth) and post-nebulization (day 22, 1 day after the last nebulization): AMs were infected with virulent R. equi using a multiplicity of infection of 10 bacteria per macrophage (infected) or kept in media only (uninfected). Supernatant was collected 48 h post-infection for determination of cytokine concentration by ELISA. Data represented as estimates of mean (95% CI); values were analyzed as ratios of R. equi-infected/uninfected cells to account for cytokine production at baseline (control uninfected). Upon ex vivo infection with R. equi, both cytokines TNF-⍺ (A) and IL-10 (B) ratios significantly increased for all groups post-nebulization, while IL-6 ratio significantly increased only after treatment with PUL-042 (C). For INF-γ, this significant increase was only observed for the 2 higher doses of PUL-042, 4× and 6× (D).

Figure 5

Internalization and intracellular killing of Rhodococcus equi by alveolar macrophages (AMs) collected via bronchoalveolar (BAL) lavage pre (day 2 after birth) and post (day 22, 1 day after the last nebulization). All 48 foals were nebulized with either glycerol 2.8% (PUL-042 0; n = 12), or varying doses of PUL-042 (2×, n = 12; 4×, n = 12; or 6×, n = 12). No foals were infected in vivo with R. equi. Alveolar macrophages were infected with 10⁶ virulent R. equi using a multiplicity of infection of 10 bacteria per macrophage (infected) or kept with media only (uninfected control). Cells were then washed and either lysed and diluted immediately (T0) for bacterial determination, or cultured for 48 h on 5% CO2 and then lysed and diluted for CFU counts (T48). There were not significant effects of treatment or age in either phagocytosis ((A); T0) or replication ((B); ratio of T48:T0). (C) CFU ratio (T48:T0) vs IFNg:IL-10. The CFU ratio (lower ratio = greater intracellular killing) was significantly (P = 0.0075) and negatively associated with the ratio of IFN-g:IL-10 expression ratios (infected/uninfected). The log10 CFU ratio decreased by 10^(−0.47397) (95% CI, 10^{(−0.8149639)} to 10^{(−0.132983)}). R-value − 0.4740.

Figure 6

Clinical parameters of foals not nebulized (group control no nebulization; n = 12), nebulized with either PUL-042 6× (139.8 µg Pam2:204 µg ODN diluted in 2.8% glycerol; group PUL-042 6×; n = 7) or glycerol 2.8% (group control nebulization; n = 3) before and after infection with 10⁶ virulent R. equi intrabronchially at 28 days of age. (A) Non-nebulized foals showed longer duration of clinical signs of pneumonia, coughing, and fever when compared to foals receiving PUL-042 6×. (B) Nebulization with PUL-042 6× had shorter duration of presence of abscesses in their lungs upon thoracic ultrasound examination when compared to both control groups, although only group control no nebulization was significantly longer. (C) Foals that did not receive any nebulization showed larger sum of ultrasonographic lesions than foals that were nebulized with either PUL-042 6× or glycerol 2.8%. Mean is represented by the symbol (circle for control no-nebulization; square for control nebulization; and triangle for PUL-042 6×) and 95% confidence interval (whiskers).