Toll-like receptor-2/6 and Toll-like receptor-9 agonists suppress viral replication but not airway hyperreactivity in guinea pigs

Abstract

Respiratory virus infections cause airway hyperreactivity (AHR). Preventative strategies for virus-induced AHR remain limited. Toll-like receptors (TLRs) have been suggested as a therapeutic target because of their central role in triggering antiviral immune responses. Previous studies showed that concurrent treatment with TLR2/6 and TLR9 agonists reduced lethality and the microbial burden in murine models of bacterial and viral pneumonia. This study investigated the effects of TLR2/6 and TLR9 agonist pretreatment on parainfluenza virus pneumonia and virus-induced AHR in guinea pigs in vivo. Synthetic TLR2/6 lipopeptide agonist Pam₂CSK₄ and Class C oligodeoxynucleotide TLR9 agonist ODN2395, administered in combination 24 hours before virus infection, significantly reduced viral replication in the lung. Despite a fivefold reduction in viral titers, concurrent TLR2/6 and TLR9 agonist pretreatment did not prevent virus-induced AHR or virus-induced inhibitory M2 muscarinic receptor dysfunction. Interestingly, the TLR agonists independently caused non-M2-dependent AHR. These data confirm the therapeutic antiviral potential of TLR agonists, while suggesting that virus inhibition may be insufficient to prevent virus-induced airway pathophysiology. Furthermore, TLR agonists independently cause AHR, albeit through a distinctly different mechanism from that of parainfluenza virus.

Figure 1.

Pam₂CSK₄ (TLR2/6) and ODN2395 (TLR9) synergistically inhibit parainfluenza virus replication in vivo. Guinea pigs were pretreated simultaneously with Pam₂CSK₄ (4 nmol) and ODN2395 (1 nmol), or with PBS vehicle, administered as an aerosol directly into the trachea (Tracheal), or as a nasal solution (Nasal), 24 hours before parainfluenza virus infection. Parainfluenza RNA from lung homogenates was quantified by RT-PCR, 4 days after infection. Both tracheal and nasal Pam2/ODN pretreatment inhibited virus replication, although the effect of nasal Pam2/ODN fell short of statistical significance (tracheal, Pam2/ODN, n = 12; PBS, n = 11; *P < 0.05; nasal, Pam2/ODN, n = 6; PBS, n = 10; ^#P = 0.08). Viral titers were normalized to 18S RNA and transformed to tissue culture infectious dose (TCID) 50 titers using a parainfluenza standard curve quantified by rhesus monkey kidney cell titration. Data shown represent the means ± standard errors of the mean.

Figure 2.

Parainfluenza virus and Pam₂CSK₄ (TLR2/6)/ODN2395 (TLR9) cause vagal-reflex airway hyperreactivity. Guinea pigs were pretreated simultaneously with Pam₂CSK₄ (4 nmol) and ODN2395 (1 nmol), or with PBS vehicle, administered as an aerosol directly into the trachea (Tracheal), or as a nasal solution (Nasal), 24 hours before parainfluenza virus or mock infection. Histamine (1–5 μg/kg, intravenous) induced dose-dependent bronchoconstrictions, measured as increases in peak pulmonary pressures (P_peak), by activating the vagal-reflex innervation of the airways. Parainfluenza infection (PBS + Virus) and Pam₂CSK₄/ODN2395 pretreatment (Pam2/ODN + Mock) independently, and concomitantly (Pam2/ODN + Virus), potentiated vagal-reflex bronchoconstriction. (A) Aerosolized pretreatment administered into the trachea (n = 4 per group). (B) Nasal solution pretreatment (n = 5 per group). Data shown represent the means ± standard errors of the mean. *P < 0.05, compared with PBS + Mock control group. **P = 0.053, compared with PBS + Mock control group.

Figure 3.

Parainfluenza virus and Pam₂CSK₄ (TLR2/6)/ODN2395 (TLR9) cause non-neuronal airway hyperreactivity. Guinea pigs were pretreated simultaneously with Pam₂CSK₄ (4 nmol) and ODN2395 (1 nmol), or with PBS vehicle, administered as an aerosol directly into the trachea (Tracheal), or as a nasal solution (Nasal), 24 hours before parainfluenza virus or mock infection. Histamine (1–5 μg/kg, intravenous) induced dose-dependent bronchoconstrictions, measured as increases in P_peak, after vagotomy by activating histamine receptors at the level of the airways. Parainfluenza virus infection (PBS + Virus) and Pam₂CSK₄/ODN2395 pretreatment (Pam2/ODN + Mock) independently, and concomitantly (Pam2/ODN + Virus), potentiated bronchoconstriction. (A) Aerosol pretreatment administered directly into the trachea (n = 4 per group). (B) Nasal solution pretreatment (n = 5 per group). Data shown represent the means ± standard errors of the mean. *P < 0.05, compared with PBS + Mock control group.

Figure 4.

Parainfluenza infection, but not Pam₂CSK₄ (TLR2/6)/ODN2395 (TLR9), causes pulmonary M2 muscarinic receptor dysfunction. Guinea pigs were pretreated with Pam₂CSK₄ (4 nmol) and ODN2395 (1 nmol) in combination, or with PBS vehicle, administered as a nasal solution 24 hours before parainfluenza virus or mock infection. Transient bronchoconstrictions were produced by electrical stimulation of the vagus nerves (8 V, 15 Hz, 2-ms duration, 3 seconds on, 40 seconds off). Gallamine (1–10 mg/kg, intravenous), an M2 muscarinic receptor antagonist, was administered after every fourth bronchoconstriction. (A) Gallamine potentiated vagally induced bronchoconstriction by inhibiting M2 muscarinic receptor function in control (PBS + Mock) and in mock-infected Pam₂CSK₄/ODN2395 pretreated guinea pigs (Pam2/ODN + Mock), but not in virus-infected guinea pigs (PBS + Virus). Pam₂CSK₄/ODN2395 pretreatment did not prevent virus-induced M2 receptor dysfunction (Pam2/ODN + Virus; n = 5 per group). (B) Gallamine blocked vagally induced decreases in heart rate by inhibiting cardiac myocyte M2 receptors. Gallamine blocked decreases in heart rate similarly in all groups (n = 5 per group). Data shown represent the means ± standard errors of the mean. *P < 0.05, compared with PBS + Mock control group.

Figure 5.

Pulmonary M3 muscarinic receptor function is unaffected by parainfluenza infection or Pam₂CSK₄ (TLR2/6)/ODN2395 (TLR9) pretreatment. Acetylcholine (1–10 μg/kg, intravenous) caused bronchoconstriction, measured as increases in P_peak, by activating airway smooth muscle M3 muscarinic receptors. Acetylcholine-induced bronchoconstriction was similar between groups (n = 5 per group). Data shown represent the means ± standard errors of the mean.

Figure 6.

Parainfluenza infection and Pam₂CSK₄ (TLR2/6)/ODN2395 (TLR9) cause pulmonary and systemic inflammatory cell changes. Guinea pigs were pretreated with Pam₂CSK₄ (4 nmol) and ODN2395 (1 nmol) in combination, or with PBS vehicle, administered as an aerosol directly into the trachea (Tracheal), or as a nasal solution (Nasal), 24 hours before parainfluenza virus or mock infection. Bronchoalveolar (BAL) and peripheral blood total and differential leukocyte counts were evaluated 4 days after virus infection. (A) Tracheal pretreatment BAL counts (n = 6 per group). (B) Nasal pretreatment BAL counts (n = 5 per group). (C) Tracheal pretreatment peripheral leukocyte counts (n = 6 per group). (D) Nasal pretreatment peripheral leukocyte counts (n = 5 per group). Data shown represent the means ± standard errors of the mean. *P < 0.05, compared with PBS + Mock control group.