In the current study, the effects of a cysLT1R antagonist, MK, on RSV-induced AHR and airway inflammation were investigated, focusing on the development of altered airway function after reinfection with RSV. The results demonstrated that RSV infection induced increased levels of cysLT release in the airway. The time pattern of cysLT release paralleled and even preceded RSV-induced AHR. MK treatment, which did not affect the levels of cysLT in BALF, decreased RSV-induced AHR and airway inflammation in primary infected adult and neonatal mice. MK administered during initial infection but not during secondary infection of neonates prevented the subsequent enhancement of AHR and the development of airway eosinophilia and mucus hyperproduction upon reinfection.
Age at primary infection is critical to the outcome of secondary RSV infection. Neonatal RSV infection was previously shown to predispose mice to develop more severe airway disease on reinfection (8
). This amplified and altered response to reinfection was characterized by the development of significant AHR associated with marked airway eosinophilia and mucus hyperproduction. IFN-γ, RSV-specific IgE, and T cells were shown to be involved in this altered response to RSV reinfection.
Central to the protective effects of MK administered during initial infection of neonates on development of AHR and lung histopathology on reinfection may be the increases seen in levels of IFN-γ. Compared with weanling mice, neonates demonstrated a lower IFN-γ response to initial RSV infection. This role of IFN-γ was confirmed in IFN-γ–deficient mice reconstituted with recombinant IFN-γ. The results showed that IFN-γ was required during initial RSV infection for the expression of protective responses against development of AHR and lung histopathology on reinfection (9
). The effects of MK treatment on IFN-γ levels have been demonstrated in other systems. In patients with asthma and in some healthy volunteers, levels of IFN-γ were increased from mononuclear cells after cysLT1R antagonism with MK (32
). In patients with allergic rhinitis, treatment with MK increased IFN-γ levels in nasal secretions (33
). Here, MK treatment during primary RSV infection increased the levels of IFN-γ after primary and secondary infection, potentially preventing the increases in Th2 responses (IL-5 and IL-13) associated with development of AHR, airway eosinophilia, and goblet cell metaplasia. These decreases in levels of Th2 cytokines after MK treatment were confirmed in in vitro
cultures of PBLN cells stimulated with RSV.
The mechanisms whereby MK treatment resulted in increased IFN-γ levels are not entirely clear. Several lines of evidence indicate that dendritic cells (DCs) and activated T cells, which express cysLT1R, are targets of cysLT. cysLT has been shown to modulate cytokine production from allergen-stimulated DCs (34
). Murine bone marrow DCs pulsed with mite antigen plus LTD4 produced higher amounts of IL-10 compared with antigen-pulsed DCs alone, whereas DCs pulsed with antigen in the presence of a cysLT1R antagonist secreted increased levels of IL-12 and decreased levels of IL-10. When these cysLT1R antagonist-treated DCs were administered intranasally into BALB/c recipients subsequently challenged with antigen, the concentrations of IFN-γ in BALF were increased. This study indicates that cysLT1 may direct DC function in a Th2-dominant manner. CysLT1R signaling may affect the initiation of immune responses by modulating DC migration. In patients with allergic asthma, pranlukast treatment attenuated the decrease in circulating myeloid DCs seen 3 hours after allergen challenge (35
). Another study in mice demonstrated that cysLT enhanced DC migration to draining lymph nodes (36
). cysLT1 may also have effects on T cells. MK treatment after TCR activation increased IFN-γ production (37
). Sensitized and challenged mice lacking leukotriene C4 synthase, the terminal enzyme for cysLT generation, exhibited reduced AHR, eosinophil infiltration, Th2 cytokine levels, and goblet cell metaplasia compared with wild-type control; Th1 cell–dependent responses remained intact (38
). Together, these findings indicate that the major activity of cysLT1 in allergen-induced inflammatory responses is to enhance Th2-type responses. T-cell responses in neonatal mice are, in general, biased toward a Th2-like phenotype (39
). In the present study, neonatal mice infected with RSV mounted Th2-dominant responses on reinfection with RSV. It appears that the root of this skewed response is an impairment of IFN-γ production at the time of initial infection (8
) and is contrasted by the response of older mice to initial infection and reinfection. Treatment with MK at the time of initial RSV infection clearly attenuated the skewing toward Th2 differentiation and was associated with increased levels of IFN-γ.
The exact mechanisms resulting in AHR are not clear, and there is no single common pathway that leads to this alteration. cysLT are potent bronchoconstrictors (41
) as well as proinflammatory mediators (42
), both of which might be involved in RSV-induced AHR. In our study, it is difficult to elucidate which one is more important in primary RSV infection in both age groups because treatment with MK decreased AHR and airway inflammation simultaneously. However, after secondary infection, when MK was administered only during primary infection, the decreased AHR in MK-treated mice after secondary infection, weeks after MK administration, cannot be attributed to decreased bronchoconstriction with MK. This conclusion is further supported by the findings that, when administered during secondary infection, MK was without effect on the development of enhanced AHR.
The dose of MK chosen in this study was based on initial dose–response experiments for preventing the development of AHR. Different doses of MK have been shown to have different effects in the mouse after exposure to allergen, including continuous subcutaneous delivery (27
). In the study by Ihaku and colleagues, only a reduction in mononuclear cells was found when lower doses (3 mg/kg) of MK were used (28
), whereas Blain and Sirois showed that the maximum inhibition of airway eosinophilia was achieved with 100 mg/kg of MK-517 (29
). Wu and colleagues demonstrated that the dose required for antiinflammatory effects was higher (25 mg/kg) than the dose required for inhibiting bronchial smooth muscle constriction (30
). One reason for the high doses apparently required in mice is that plasma clearance of MK is considerably higher in mice than in humans (30
). It is not possible to rule out the potential for off-target effects of MK at the doses used that may be independent of cysLT1R blockade. In support of the role for leukotrienes mediating the RSV-induced changes in lung function and airway inflammation, other leukotriene synthesis inhibitors have shown efficacy in a primary RSV infection model (26
The cellular sources of cysLT in mice triggered by RSV infection are unclear. Similar to the asthmatic response, a variety of inflammatory cells have the capacity to produce cysLT. These cells are of myeloid origin and are resident in the lung (e.g., mast cells, macrophages) or are recruited to the lung after RSV infection (e.g., eosinophils, neutrophils, lymphocytes) (43
). In this study, we found that the levels of cysLT were significantly increased in BALF after primary RSV infection in both age groups. The increase in cysLT levels was even more pronounced after secondary RSV infection. This may have been the result of the increased accumulation of inflammatory cells and in particular eosinophils after secondary RSV infection. In association with alterations in many other parameters, including a significant reduction in airway eosinophilia, the levels of cysLT in the BALF of secondary infected mice were also reduced by MK treatment in the neonatal period. Airway eosinophilia may not be the sole or primary source for the increased cysLT levels in infected mice. Primary airway epithelial cells can release appreciable amounts of LTC4 when activated (44
). Because epithelial cells are activated by RSV but do not express the cysLT1R after infection with RSV (45
), the continued production of some cysLT may occur in the absence of inflammation. Reinfection with RSV may have induced airway epithelial cells to release more cysLT when compared with primary RSV infection.
The data indicate that cysLT release and the consequences are to some extent linked, but it is possible that MK acts in different ways during primary and secondary infection. Based on the data accumulated in this model, the early exposure of neonatal mice to RSV resulted in a Th2 skewing of the response to reinfection (8
). In the absence of MK treatment during primary infection, the presumption is that cysLT acted on a number of cell types expressing a cysLT receptor, leading to their recruitment and activation. It is likely that this response conditioned the neonatal mice in a way that resulted in the Th2 skewing on reinfection. MK treatment blocked these responses (e.g., airway eosinophilia) at the receptor level, whereas cysLT levels remained unchanged. After secondary infection, it is unlikely that the cysLT receptors remained blocked by the MK treatment administered 5 weeks earlier. More likely are the findings that MK treatment during primary infection modified the neonatal response to RSV, preventing Th2 skewing by enhancing IFN-γ release. This prevention of Th2 skewing and increases in IFN-γ were sustained on reinfection.
The failure of MK treatment during secondary infection to alter the development of enhanced AHR and airway eosinophilia suggests that these altered responses to secondary infection may be governed by pathways that are (now) independent of cysLT/cysLT1R interactions, unlike the responses to primary infection in neonatal and adult mice. These findings may have important clinical relevance, emphasizing the importance of preventing the responses during initial encounters with RSV.
These findings confirm that the early encounter with RSV sets the stage for the responses to reinfection and that MK treatment of young mice during primary infection significantly alters their responses to reinfection. MK has been studied in the prophylaxis against asthma exacerbations with effects shown in the fall and winter compared with placebo (46
). Although the associations between RSV infection and triggering of asthma are not clear, it appears that severe RSV infection requiring hospitalization may show such an association (49
). In these cohorts, there is no comment on whether the infants had been reinfected.
In summary, the results of this study support the notion that cysLT production at the time of initial RSV infection may be one of the critical factors that determine the outcomes of subsequent reinfection with RSV. Under the conditions described, MK may act beyond cysLT1R blockade; nonetheless, treatment with MK attenuated the initial responses to primary RSV infection and altered the consequences of RSV reinfection in mice initially infected as neonates. These findings identify an important role for cysLT in RSV-induced airway responses and provide novel insights into prophylactic approaches for the prevention of RSV-mediated long-term sequelae in infants with severe RSV bronchiolitis.