In the present study, we described a neonatal mouse model of influenza A infection. In this model, BALB/c mice were infected with influenza virus at 7 d of age and allowed to mature. The mice developed acute, severe pulmonary inflammation, which remained unresolved four months later and long after influenza virus was no longer detectable. This correlated with significant increases in pulmonary resistance in response to increasing doses of methacholine (i.e. airway hyperreactivity) and decreases in compliance at four months. Histopathologic analysis of the lungs from adult mice infected as neonates revealed emphysematous-type lesions characterized by airspace enlargement and destruction of alveolar walls. Adoptive transfer of adult CD8+ T cell into the pups before infection with influenza reversed the effects of neonatal influenza infection as evidenced by enhanced pulmonary function (returning airway resistance to SHAM levels) and reduced pulmonary inflammation and reduced viral load compared to pups receiving neonatal CD8+ T cells. Adoptive transfer of adult CD8+ T cells deficient in IFN-γ indicated that IFN-γ was critical in determining disease outcome.
The inflammatory cells in the BALF during the acute phase (5 hpi to 10 dpi) and chronic phase (109 dpi) included monocytes/AMs, lymphocytes, and neutrophils. Interestingly, although there were more cells in the BALF at 10 dpi than at 109 dpi (~50% more), the composition of the BALF was not significantly altered (i.e. both were comprised of ~80% of monocytes/AMs and 20% of lymphocytes and neutrophils) suggesting the continued presence or secretion of chemokines responsible for neutrophil and lymphocyte recruitment even in the absence of detectable infectious virus or viral antigen. At 109 dpi, we were unable to detect differences in cytokines between mice infected as neonates and SHAM controls; however, the role of other cytokines or inflammatory mediators (e.g. leukotrienes, etc.) cannot be excluded.
In addition to the chronic inflammation and airway hyperreactivity to MeCh, histopathologic analysis of the lungs from adult mice originally infected as neonates revealed emphysematous-type lesions within the lung architecture. Intriguingly, there were no differences in baseline resistance between this group and SHAM controls at 109 dpi despite significant differences in lung architecture and inflammatory state. The reason(s) for this are unclear, but may represent masking due to the persistent inflammation or a limitation of the model chosen (i.e. single-compartment) to measure respiratory mechanics.
In general, the immune system of a neonate is quite different from that of an adult, in that the innate and adaptive immune responses are immature (28
). Naïve, neonatal T cells are also functionally distinct from adult T cells (30
); and effector, neonatal T cells are less able to lyse antigen bearing cells or produce cytokines (31
). Although neonatal T cells are able to mount comparable proliferative responses to mitogens (32
), they have intrinsically lower levels of CD3/TCR, adhesion molecules, and costimulatory molecules (33
). Data from our studies indicate that recruitment of T cells is different in the infected neonates versus the adult and although both T cell populations are induced in the neonate, CD8+ T cell numbers are doubled in the adults. This suggested that cell number alone may have been responsible for the pathophysiological impact of neonatal influenza infection. Although adoptive transfer of neonatal CD8+ T cells prior to infection did help to control the infection (i.e. reduced viral load and improved pulmonary function compared to neonates infected with influenza); it was not as effective as adoptive transfer of adult CD8+ T cells (i.e. further reduction in viral load and pulmonary function equivalent to that of SHAM). These data suggest that neonatal CD8+ T cells are functional impaired compared to adult CD8+ T cells.
In addition to the lower magnitude of the T cell response in the neonates, the number of IFN-γ+ CD8+ T cells was significantly lower than that of their adult counterparts after influenza infection. Previous studies have showed that IFN-γ plays an important role in recovery from influenza infection by helping to clear the virus (34
) and that adoptive transfer of Tc1 cells (IFN-γhi
cytotoxic T cells) promotes clearance of the influenza virus, while transfer of Tc2 cells (IFN-γlo
cytotoxic T cells) does not affect viral clearance (36
). Our data confirmed that IFN-γ produced by CD8+ T cells was important to effectively clear influenza from the neonatal lung, since mice receiving IFN-γ deficient adult CD8+ T cells showed higher viral loads than mice receiving wild-type adult CD8+ T cells. Moreover, adoptive transfer of IFN-γ deficient CD8+ T cells totally abolished the benefits observed upon administration of adult CD8+ T cells, as demonstrated by increased airway hyperreactivity, BALF cellularity, and lung histopathology (similar to neonatal infection controls). In total, our data further demonstrate the importance of IFN-γ in the resolution of infection and inflammation initiated upon infection of neonatal mice.
Viral load and immune function are inescapably linked. Also, it is readily apparent that CD8+ T cells do not directly affect disease outcome and that they alter the course of pathogenesis by acting against virus-infected cells (i.e. decreasing viral load) through production of IFN-γ. This contention is strengthened by our observation that introduction of poorly functional neonatal CD8 T-cells does not ameliorate disease, or act against virus-infected cells, thereby permitting a higher viral load in the host. Mice receiving wild-type adult CD8+ T cells exhibited lower viral loads, improved pulmonary function, a reduction in total BALF cellularity, and a reduction in pulmonary inflammation compared to mice infected as adults.
Influenza and another common respiratory virus, RSV, infect the same human population (infants) but elicit different pulmonary diseases. It has been reported that Th2 (IL-4+ T helper cells) responses dominate in neonatal immunity; while Th1 (IFN-γ+ T helper cells) responses dominate in adults (37
). However, studies from our lab and other groups clearly demonstrate that the immune response initiated by neonates is more complex (20
). A previous study showed that RSV infected neonatal mice mount a Th2 biased response when rechallenged as adults with RSV (18
). Although a mixture of Th1 and Th2 cells is elicited in lungs during reinfection, there were significantly more (4 fold) Th2 cells in lungs compared to mice primarily infected as adults (18
). Data from our lab showed that even at primary infection, neonatal RSV infection mounts a Th2-skewed response (20
) compared to influenza infection (data presented here). In fact, both infections mount a mixed Th1/Th2 response. Following influenza infection about 5 fold more Th1 cells than Th2 cells were recruited to the lungs, while RSV infection recruited similar numbers of Th1 and Th2 cells. These data suggest that the immune response initiated in neonates is not predestined toward a Th2 response, as previously implied, and appears to depend on the antigen encountered. Besides the differences in responses of helper T cells to RSV and influenza, both viruses induce a weak CD8+ T cell response similar in magnitude and function. Finally during neonatal RSV infections, although airway remodeling is present (i.e. increased basement membrane thickness, smooth muscle hypertrophy, subepithelial fibrosis), there is relatively no tissue destruction (20
). In contrast, neonates infected with influenza exhibited a tremendous amount of tissue destruction, which may be the principal determinant of the severity of airway symptoms. Taken together, there is similarity and disparity in the immune responses induced by RSV and influenza, and the immune and cytopathic differences may explain the specific pulmonary diseases elicited by these two viruses.
In summary, our data demonstrate that infection of newborn mice with influenza has long-term consequences for the host inducing diffuse emphysematous changes in the lung and marked pulmonary inflammation. These alterations were persistent and associated with increased airway resistance and reduced compliance. The adaptive T cell response was markedly reduced in the neonates, with the most striking difference being observed among the CD8+ T cell population. Our adoptive transfer data suggest that the immaturity of this cell population is an important factor in determining disease outcome in the context of the pulmonary microenvironment. These data, along with recent data suggesting that one lung infection has the potential to modify immunity for extended periods of time(42
), emphasize the importance of delaying the time of initial influenza infection, and therefore the importance of vaccination in infants and young children. Future studies to elucidate the molecular mechanisms responsible for the persistent inflammation and structural alterations observed with neonatal influenza infection should identify important therapeutic targets capable of controlling long-term complications due to viral bronchiolitis in infancy. Our observations (i.e. that CD8+ T cell responses in neonate are functional different than that of adults) have significant implications for human infants beyond just influenza infection including infant responses to nosocomial infections and even responses to vaccination.