The relative lack of inflammatory responses in AAV-transduced hosts compared to the acute toxicity and inflammation induced by adenovirus vectors prompted us to embark on the studies in this report (17
). Although these observations have been documented in numerous studies in vivo (13
), the potential molecular mechanism(s) underlying these divergent responses is not clear. The results of our studies in vitro showed that adenovirus but not AAV vectors induced the expressions of various chemokines in two epithelium-derived cell lines, REC and HeLa. The differential induction of chemokines was evident in vitro despite exposure of cells to AAV titers that were 40-fold higher than the lowest titer of adenovirus required to initiate the response. Since cells cultured in vitro do not reflect the response in the whole animal, mice were used to examine whether findings in culture extended to an in vivo model. We found that AAV vectors in vivo induced only transient chemokine and cytokine expression and leukocyte recruitment in the liver. This response was not associated with significant toxicity or inflammation. In contrast, the chemokine and cytokine induction following exposure of mice to adenovirus vectors was rapid, marked, and sustained. These features stand in direct contrast to what is observed with AAV and confirm the differences in host responses to these two vectors.
The early host response to viral infection involves interaction between two components, the infected target cell and the innate immune system (14
). We and others have previously demonstrated with epithelium-derived target cells in vitro that adenovirus vectors induce the expression of chemokines (1
). The molecular basis for adenovirus vector induction of host inflammatory genes has been studied in nonmacrophage target cells. For example, adenovirus transduction of HeLa cells activates Raf-1 and the extracellular signal-regulated kinase signaling pathway. These events trigger expression of the C-X-C chemokine IL-8 (6
). It was previously shown in epithelium-derived REC cells that the nuclear translocation of NF-κB is an important component of adenovirus vector induction of the C-X-C chemokine IP-10 (5
). These findings illustrate the ability of adenovirus vectors to induce the expression of host inflammatory genes.
Unlike the situation with adenovirus vectors, the impact of AAV vector transduction in target cells has not been extensively studied. Recently, Stilwell and Samulski reported the effects of AAV-2 and adenovirus vectors on cellular gene expression in lung fibroblasts in studies using DNA microarray technology (J. Stilwell and R. J. Samulski, •••, Mol. Ther., p. 131, 2001.). Their findings parallel the results in this report. AAV demonstrated minimal effects on cellular gene expression in transduced cells, while adenovirus vectors increased the mRNA levels of many genes including those encoding cytokines and chemokines and stress response genes. Unlike the adenovirus, AAV vectors may lack a feature or features that enable them to activate signal transduction pathways following viral cell entry. It is possible that differences in surface receptor usage, virus internalization, or intracellular trafficking may result in differential activation of signaling pathways by these two vectors. For example, there is evidence suggesting that AAV particles enter the nucleus prior to uncoating and this pathway differs from the entry of adenovirus particles (2
). Furthermore, AAV virions may need to be routed as far as the late endosome before penetration into the cytosol. This route contrasts with the early endosome escape during adenovirus infection (10
). The inability of AAV vectors to induce the expression of host genes in target cells may underlie the truncated inflammatory response induced by these agents in vivo.
The target cell response acts in collaboration with the innate immune system in the early host response to invading pathogens, including viruses (14
). Effector cells of the innate immune system include macrophages, neutrophils, and natural killer cells that serve as the primary defense to infection. Adenovirus vectors efficiently activate the innate immune system, a response that leads to acute inflammation of transduced tissues and reduced gene transfer efficiency (22
). Resident macrophages such as Kupffer cells in the liver and alveolar macrophages in the lung are significant components of the innate immune system that mediate the acute inflammatory response to adenovirus vectors (32
). In addition to performing important phagocytic and antigen presenting functions, macrophages secrete cytokines and chemokines in response to viral infection, which in turn trigger multiple effects (22
). These effects include leukocyte recruitment to infected tissues and the participation of other effector cells of the immune system, resulting in a cascade of events that propagate the inflammatory response (30
). The results of our studies in vivo show a Kupffer cell-dependent activation of innate immune responses following AAV vector administration. While this outcome is unexpected, it is not surprising given the role of Kupffer cells in the first line of defense against invading pathogens in the liver (20
). Although AAV vectors induced transient chemokine and cytokine expression and leukocyte recruitment to the liver, this response did not result in detectable tissue damage or inflammation. In contrast, the induction of chemokines and cytokines by adenovirus vectors was greater in magnitude, prolonged, and associated with widespread liver inflammation. These findings suggest that a sustained increase in chemokine and cytokine expression appears to be required to trigger more-severe inflammation in the liver.
The depletion of Kupffer cells abolished AAV but not adenovirus vector-induced chemokine and cytokine expression in the liver. Yet, AAV vector transduction of the liver was not affected by Kupffer depletion, suggesting that AAV vectors did not activate other cells in the liver, such as hepatocytes. The differential induction of chemokines in vivo by AAVlacZ and AdlacZ in the absence of Kupffer cells is consistent with our in vitro data demonstrating a lack of chemokine induction by AAV vectors in nonmacrophage target cells. While macrophages may simply be more sensitive than epithelial cells to AAV transduction, these results raise the possibility that AAV vectors interact with macrophage and nonmacrophage cells via different mechanisms.
The induction of chemokines and cytokines by AAV vectors in vivo is likely not due to viral gene or transgene expression. Recombinant AAV vectors lack wild-type viral genes, and furthermore, lacZ
expression in AAVlacZ-transduced livers was not detectable within 24 h (data not shown). The differential activation of chemokines and cytokines by adenovirus and AAV vectors is also not likely the result of adenovirus gene expression. Transcription-defective adenovirus particles have been shown to induce chemokine and cytokine expression in vitro and induce acute inflammation in vivo (5
Viral vector-induced immunity and toxicity limit the success of human gene therapy. Our results demonstrate, for the first time, the involvement of the innate immune system in the host response to AAV vectors. While AAV vectors transiently activate cells of the innate immune system, they have a limited capacity to trigger the expression of proinflammatory genes and induce inflammation in transduced tissues. The impact of the innate immune system on AAV-mediated gene transfer remains to be determined. Since the innate immune system significantly reduces adenovirus gene transfer efficiency (35
), the activation of innate immune responses may also affect AAV-mediated gene delivery. Understanding the molecular mechanism underlying the differential activation of innate immune responses between adenovirus and AAV vectors is expected to facilitate the design of safer, more-effective vectors for human gene therapy.