The present studies identify a neuroanatomic context in which ANS activity can interact with SIV-infected cells to facilitate lentiviral replication in vivo. In experimentally infected rhesus macaques, levels of active SIV replication were significantly higher in the vicinity of catecholaminergic neural fibers within the lymph node parenchyma. Compared to regions containing no catecholaminergic innervation, the odds of SIV replication were approximately fourfold higher in lymph node tissue units containing one or more parenchymal catecholaminergic varicosities. This relationship held at the level of the lymph node as a whole and within distinct anatomic subregions (medulla and cortex). Such results are consistent with the hypothesis that norepinephrine released from ANS neural fibers might enhance viral load in HIV-1 patients (10
) and with in vitro studies defining specific molecular mechanisms by which norepinephrine can accelerate HIV-1 replication (7
). The present data extend those findings to identify a specific anatomic nexus in which ANS signaling can interact with virally infected cells to influence the lentiviral replication cycle in vivo. In addition to providing a lymphoid tissue context for catecholamine effects on viral replication, these findings also suggest that adrenergic interventions aimed at blocking such interactions must be capable of penetrating into secondary lymphoid organs to be effective.
The most parsimonious explanation of the spatial association between ANS neural fibers and SIV replication is that catecholamines can accelerate SIV replication via the same molecular mechanisms previously identified for HIV-1 in vitro (7
). However, other mechanisms could potentially contribute to spatial association. For example, catecholamine-induced signaling through the PKA pathway is known to inhibit cytotoxic T-cell activity (17
). This could impair cellular immunity in the vicinity of catecholaminergic varicosities and thus extend the duration of productive viral gene expression. It is also possible that catecholamines might selectively recruit or retain cells already supporting SIV replication via PKA-mediated effects on chemokine receptors or adhesion molecules (3
). Although theoretically possible, no previous studies have demonstrated chemotactic attraction of activated or virally infected leukocytes to catecholaminergic nerve fibers. Another potential mechanism for colocalization is the selective recruitment of catecholaminergic fibers by cellular processes associated with SIV replication. Activated leukocytes produce neurotropic factors and cytokines that could support the growth or maintenance of neural fibers (1
). This hypothesis might account for the localized association between catecholaminergic varicosities and SIV replication within a given lymph node, but it is not consistent with the strong inverse relationship observed between SIV replication frequencies and overall innervation density across different lymph nodes. In comparisons among SIV-infected tissues with different levels of viral replication, and in comparisons between SIV-infected and -uninfected tissues, higher rates of SIV replication were consistently associated with a lower density of catecholaminergic varicosities. Thus, the spatial colocalization observed within a given lymph node is not likely to be mediated by SIV-induced enhancement of local innervation. Another possibility is that changes in lymph node architecture induced by SIV pathogenesis could act to cosegregate SIV-expressing cells in the same vicinity as catecholaminergic neural fibers (e.g., by selectively decreasing the size of anatomic subcompartments, such as the paracortex, that contain high levels of both catecholaminergic nerve fibers and SIV replication). However, statistical analyses that controlled for differential distribution of SIV-expressing cells and parenchymal catecholaminergic varicosities across anatomic subregions continued to show a strong spatial relationship between those two parameters within structurally homogenous anatomic subcompartments. Moreover, the use of a stereologically based density estimate (SIV-expressing cells per 250-μm2
tissue unit) ensured that changes in the overall cellularity or size of the lymph node cannot account for the relationships observed (see reference 26
for more detail on statistical control for stereological bias). Finally, empirical analysis of cellularity showed that the mid-stage SIV infection analyzed here was not associated with any significant decrease in cell density that might act to cosegregate ANS neural fibers and SIV-replicating cells (in fact, a slight and nonsignificant increase in cell density was noted). These results show that SIV gene expression is selectively increased in the vicinity of catecholaminergic varicosities within the lymph node parenchyma and that such relationships are independent of any overall change in innervation or SIV gene expression that might result from global changes in lymph node structure or cellularity. Given the lack of empirical support for alternative explanations, catecholamine-induced stimulation of viral replication represents the best-substantiated explanation for the colocalization observed in these studies. However, future studies involving experimental manipulation of such interactions will be required to definitively identify the mechanism by which ANS nerve processes facilitate SIV replication in vivo.
The quantitative depletion of catecholaminergic varicosities in SIV-infected lymph nodes suggests that lentiviral pathology might potentially alter the distribution of neural fibers in lymphoid tissue. This finding is independent of the relationship between catecholaminergic varicosities and local SIV replication rates within a given lymph node and suggests a distinct dynamic in which differences in SIV pathogenesis across lymph nodes might influence the overall density of ANS innervation. SIV-induced depletion of lymph node innervation was quantitative, not qualitative, and the majority of SIV-infected tissues still retained parenchymal catecholaminergic varicosities that were associated with localized increases in SIV RNA expression. This quantitative reduction in ANS innervation density is consistent with data from previous studies that showed a depletion of autonomic nerve fibers from the spleens of mice infected with the LP-BM5 mixture of lymphotropic murine retroviruses (20
). The mechanisms by which lymphotropic viruses alter lymphoid tissue innervation are unknown, but at least two broad possibilities exist. One involves neurotoxic effects of viral proteins such as HIV-1 Tat and gp120, which have been shown to induce apoptosis in central nervous system neurons (33
) and may cleave the lymphoid chemokine SDF-1 into a neurotoxic by-product (39
). A second broad possibility is that cytokines or other signaling molecules released by activated/apoptotic lymphocytes might alter neural architecture. A similar dynamic has been observed in rheumatoid arthritis, where increased expression of semaphorin 3C is associated with a decreased density of catecholaminergic neurons in synovial tissue (24
). Regardless of the exact molecular mechanisms involved, the present data suggest that progressing SIV infection may alter autonomic innervation in ways that could potentially affect neuroimmune and neurovirologic interactions. We did not analyze lymph nodes during early-stage acute infection, but the observed depletion of ANS innervation from mid-stage lymphoid tissue with significant viral burden suggests that ANS activation might exert even more pronounced effects on SIV gene expression early in infection, when lymphoid innervation remains dense and viral replication interacts with an emerging immune response to establish the viral load set point. This hypothesis would be consistent with data showing elevated viral load set points in both SIV-infected animals experiencing social stress (5
) and HIV-1 patients with high levels of ANS activity (10
). Under that scenario, ANS activity early in infection could exert a long-term influence on lentiviral disease progression by altering the dynamic equilibrium between viral replication and antiviral immune responses. Regardless of their potential role in acute viral dynamics, catecholaminergic neural fibers show a strong anatomic relationship to SIV replication within the chronically infected tissues analyzed here. Such relationships are independent of the effect of SIV infection on lymph node cellularity and architecture, and they suggest that ANS activity in lymphoid organs might represent a potential target for adjunctive therapies aimed at limiting long-term physiologic support for lentiviral replication in vivo.