Indirect evidence has implicated microbial translocation from the gut as a factor contributing to pathological immune activation in chronic HIV/SIV infection, but direct evidence of translocation and demonstration of a plausible underlying mechanism have been lacking. Using an unbiased, comprehensive approach for quantitative and qualitative immunohistologic analysis of randomly selected tissue specimens obtained from non-human primates at various times relative to SIV infection, we have shown that: 1) microbial products can be found in the LP of the large bowel, in draining and distant lymph nodes, and in the liver of chronically SIV-infected RMs; 2) microbial translocation is associated with breakdown of the integrity of the epithelial barrier of SIV-infected RMs; 3) the extent of epithelial breakdown correlates with the extent of microbial translocation; 4) epithelial barrier breakdown and microbial translocation begin to be apparent during the late acute phase of infection (14 dpi); 5) the presence of microbial products in multiple anatomical sites is associated with expression of IFN-α and IL-18 in the absence of detectable local viral replication in the LP, consistent with direct induction of immune activation; 6) macrophages in chronically SIV-infected RMs appear dysfunctional with respect to their ability to phagocytose translocated microbial products; and 7) neither epithelial barrier breakdown nor infiltration of microbial products into the LP occur during the chronic phase of SIV infection of SMs.
We provide two lines of evidence linking microbial translocation to immune activation. First, we show that in pathogenic SIV infection of RMs, damage to the integrity of the epithelial barrier of the GI tract is associated with microbial translocation, and that microbial translocation is linked to local immune activation, based on co-localization of microbial products and production of the immunoinflammatory cytokines IFNα and IL-18, including in lymph nodes anatomically distant from the GI tract. Second, in marked contrast, in SIV-infected SMs where immune activation is quickly resolved in the acute stage of infection 
, and chronic infection is not accompanied by persistent immune activation, we found neither damage to the intestinal barrier nor microbial translocation.
Recent in vitro
studies with peripheral blood lymphocytes from SMs have been interpreted to suggest that a lack of type I IFN cytokine response to SIV RNA accounts for the typically nonprogressive nature of SIV infection in SMs 
. Furthermore, these data have been used to suggest that the raised plasma LPS levels observed in chronically SIV-infected RMs and HIV-infected humans, are simply markers of damage to the GI tract and that the microbial translocation that is reflected in elevated plasma LPS levels does not contribute significantly to causing systemic immune activation 
. However not only were LPS levels increased in chronically-infected individuals, but sCD14 and LPS binding protein levels were also increased 
. These data strongly suggested that LPS was directly stimulating the immune system in vivo
. In the present study, we show directly that damage to the integrity of the epithelial barrier of the GI tract allows microbial products to infiltrate into the LP and this infiltration is associated with local immune activation demonstrated by co-localization of microbial products within the LP with IFNα and IL-18 in MesLN. As only limited viral replication is demonstrable in the LP of the GI tract of chronically SIV-infected RMs, the damage to the GI epithelial lining, microbial translocation and local immune activation are unlikely to be caused by the direct effects of local viral replication. Rather, where rare infected cells are seen in the LP underlying damaged mucosa, it is more likely that the chronic immune activation, due to translocated microbial products, has provided activated CD4+
T cell targets for the virus.
Taken together, these data suggest that microbial translocation, resulting from damage to the GI tract epithelial barrier and impaired macrophage-mediated phagocytosis, results in immune activation during the chronic phase of HIV/SIV infection of humans and RMs. Importantly, we found a significant correlation between the extent of damage to the epithelial barrier of the colon and the amount of LPS within the underlying mucosa and the extent of translocated microbial products in draining and remote lymph node tissues. The extent of microbial constituents present in lymph node tissues correlated with the extent of local evidence of immune activation, further substantiating the link. Moreover, while we find that microbial translocation begins during the acute phase of infection, our previous work had indicated that elevated levels of microbial products were not seen in plasma until the chronic phase 
. Our data suggest that microbial products are localized within tissue macrophages during the acute phase thus limiting their circulation.
The causes of damage to the integrity of the epithelial barrier of the GI tract are likely to be multifaceted, but in the chronic stages of SIV infection seem unlikely to be due to direct virotoxic effects, given the lack of association with very low levels of demonstrable local viral replication in the LP relative to the extensive epithelial damage. One possible mechanism may be related to the preferential loss of Th17 cells in the GI tract in progressive immunodeficiency lentiviral infections 
, because Th17 cells produce cytokines important for enterocyte proliferation and antibacterial defensins 
and IL-17 has recently been shown to suppress Th1-mediated damage to gut epithelium. Importantly, preservation of this T cell subset in the gut of chronically SIVsmm-infected SMs and SIVagm-infected AGMs 
and the sparing of the epithelial barrier of SIVsmm infected SMs we show here supports this mechanism.
We speculate that the association we found between immune activation, microbial translocation and chronic stages of SIV infection, and similarly, later stages of HIV-1 infection, reflects damage to the structural integrity of the GI tract and a potential “deficiency” of the GI tract macrophage-phagocytic system. Our observation that intestinal macrophages from SIV-infected RMs, which are generally not proinflammatory 
, are unable to clear translocated microbial products, within the LP, and could lead to increased proinflammatory responses locally are supported by several groups findings that showed: i) impaired monocyte phagocytosis in HIV-infected individuals 
; ii) reduced LPS-mediated enhancement of phagocytosis in monocytes HIV-infected individuals compared to healthy donors 
; and iii) significantly higher colonic mucosa proinflammatory mRNA expression levels (e.g. TNF-α, IFN-γ, and IL-6) in HIV-infected patients than in control patients 
. These data certainly warrant further investigation into the functional properties of tissue macrophages from HIV/SIV-infected individuals and the mechanisms underlying their apparent dysfunction. While increased microbial translocation begins in the late acute stage of SIV infection, it was not until the later stages of infection that the capacity of macrophages for clearance was apparently affected, suggesting that microbial translocation has an increasing major contribution to immune activation as the host progresses towards disease. The evidence for this model comes from images of MesLN and AxLN stained for bacterial products in the chronic stage that showed dramatically increased extracellular bacterial constituents in the late AIDS stage of SIV infection, versus the mainly cellular staining of LPS at earlier stages. Taken together, these data strongly suggest that in SIV infection of RMs, and by extension, HIV infection of humans, damage to the epithelial barrier of the GI tract leads to levels of microbial translocation that exceed the capacity of host defense mechanisms to sequester away microbial constituents from secondary lymphatic tissues, resulting in persistent immune activation that contributes importantly to pathogenesis during the chronic phase of infection. Understanding the factors underlying damage to the integrity of the epithelial barrier and macrophage deficiencies that we report may lead to novel therapeutic interventions that aim to reduce microbial translocation and the deleterious effects of the consequent immune activation.