New Studies of Microbial Translocation
By Richard Jefferys
Immune activation has become a central culprit in the latest theories of how HIV causes disease. But despite increasing research on the question, the sources of immune activation continue to puzzle scientists.
The term immune activation is frequently encountered in the context of HIV research, but exactly what it means can be unclear. In a general sense, immune activation can be thought of as the mobilization of immune system cells that occurs during a battle with a pathogen. In most cases, immune activation is transient and subsides as the immune system either eliminates the pathogen or brings it under long-term control. What makes HIV (both HIV-1 and HIV-2) infection different is that immune activation does not fully resolve after initial infection but instead persists, eventually increasing as CD4 T-cell counts decline and progression to AIDS occurs.
A couple of years ago, Jason Brenchley and his colleagues at the National Institute of Allergy and Infectious Disease identified what may be a key contributor to immune activation in HIV infection: the leakage of normally “friendly” gut bacteria out of the gastrointestinal tract and into systemic circulation, a phenomenon called microbial translocation. Over the past several months, a number of new research reports have confirmed and extended Brenchley’s main findings. In parallel, an immunology study in mice has hinted that the causes of microbial translocation in HIV infection may be more complex than originally thought. This new research has potential implications for both pathogenesis and treatment.
Activated immune system cells express certain molecules on their surface, which can be measured to get an idea of the magnitude of immune activation occurring in an individual. In HIV infection, the expression of the molecule CD38 by CD8 T cells has proven the most useful measure of immune activation; elevated levels were cited in the very first AIDS case reports in 1981 (at that time CD38 was known as T10). In the early 1990s, UCLA researcher Janis Giorgi showed that levels of CD38 expression on CD8 T cells correlate with HIV disease progression. Several studies have since reported a closer association between CD38 expression and progression than is seen with viral load measurements. In monkeys, immune activation has also emerged as the key factor in determining whether infection with SIV (HIV’s simian sibling) causes disease; some monkey species develop persistent immune activation and progress to AIDS, while others show little or no immune activation and do not develop disease despite relatively high viral loads.
Despite this appreciation of the role immune activation plays in pathogenesis, the mechanisms by which it occurs are yet to be fully elucidated. At the time of initial infection, activation appears largely driven by responses to HIV. The immune system then typically gains some degree of control over HIV replication and activation subsides to a set point that correlates with the viral load set point and also predicts the pace of subsequent disease progression. However, in just about all HIV-infected people (including most long-term nonprogressors), immune activation levels remain significantly higher than those seen in uninfected individuals. Although it is likely that immune responses to HIV contribute to persistent activation, HIV antigens alone are insufficient to explain the phenomenon. Other persistent coinfections (such as hepatitis B or C) have been shown to contribute, but immune activation and disease progression still occurs in individuals who are not coinfected.
The search for additional causes of immune activation led to Jason Brenchley’s study, which was published in Nature Medicine in 2006. Brenchley found that people with progressing HIV infection and AIDS (but not acute or early stage HIV infection) have significantly elevated levels of bacterial products called lipopolysaccharides (LPS) in their bloodstream compared to uninfected individuals, and that levels of LPS correlated with CD38 expression on CD8 T cells. As the research paper notes, levels of LPS are a widely accepted indicator of microbial translocation, which has been reported to occur in a number of other settings including burn injuries, gastrointestinal surgeries and after the use of T cell–depleting cancer chemotherapies. Brenchley also confirmed the presence of biologically active LPS in the bloodstream of people with HIV by demonstrating that soluble CD14 levels were increased in parallel; CD14 is a molecule from immune system cells called monocytes that is both secreted and shed from the cell surface after stimulation by LPS. Brenchley and colleagues speculated that the likely cause of microbial translocation was the rapid, early depletion of gut CD4 T cells that occurs during acute HIV infection.
Microbial Translocation and Immune Reconstitution on Antiretroviral Therapy
One potential issue with using levels of LPS as a marker of microbial translocation is that it is not a direct measurement of the presence of bacteria. An alternative approach is to use polymerase chain reaction (PCR) to look for bacterial DNA in the bloodstream. Jason Brenchley has presented this type of analysis at conferences, reporting that bacterial DNA (called 16s DNA) correlates with levels of LPS, but these data have yet to be published. Consequently, a research letter by Giulia Marchetti and colleagues in the October 1, 2008, issue of the journal AIDS represents the first published direct evidence of microbial translocation in HIV infection.
The aim of Marchetti’s study was to assess whether microbial translocation impacts CD4 T-cell recovery after starting ART. The researchers recruited 24 individuals dubbed “immunological nonresponders” (INRs) who were compared to 11 people with good CD4 reconstitution (“full responders” or FRs) and 12 controls with advanced, untreated HIV infection. An initial evaluation of plasma levels of LPS showed that they were significantly higher in both INRs and individuals with untreated, advanced HIV infection compared to FRs.
Echoing Brenchley’s data, levels of LPS also correlated with markers of immune activation. The PCR technique was then employed to measure bacterial 16s DNA in samples, and sequencing experiments were conducted in order to confirm that the DNA was derived from gut bacteria species. Using this method, Marchetti and colleagues showed that 16s DNA could be isolated from 5 out of 7 INRs and 6 out of 12 individuals with advanced HIV infection, but none of the 7 FRs evaluated.
The results are consistent with the idea that microbial translocation contributes to immune activation in HIV infection and also indicate that it is associated with poor immune reconstitution despite viral suppression. However, in terms of the mechanism by which microbial translocation may be occurring, Marchetti and colleagues raise an interesting issue when discussing their results. They note microbial translocation may contribute to the lack of an immunological response following viral suppression by antiretroviral therapy (ART), but also point out that—conversely— persistently low T-cell counts might be causing microbial translocation. In their more technical language, “bacterial translocation might be favored in INRs by reduced T-cell-mediated competence failing to provide full immune control in mucosa and mesenteric lymph nodes, thus permitting peripheral egress and survival of bacteria.”
This distinction may be important. Poor immunological responses to ART have been attributed to elevated levels of immune activation in several prior studies. But Marchetti and colleagues are suggesting that causality could go in the opposite direction as well: a failure to restore CD4 T cells may allow persistent microbial translocation, which in turn causes persistent immune activation.
Lessons from Mice
In support of the idea that a lack of CD4 T cells can cause microbial translocation, Marchetti and colleagues cite a basic immunology study from way back in 1980. The paper reports that microbial translocation occurs in mice bred to lack a thymus (an organ that essentially serves as a T-cell training camp), and is reduced when these mice receive thymic grafts (which restore their ability to make naive T cells). Complementary findings were reported in a more recent mouse study published by the journal PNAS in June 2008. Christine Bourgeois and colleagues from Brigitta Stockinger’s laboratory in London reported that blocking the export of new naive T cells from the thymus led to microbial translocation, which in turn exacerbated the depletion of existing naive T cells by causing immune activation. Naive CD4 T cells were particularly sensitive to activation in this setting, leading to a skewing of the CD4/CD8 ratio. The researchers note the similarity with other settings in which naive T-cell depletion is observed, such as aging and HIV infection, and conclude that “continued replenishment with cells from the thymus seems to be required to maintain efficient gut mucosal defense.”
Taken together, these data may suggest that the slow, progressive depletion of naive CD4 T cells that occurs over the course of HIV infection could also contribute to microbial translocation. Under this scenario, even complete HIV suppression might be unable to prevent persistent immune activation if naive CD4 T-cell reconstitution is poor. Aging would also represent another complicating factor, as thymus function declines dramatically in adulthood and naive T-cell output slows to a relative trickle by the sixth decade of life.
One means to address whether the data from basic immunology studies have any relevance to humans (which is extremely uncertain, given the large differences between mice and people) may be through studies of immunebased therapies with the potential to accelerate naive T-cell reconstitution. Interleukin-7 is one such candidate and phase I trials are ongoing. A pilot study published by Laura Napolitano in the Journal of Clinical Investigation has shown that human growth hormone (considered inappropriate for further development due to potential toxicity) can increase naive T cell levels in people with HIV and it also significantly decreased CD38 expression on CD4 and CD8 T cells. A growth hormone derivative, tesamorelin, is now being developed as a treatment for lipodystrophy but the immunological effects have yet to be evaluated.
Hopefully, further research with these types of interventions will help answer the question of whether naive T-cell reconstitution can contribute to reducing microbial translocation and immune activation. Since poor immune reconstitution despite ART is also associated with a significantly elevated risk of clinical illness and death, the issue is not just academic; it is possible that a successful immune-based therapy could produce measurable health benefits.
Studies Suggest Microbial Translocation Contributes to Liver Disease and Neurological Impairment in HIV Infection
Systemic lipopolysaccharides (LPS) resulting from microbial translocation have been associated with alcohol-induced liver disease, via interactions with liver macrophages (called Kupffer cells) that promote production of proinflammatory and profibrogenic cytokines. This led Ashwin Balagopal and colleagues from the Johns Hopkins Medical Institutions to study the role of microbial translocation on liver disease progression among individuals coinfected with HIV and hepatitis C. Their results, published in the July issue of the journal Gastroenterology, showed that CD4 Tcell depletion is strongly associated with microbial translocation. In a cohort of 29 individuals with pre- and post-HIV seroconversion samples available, a decline in peripheral blood CD4 counts to less than 200 was associated with significantly higher levels of LPS compared to participants whose CD4 T cell counts remained above that threshold.
In a larger group of 88 individuals with either cirrhosis or minimal liver disease, progression of liver disease was found to occur 19 times more frequently among individuals with levels of LPS in the highest quartile compared to those with levels in the two lowest quartiles. The study authors supplemented this analysis with a look back at stored samples from 53 members of this cohort for whom samples were available from at least eight years prior to the ascertainment of their liver disease status. Compared with baseline, a statistically significant elevation in levels of LPS became evident during the year prior to the development of liver damage, but not before. In discussing these results, the researchers point out that untangling the cause-and-effect relationship between microbial translocation and liver disease will require additional studies, as their data cannot completely rule out the possibility that liver disease caused the observed microbial translocation, as opposed to the other way around. They also stress that causality could plausibly go in both directions.
In the journal PLoS One in June of 2008, Petronela Ancuta and colleagues from the Dana-Farber Cancer Institute in Boston published data showing that microbial translocation is associated with the development of HIVassociated dementia (HAD). The impetus for the study came from prior findings that LPS induce the activation of immune system cells called monocytes and thus increases monocyte trafficking into the brain; these events appear key in precipitating HAD. Ancuta evaluated levels of LPS in a cohort of 119 people with AIDS and found that they were associated with HAD independently of HIV viral loads and CD4 counts. The researchers also discovered that intravenous heroin use, alcohol use, and hepatitis C coinfection were all associated with higher levels of LPS in their cohort.
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